WO2018058489A1 - Cacna1h衍生的肿瘤抗原多肽及其应用 - Google Patents

Cacna1h衍生的肿瘤抗原多肽及其应用 Download PDF

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WO2018058489A1
WO2018058489A1 PCT/CN2016/100978 CN2016100978W WO2018058489A1 WO 2018058489 A1 WO2018058489 A1 WO 2018058489A1 CN 2016100978 W CN2016100978 W CN 2016100978W WO 2018058489 A1 WO2018058489 A1 WO 2018058489A1
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cancer
polypeptide
tumor
cell
nucleic acid
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PCT/CN2016/100978
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French (fr)
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李光磊
侯勇
罗顺涛
林秀妹
安婷
李波
张伟
赵正琦
程震
李汉东
杨乃波
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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 CN201680089572.9A priority Critical patent/CN109790224A/zh
Priority to PCT/CN2016/100978 priority patent/WO2018058489A1/zh
Priority to US16/338,126 priority patent/US11548925B2/en
Publication of WO2018058489A1 publication Critical patent/WO2018058489A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
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    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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
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    • A61K39/4615—Dendritic cells
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    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • 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
    • G01N33/57496—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 intracellular compounds
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H10/00—ICT specially adapted for the handling or processing of patient-related medical or healthcare data
    • G16H10/40—ICT specially adapted for the handling or processing of patient-related medical or healthcare data for data related to laboratory analysis, e.g. patient specimen analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • A—HUMAN NECESSITIES
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    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to the field of diagnostic, prophylactic and immunotherapeutic techniques for cancer, and in particular to a tumor antigen polypeptide resulting from mutation of the CACNA1H gene, related products, and their medical use.
  • 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 [4,5 ] .
  • Immunotherapy enhances the tumor microenvironment's anti-tumor immunity by regulating the immune system of the body, 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 [ 5,6] .
  • Tumor immunotherapy vaccines mainly include tumor cell vaccine, dendritic cell vaccine, protein & peptide vaccine, nucleic acid vaccine, genetic engineering vaccine and anti-idiotypic tumor vaccine [7] .
  • 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 CTL-specific killing, among which CTL-specific killing plays a large role in tumor immune response.
  • 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 represented by peptide-specific DC-CTL, and TIL adoptive reinfusion, has a good therapeutic effect [8,9] .
  • 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 CACNA1H gene encodes a 2353 amino acid-length protein gene with a molecular weight of 259,163 daltons encoding a T-type calcium channel Cav3.2, which acts as a subunit of a voltage-sensitive calcium channel and is involved in the transport of calcium ions into excitable cells. Participate in multiple processes of calcium dependence, including muscle contraction, release of hormones or neurotransmitters, gene expression, cell movement, division and death.
  • the object of the present invention is to provide a tumor antigen polypeptide caused by mutation of a CACNA1H gene, a nucleic acid encoding the tumor antigen polypeptide, a nucleic acid construct comprising the nucleic acid, an expression vector, a host cell, and an antigen presenting cell of the tumor antigen polypeptide and Immune effector cells, and their medical use.
  • the mutation of the CACNA1H gene causes an amino acid sequence change such that the amino acid at position 1428 is mutated from arginine to methionine.
  • the mutated CACNA1H gene is capable of high level expression in tumor tissues and, therefore, enables expression of related polypeptides at high levels in tumor tissues. So far, tumor-specific polypeptide sequences caused by the above mutation of the CACNA1H gene have not been reported at home and abroad, and the polypeptide has not been used for immunotherapy research of tumors; the polypeptide has tumors because it is expressed only in mutant tumor tissues. Tissue specificity is therefore of great importance for tumor detection, early prevention, and immunotherapy in patients.
  • the invention provides an isolated polypeptide, or an immunologically active fragment thereof, selected from the group consisting of:
  • the inventors of the present invention found that the mutation of the CACNA1H gene from the melanoma database resulted in the mutation of the amino acid encoded by the 1428 site from arginine (Arg, R) to methionine (Met, M), and predicted by computer prediction software.
  • the mutant polypeptide sequence has high affinity with HLA-A, especially HLA-A0201, which consists of 9 amino acids with a molecular weight of 974.23 Daltons and a full length sequence of: TLISSLMPI (ie, SEQ ID NO: 2).
  • the polypeptide was synthesized by chemical synthesis and tested by T2 affinity to confirm that the polypeptide does have high affinity with HLA-A, especially with HLA-A0201.
  • ELISPOTs The in vitro immunogenicity assay (ELISPOTs) confirmed that the polypeptide can induce antigen-specific T cells to secrete IFN- ⁇ cytokines, which can induce activation of immune cells; and LDH release experiments confirmed that CD8+ T cells can be specific sexual recognition presents the target cells of the polypeptide and kills the target cells.
  • a polypeptide formed by substitution, addition or deletion of one or more amino acid residues based on the amino acid sequence of (a) a polypeptide may have the above-described function of (a) a polypeptide, ie, with HLA -A, especially with HLA-A0201, has high affinity and can be specifically recognized by CD8+ T cells, resulting in a specific immune response; with cytotoxic T lymphocyte inducing ability.
  • the substitution, addition or deletion of the one or more amino acids is a substitution of amino acid position 2 and/or amino acid 9 of the amino acid sequence shown in SEQ ID NO: 2;
  • substitution, addition or deletion of the one or more amino acids is such that the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 is substituted with M and/or the amino acid at position 9 is substituted with L or V;
  • the (b) polypeptide has SEQ ID NO: 3 (ie, TLISSLMPL), SEQ ID NO: 4 (ie, TLISSLMPV), SEQ ID NO: 5 (ie, TMISSLMPI), SEQ ID NO: The amino acid sequence shown in 6 (ie, TMISSLMPL) or SEQ ID NO: 7 (ie, TMISSLMPV).
  • polypeptides of the present invention can be synthesized according to methods used in conventional peptide chemistry, including, for example, the methods described in Peptide Synthesis, Interscience, New York, 1966; the polypeptides of the present invention can also be prepared by conventional genetic engineering.
  • the polypeptide encoding the polypeptide can be prepared using conventional DNA synthesis and genetic engineering methods to prepare the polypeptide; that is, the polypeptide can be prepared by inserting the above nucleotide into a commonly used expression vector; The recombinant expression vector transforms the host cell; the resulting transformant is cultured; and the polypeptide is collected from the culture. This can be done, for example, by the method described in Molecular Cloning, T. Maniatis et al., CSH Laboratory (1983). The polypeptide obtained by the above method can be confirmed by reversed-phase high performance liquid chromatography-mass spectrometry.
  • the invention provides an isolated nucleic acid encoding the isolated polypeptide of the first aspect.
  • the invention provides a nucleic acid construct comprising the nucleic acid of the second aspect, and one or more control sequences operably linked thereto for directing production of the polypeptide in an expression host.
  • the invention provides an expression vector comprising the nucleic acid construct of the third aspect.
  • the invention provides a host cell, wherein the nucleic acid construct of the third aspect or the expression vector of the fourth aspect is transformed or transfected.
  • the present invention provides an antigen presenting cell which presents the isolated polypeptide of the first aspect on the cell surface.
  • the invention provides a method of producing an antigen presenting cell of the sixth aspect, comprising: The step of contacting the polypeptide of the first aspect with a cell having antigen-presenting ability, or comprising: the nucleic acid of the second aspect, or the nucleic acid construct of the third aspect, or the fourth aspect The step of introducing the expression vector into a cell having antigen presentation ability;
  • the cell having antigen presenting ability is a dendritic cell.
  • the present invention provides an immune effector cell which recognizes the polypeptide of the first aspect or an antigen presenting cell which recognizes a polypeptide as described in the first aspect on the cell surface.
  • the present invention provides the method of producing an immune effector cell according to the eighth aspect, comprising: the step of contacting the antigen presenting cell according to the sixth aspect with an immunogenic effector cell;
  • the immunogenic effector cell is a T cell, preferably a CD8+ T cell.
  • the present invention provides a targeted immune cell population formed by mixing and co-cultivating antigen presenting cells with lymphocytes.
  • the present invention provides a conjugate comprising the polypeptide of the first aspect and an anticancer drug.
  • the invention provides an antibody which specifically recognizes the polypeptide of the first aspect.
  • the present invention provides a method of preparing an antibody comprising:
  • the antibody of interest is purified from the serum.
  • the present invention provides a vaccine for treating or preventing cancer in a patient, comprising the polypeptide of the first aspect, or comprising the nucleic acid of the second aspect, or comprising the third aspect
  • the cancer is a cancer that expresses the polypeptide of the first aspect
  • the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.
  • the present invention provides a pharmaceutical composition for treating or preventing cancer in a patient, comprising the polypeptide of the first aspect, and a pharmaceutically acceptable carrier.
  • the present invention provides the use of a polypeptide according to the first aspect, in the preparation of an antibody for preventing or treating a tumor,
  • 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 and brain tumor.
  • the present invention provides the use of the polypeptide of the first aspect, in the preparation of a medicament for preventing or treating a tumor,
  • 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 and brain tumor.
  • the present invention provides the use of a polypeptide according to the first aspect, in the preparation of a vaccine for preventing or treating a tumor,
  • 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 and brain tumor.
  • the present invention provides the use of the polypeptide of the first aspect or the detection reagent of the nucleic acid of the second aspect, in the preparation of a kit for diagnosing cancer in a patient;
  • the cancer is a cancer that expresses the polypeptide of the first aspect
  • the cancer is selected from the group consisting of lung cancer, melanoma, breast cancer, nasopharyngeal cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, skin cancer, prostate cancer, cervical cancer, leukemia and brain tumor.
  • the invention provides a kit for diagnosing cancer in a patient, comprising the polypeptide of the first aspect or the detection reagent of the nucleic acid of the second aspect.
  • the present invention provides a method of treatment comprising:
  • An effective amount of the polypeptide of the first aspect, the antigen presenting cell of the sixth aspect, the immune effector cell of the eighth aspect, the targeted immune cell population of the tenth aspect, The conjugate according to the eleventh aspect, the antibody according to the fourteenth aspect, or the pharmaceutical composition according to the fifteenth aspect.
  • the present invention provides a diagnostic method comprising:
  • 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 and brain tumor.
  • the tumor antigen polypeptide TLISSLMPI of the present invention is produced by a tumor-specific mutation of the CACNA1H gene, which is absent in normal human tissues in which the mutation does not occur, and is present only in the tumor tissue of the patient in which the mutation occurs; since it exists only In patients with tumor tissue, but not in normal tissues, so its specificity is high, and the specificity of the immune response caused is also high, which can cause specific immune response against tumors; when it is used as a tumor vaccine, It is safer than other tumor polypeptide vaccines, has few side effects, rarely causes serious immune reactions, and is suitable for industrial production because of its simple structure and easy artificial synthesis.
  • variable form of the above polypeptide has an enhanced binding to HLA-A0201 and is associated with T cells.
  • the specificity does not change, therefore, these variable forms have the same activation-specific T immunity as the polypeptide TLISSLMPI.
  • the polypeptide TLISSLMPI or a variant thereof can be used as a target or vaccine for tumor biotherapy directed to the simultaneous expression of HLA-A0201 and the mutant polypeptide.
  • the polypeptide TLISSLMPI or a variable form thereof can be used for the prevention and treatment of tumors by using a polypeptide + adjuvant, or a polypeptide-supported DC vaccine, or a polypeptide-specific DC-CTL, a DC-CIK vaccine, etc.; Polypeptide sequences of lung cancer, 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.
  • polypeptide of the present invention is present only in tumor tissues, and whether the free polypeptide is present in the serum by mass spectrometry, it can be used as a tumor marker for the diagnosis of tumors.
  • Figure 1 is a graph showing the results of flow cytometry for detecting the affinity of the polypeptide of the present invention with T2.
  • Figure 2 shows a graph of the results of immunocyte-specific killing of target cells presenting a polypeptide of the present invention.
  • Figure 3 shows tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention
  • Figure 3A shows the use of adjuvant, adjuvant + wild type polypeptide (TLISSLRPI, SEQ ID NO: 1), adjuvant + mutation
  • TLISSLRPI wild type polypeptide
  • FIG. 3B shows the survival of the mouse after treatment with an adjuvant, an adjuvant + a wild type polypeptide, an adjuvant + a mutant polypeptide TLISSLMPI or a variant polypeptide thereof rate.
  • Figure 4 shows the tumor growth inhibitory effect and mouse survival rate after immunotherapy using the polypeptide of the present invention
  • Figure 4A shows treatment with a DC-loaded wild-type polypeptide, a DC-loaded mutant polypeptide (TLISSLMPI) or a variant polypeptide thereof
  • Figure 4B shows mouse survival after treatment with DC-loaded wild-type polypeptide, DC-loaded mutant polypeptide TLISSLMPI or a variant thereof.
  • Figure 5 shows the tumor growth inhibitory effect and mouse survival rate after immunotherapy using the polypeptide of the present invention
  • Figure 5A shows the use of a wild-type polypeptide (TLISSLRPI), a mutant polypeptide (TLISSLMPI) or its variable
  • TLISSLRPI wild-type polypeptide
  • TLISSLMPI mutant polypeptide
  • FIG. 5B shows DC cells infected with a lentiviral vector carrying a wild-type polypeptide (TLISSLRPI), a mutant polypeptide (TLISSLMPI) or a variable form thereof Survival rate of mice after treatment.
  • Figure 6 shows tumor growth inhibition effect and mouse survival rate after immunotherapy using the polypeptide of the present invention
  • Figure 6A shows the use of DC-loaded wild-type polypeptide (TLISSLRPI) + CTL, DC-loaded mutant polypeptide (TLISSLMPI) or Tumor growth inhibitory effect after variable form polypeptide + CTL treatment
  • Figure 6B shows treatment with DC-loaded wild-type polypeptide (TLISSLRPI) + CTL, DC-loaded mutant polypeptide (TLISSLMPI) or its variable form polypeptide + CTL Mouse survival rate.
  • the affinity of the polypeptide is predicted by the following procedure:
  • the affinity prediction of the polypeptide was performed using a self-developed "tumor DNA and RNA sequencing-based mutant polypeptide binding ability prediction software" (software copyright number: 2016SR002835).
  • the predicted results are expressed as IC50 scores, an IC50 of less than 500 indicates that the polypeptide has affinity, and an IC50 of less than 50 indicates that the polypeptide has high affinity.
  • the wild type polypeptide TLISSLRPI (SEQ ID NO: 1), the mutant polypeptide of the present invention TLISSLMPI and its five variable form polypeptides (also belonging to the present invention) were obtained by standard solid phase synthesis and purified by reverse phase HPLC. The purity (>90%) and identity of the polypeptide were determined by HPLC and mass spectrometry, respectively.
  • the affinity of the wild-type polypeptide and several mutant polypeptides of the present invention to HLA-A0201 was predicted using a mutant polypeptide binding ability prediction software based on tumor DNA and RNA sequencing, and the predicted score is shown in Table 1 below.
  • the IC50 score of the mutant polypeptide of the present invention was less than 50 nM as predicted by computer software, indicating that the mutant polypeptide of the present invention has high affinity with HLA-A0201.
  • T2 cells a lymphocyte, a tumor cell line, and an HLA-expression
  • T2 cells are cell lines deficient in the antigenic polypeptide transporter (TAP) required for endogenous antigen presentation, and are HLA-A2-positive T and B lymphocyte hybridoma cells, which can be used to study polypeptides and HLA-A2.
  • TEP antigenic polypeptide transporter
  • the cells were harvested by centrifugation at 200 g for 5 minutes. After the collected cells were washed twice with PBS, the cells were directly incubated with anti-HLA-A 0201 FITC monoclonal antibody and maintained at 4 ° C for 30 minutes.
  • flow cytometry BD FACSJazz TM
  • software to detect and analyze the mean fluorescence intensity (MFI).
  • MFI mean fluorescence intensity
  • FI [MFI sample- MFI background ]/MFI background ;
  • the MFI background represents a peptide-free value
  • FI > 1.5 indicates that the peptide has a high affinity for the HLA-A0201 molecule
  • 1.0 ⁇ FI ⁇ 1.5 indicates that the peptide has a medium affinity for the HLA-A0201 molecule
  • 0.5 ⁇ FI ⁇ 1.0 indicates that the peptide is low affinity for HLA-A0201 molecule.
  • Example 3 Mutant polypeptide of the present invention stimulates expansion of CD8+ T cells in vitro
  • PBMC cells from HLA-A0201 subtype-positive volunteers 2 ⁇ 10 7 PBMC cells were isolated, and CD8+ T cells were isolated by adherent separation of monocytes (posted for 3 h) and CD8 magnetic beads.
  • GM-CSF 1000 U/ml
  • IL-4 1000 U/ml
  • IFN- ⁇ 100 U/ml
  • CD40L 100 U/ml
  • the mutant polypeptide TLISSLMPI or any of its five variable form polypeptides induces adherent cells to be polypeptide-specific mature DC cells.
  • the mature DC cells carrying the polypeptide were irradiated, co-cultured with CD8+ T cells of the volunteers, and IL-21 was added. After 3 days, IL-2 and IL-7 were added, and then supplemented on the 5th and 7th days. IL-2 and IL-7 (final concentrations of IL-21, IL-2 and IL-7 were 30 ng/ml, 5 ng/ml and 10 ng/ml, respectively), and co-cultured cells were counted on day 10 and performed. Subsequent ELISPOTs and LDH testing. The count results are shown in Table 3 below.
  • Example 4 ELISPOTs method to verify that the mutant polypeptide of the present invention activates CD8+ T cell immune response
  • an ELISPOTs detection kit (Cat. No.: 3420-4 AST-10, MABTECH) was used to verify that the polypeptide of the present invention activates the immune response of CD8+ T cells.
  • CD8+ T cells can specifically recognize the complex of HLA-A0201 and polypeptide, and the difference in polypeptide sequence is different from the T cell population of the complex of HLA-A0201. Since T2 cells express HLA-A0201, CD8+ T cells can specifically recognize T2 cells loaded with polypeptides. After specifically recognizing the complex of HLA-A0201 and polypeptide, polypeptide-specific CD8+ T cells can be activated and secreted again. IFN- ⁇ interferon.
  • CD8+ T cells are activated by secreted IFN- ⁇ interferon can be ELISPOTs
  • the antibody on the plate is captured, and the antibody that finally recognizes IFN- ⁇ can develop a color by degrading the substrate by an enzyme coupled to the antibody, eventually producing spots.
  • the number of spots represents the number of cells that are activated to secrete IFN- ⁇ interferon.
  • the cells cultured in Experimental Example 3 were separately cultured with T2 cells loaded with the mutant polypeptide TLISSLMPI and the wild type polypeptide TLISSLRPI of the present invention into ELISPOTs plates, and ELISPOTs were detected 20 hours later (see kit instructions). Finally, the spots produced by the ELISPOTs test are counted.
  • the immunogenicity of the test polypeptide is as follows: number of spots (test polypeptide) / number of spots (unrelated polypeptide) > 2; that is, the number of spots caused by the test polypeptide exceeds twice the number of unrelated polypeptide spots, indicating that the test polypeptide has an immunogen Sex.
  • polypeptide of the present invention and its variable form are immunogenic and can specifically activate the CD8+ T cell immune response.
  • Example 5 LDH release assay demonstrates the specificity of CD8+ T cells for target cells presenting a polypeptide of the invention Killing activity
  • the cells cultured in Experimental Example 3 were co-cultured with the mutant polypeptide of the present invention or the wild type polypeptide or the T2 cell without the polypeptide, and the largest release well, the volume correction well, the medium control well, and the spontaneous release well were set in the experiment.
  • three replicate wells were set in each group.
  • 50 ⁇ l of the co-cultured cell supernatant was taken out and added to 50 ⁇ l of LDH substrate mixture.
  • the cell supernatant catalyzes the LDH substrate reaction, finally reads the 490 nm wavelength and the 680 nm reference wavelength, and calculates the killing activity of the target cell killing T2 according to the control well.
  • the test results are shown in Figure 2 and Table 5 below.
  • 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%
  • T cells specifically recognize and kill target cells that present experimental peptides
  • the mutant polypeptide TLISSLMPI of the present invention and its variable form polypeptide-activated T cells can kill T2 which presents a mutant polypeptide at a target ratio of 1:1 or 1:10.
  • Example 6 Construction and packaging of a mutant polypeptide TLISSLMPI and its variable form polypeptide recombinant lentivirus
  • pHBLV-Puro-TLISSLRPI pHBLV-Puro-TLISSLMPI
  • pHBLV-Puro-TLISSLMPL pHBLV-Puro-TLISSLMPV
  • pHBLV-Puro-TMISSLMPI pHBLV-Puro-TMISSLMPL
  • pHBLV-Puro-TMISSLMPV pHBLV-Puro-TMISSLMPV.
  • the seven lentiviral plasmids were co-transfected into 293T cells with pSPAX2 and pMD2G helper plasmids, and the wild type polypeptide TLISSLRPI, the mutant polypeptide TLISSLMPI and its variable form polypeptide lentivirus were packaged.
  • the human non-small cell lung adenocarcinoma cell line NCI-H2087 was purchased from ATCC 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 100 U/ml streptomycin, and cultured in an incubator at 37 ° C, 5% CO 2 .
  • the TLISSLMPI lentivirus packaged in Example 6 was transfected into the H2087 cell line, and the surviving H2087 cell line was continuously screened with Puromycin antibiotic, and finally the H2087 cell line expressing the TLISSLMPI polypeptide was established and designated as the H2087-TLISSLMPI cell line.
  • the healthy volunteers were collected from 600 to 900 ml of anticoagulated peripheral blood. Ficoll was used to separate peripheral blood mononuclear cells (PBMC), and the cells were collected for use. 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. Mice after 4 weeks of selection were prepared to inoculate a human lung cancer cell line model.
  • PBMC peripheral blood mononuclear cells
  • Example 9 Establishment of a subcutaneous xenograft model of H2087-TLISSLMPI
  • the human non-small cell lung adenocarcinoma cell line H2087-TLISSLMPI established in Example 7 was cultured in DMEM medium containing 10% fetal calf serum, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C. Incubate in a 5% CO 2 incubator. H2087-TLISSLMPI tumor cells were collected, centrifuged at 1500 rpm for 5 min, and the tumor cells were washed 3 times with sterile physiological saline.
  • mice After 7 days, the mice were able to reach the tumor of about rice size subcutaneously.
  • the NOD/SCID mice of the H2087-TLISSLMPI subcutaneous tumor model were treated with DC-CTL vaccine.
  • the H2087-TLISSLMPI subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were treated with peptide + complete Freund's adjuvant, or polypeptide + DC vaccine, or lentivirus-infected DC cell vaccine, and DC-CTL vaccine, respectively.
  • the volume of the tumor and the survival rate of the mice were recorded every 2 days.
  • the H2087-TLISSLMPI subcutaneous tumor model NOD/SCID mice immunized for 4 weeks were randomly divided into 8 groups: adjuvant + wild type polypeptide group, adjuvant group, adjuvant + TLISSLMPI polypeptide group and adjuvant + 5 kinds of variable Formal peptide group, 6 in each group.
  • the first immunization dose of the above polypeptide was 100 ⁇ g/mouse.
  • the polypeptide was resuspended in PBS, and then mixed with 150 ⁇ l/micro mouse Freund's complete adjuvant, adjusted to 300 ⁇ l/cell with PBS, and injected into the back subcutaneously.
  • the polypeptide, the mutant polypeptide TLISSLMPI and its five variable form polypeptides were incubated for 4 hours and washed three times with physiological saline.
  • the DC of the loaded polypeptide was adjusted to (4.0 ⁇ 0.5) ⁇ 10 7 /ml with physiological saline for subsequent experiments.
  • Tumor-bearing mice were randomly divided into 7 groups: DC-loaded wild-type polypeptide group, DC-loaded mutant polypeptide TLISSLMPI, and DC-loaded with 5 variable-form polypeptides, 6 in each group.
  • a DC-loaded wild-type polypeptide, a DC-loaded TLISSLMPI polypeptide, and a DC-loaded cell suspension of its five variable form polypeptides were prepared.
  • the tumor-bearing mice were intradermally injected into the inner thigh of the inguinal region, 0.1 ml per side, and once a week. The dose was (4.0 ⁇ 0.5) ⁇ 10 6 cells/time, and a total of 2 injections.
  • Example 6 (Constructed as in Example 6). After 24 h, the virus culture solution 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 cultured at 37 ° C, 5% CO 2 . Cultivate in the box. After 16 h, the DC cells were adjusted to (4.0 ⁇ 0.5) x 10 7 / ml for subsequent experiments.
  • Tumor-bearing mice were randomly divided into 7 groups: wild-type polypeptide-DC group, TLISSLMPI polypeptide-DC group, and 5 variable-form polypeptide-DC groups, 6 in each group.
  • a DC-loaded wild-type polypeptide, a DC-loaded TLISSLMPI polypeptide, and a DC-loaded cell suspension of its five variable form polypeptides were prepared.
  • Immunologically reconstituted tumor-bearing mice were injected intradermally into the inner thigh of the inguinal region, 0.1 ml per side, and once a week. The dose was (4.0 ⁇ 0.5) ⁇ 10 6 cells / time, and a total of 2 injections.
  • Fig. 5 show that the recombinant polypeptide of the mutant polypeptide TLISSLMPI or its five variable form polypeptide genes has a significant tumor suppressing effect and prolongs the mouse relative to the wild type polypeptide group. The survival period, but the wild-type polypeptide does not respond to the tumor.
  • Example 13 Preparation of a polypeptide-specific CTL vaccine and a treatment regimen using the vaccine in vivo
  • PBL peripheral blood lymphocytes
  • CD8+T 1:4.
  • PI number of cells after expansion / number of cells inoculated.
  • cells i.e., cytotoxic T lymphocytes (CTL)
  • CTL cytotoxic T lymphocytes
  • the cells were resuspended in saline, the volume was 0.2 ml, and the cells were reinfused through the tail vein.
  • the number of cells transfused in each tumor model mouse was about 1 ⁇ 10 8 cells.
  • the vital signs of the mice were observed and the vertical and horizontal dimensions of the tumors were measured with vernier calipers every 2 days.
  • the results are shown in Figure 6.
  • the results in Figure 6 show that the mutant polypeptide TLISSLMPI or its five variable form polypeptide-activated DC-CTL vaccines have significant tumor suppressive effects and prolonged mice compared to the wild-type polypeptide group. Survival period.

Abstract

本发明提供了一种具有如SEQ ID NO：2所示氨基酸序列的肿瘤抗原多肽或其变体，其编码核酸，包含该编码核酸的核酸构建体，表达载体，宿主细胞，在细胞表面呈递该肿瘤抗原多肽的抗原呈递细胞及其免疫效应细胞。本发明还提供了所述多肽，核酸，抗原呈递细胞或免疫效应细胞在诊断，预防，治疗癌症中的应用。

Description

CACNA1H衍生的肿瘤抗原多肽及其应用 技术领域
本发明涉及癌症的诊断、预防及免疫治疗技术领域，具体地，本发明涉及一种由CACNA1H基因突变导致的肿瘤抗原多肽，其相关产品，以及它们的医药用途。
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[1]。《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[2]。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[3]。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[4，5]。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[5，6]。
肿瘤免疫治疗疫苗主要包括肿瘤细胞疫苗、树突状细胞疫苗、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[7]。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TIL过继回输等方法为代表，具有良好的治疗效果[8，9]。
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。
CACNA1H基因编码一个2353个氨基酸长度，分子量大小为259,163道尔顿的蛋白基因编码T型钙通道Cav3.2，作为电压敏感的钙离子通道的组成亚基，参与转运钙离子进入易兴奋细胞，并参与钙离子依赖的多个过程，包括肌肉收缩，激素或神经传递素的释放，基因表达，细胞运动，分裂与死亡等。
参考文献：
[1]World Health Organization.Globocan 2012：Estimated cancer incidence，mortality and prevalence worldwide in 2012.
[2]Bernard W.Stewart CPW.World Cancer Report 2014.2014.
[3]哈小琴，张尚弟，杨志华，张俊.肿瘤生物治疗的新技术--靶向基因治疗.解放军医药杂志2014；26：24-7.
[4]Mellman I，Coukos G，Dranoff G.Cancer immunotherapy comes of age.Nature 2011；480：480-9.
[5]Chen DS，Mellman I.Oncology meets immunology：the cancer-immunity cycle.Immunity 2013；39：1-10.
[6]Currie GA.Eighty years of immunotherapy：a review of immunological methods used for the treatment of human cancer.British journal of cancer 1972；26：141-53.
[7]李亭葶，李晖，王熙才.肿瘤疫苗在肿瘤治疗中的研究进展.现代肿瘤医学2013；21：2351-3
[8]Tran E，Turcotte S，Gros A，et al.Cancer immunotherapy based on mutation-specific CD4+T cells in a patient with epithelial cancer.Science.2014.344(6184)：641-5.
[9]Cobbold M，De La Pena H，Norris A，et al.MHC class I-associated phosphopeptides are the targets of memory-like immunity in leukemia.Sci Transl Med.2013.5(203)：203ra125.
发明内容
本发明的目的在于提供一种由CACNA1H基因突变导致的肿瘤抗原多肽，编码该肿瘤抗原多肽的核酸，包含该核酸的核酸构建体、表达载体、宿主细胞，以及该肿瘤抗原多肽的抗原呈递细胞和免疫效应细胞，以及它们的医药用途。
CACNA1H基因的突变，引起其氨基酸序列的改变，使得其第1428位点的氨基酸由精氨酸突变为蛋氨酸。突变后的CACNA1H基因能够在肿瘤组织中高水平表达，因此，也使得相关多肽能够在肿瘤组织内高水平的表达。迄今为止国内外尚未报道过由CACNA1H基因的上述突变导致的肿瘤特异性多肽序列，也未见所述多肽用于肿瘤的免疫治疗研究；所述多肽因其只表达于突变的肿瘤组织，具有肿瘤组织特异性，因此对于患者的肿瘤检测，早期预防，以及免疫治疗，具有非常重要的意义。
在第一方面，本发明提供了一种分离的多肽或其免疫学活性片段，所述多肽选自：
(a)由SEQ ID NO：2所示氨基酸序列组成的多肽；
(b)由SEQ ID NO：2所示氨基酸序列经过一个或多个氨基酸残基的取代、添加和/或缺失而形成的，且具有细胞毒性T淋巴细胞诱导能力的多肽；以及
(c)(a)多肽或(b)多肽的变体或衍生物，其具有细胞毒性T淋巴细胞诱导能力。
本发明的发明人从黑色素瘤的数据库中发现CACNA1H基因的突变导致其编码的1428位点的氨基酸由精氨酸(Arg，R)突变为蛋氨酸(Met，M)，并通过计算机预测软件，预测了该突变多肽序列与HLA-A、尤其与HLA-A0201具有高亲和力，该多肽由9个氨基酸组成，分子量为974.23道尔顿，全长序列为：TLISSLMPI(即，SEQ ID NO：2)。通过化学合成的方法合成该多肽，并经T2亲和力测试，证实该多肽确实与HLA-A、尤其与HLA-A0201具有高亲和能力。经体外免疫原性实验(ELISPOTs)验证发现，该多肽可以诱导抗原特异性的T细胞分泌IFN-γ细胞因子，可以引起免疫细胞的激活反应；以及经LDH释放实验证实，CD8+T细胞可以特异性识别呈递该多肽的靶细胞并杀伤该靶细胞。
发明人还发现，在(a)多肽的氨基酸序列基础上进行一个或多个氨基酸残基的取代、添加或缺失所形成的(b)多肽可具有(a)多肽的上述功能，即，与HLA-A、尤其与HLA-A0201具有高亲和力，并可被CD8+T细胞特异性识别，从而引起特异性免疫响应；具有细胞毒性T淋巴细胞诱导能力。
优选地，所述一个或多个氨基酸的取代、添加或缺失为如SEQ ID NO：2所示氨基酸序列的第2位和/或第9位氨基酸的取代；
进一步优选地，所述一个或多个氨基酸的取代、添加或缺失为如SEQ ID NO：2所示氨基酸序列的第2位氨基酸取代为M和/或第9位氨基酸取代为L或V；
更进一步优选地，所述(b)多肽具有如SEQ ID NO：3(即，TLISSLMPL)、SEQ ID NO：4(即，TLISSLMPV)、SEQ ID NO：5(即，TMISSLMPI)、SEQ ID NO：6(即，TMISSLMPL)或SEQ ID NO：7(即，TMISSLMPV)所示的氨基酸序列。
经实验验证发现，与(a)多肽相比，与HLA-A、尤其与HLA-A0201的结合力增强，而其与T细胞之间的特异性不变。因此，所述(b)多肽与(a)多肽均具有相同的激活特异性T免疫能力。
本发明的多肽可依照在常规肽化学中使用的方法合成，这些方法包括例如在下列文献中描述的方法：Peptide Synthesis，Interscience，New York，1966；也可以通过常规的基因工程制备本发明的多肽。例如，可使用常规DNA合成和基因工程方法制备编码所述多肽的核苷酸来制备所述多肽；即，通过下述方法制备所述多肽：将上述核苷酸插入常用的表达载体；用得到的重组表达载体转化宿主细胞；培养得到的转化体；和从培养物中收集所述多肽。可参照例如以下文献中描述的方法进行：Molecular Cloning，T.Maniatis等人，CSH Laboratory(1983)。通过上述方法获得的多肽，可通过反向高效液相色谱-质谱的方法进行确认。
第二方面，本发明提供了一种分离的核酸，其编码如第一方面所述的分离的多肽。
第三方面，本发明提供了一种核酸构建体，其包含如第二方面所述的核酸，以及与之可操作连接、可指导多肽在表达宿主中生产的一个或多个控制序列。
第四方面，本发明提供了一种表达载体，其包含如第三方面所述的核酸构建体。
第五方面，本发明提供了一种宿主细胞，其中转化或转染了如第三方面所述的核酸构建体或如第四方面所述的表达载体。
第六方面，本发明提供了一种抗原呈递细胞，其在细胞表面呈递如第一方面所述的分离的多肽。
第七方面，本发明提供了产生如第六方面所述的抗原呈递细胞的方法，其包括： 将如第一方面所述的多肽与具有抗原呈递能力的细胞接触的步骤，或者包括：使如第二方面所述的核酸、或者如第三方面所述的核酸构建体、或者如第四方面所述的表达载体导入具有抗原呈递能力的细胞中表达的步骤；
优选地，所述具有抗原呈递能力的细胞为树突细胞。
第八方面，本发明提供了一种免疫效应细胞，其可识别如第一方面所述的多肽或者识别在细胞表面呈递如第一方面所述的多肽的抗原呈递细胞。
第九方面，本发明提供了产生如第八方面所述的免疫效应细胞的方法，其包括：将如第六方面所述的抗原呈递细胞与具有免疫效应能力的细胞接触的步骤；
优选地，所述具有免疫效应能力的细胞为T细胞，优选为CD8+T细胞。
第十方面，本发明提供了一种靶向性免疫细胞群，其由抗原呈递细胞与淋巴细胞混合并共培养而形成。
第十一方面，本发明提供了一种缀合物，其包含如第一方面所述的多肽和一种抗癌药。
第十二方面，本发明提供了一种抗体，所述抗体特异性识别如第一方面所述的多肽。
第十三方面，本发明提供了一种制备抗体的方法，其包括：
利用如第一方面所述的多肽对动物进行免疫接种；
采集经免疫接种的动物血清；以及
从所述血清中纯化出目的抗体。
第十四方面，本发明提供了一种在患者中治疗或预防癌症的疫苗，其包括如第一方面所述的多肽，或包括如第二方面所述的核酸，或包括如第三方面所述的核酸构建体，或包括如第四方面所述的表达载体，或包括如第六方面所述的抗原呈递细胞，或包括如第八方面所述的免疫效应细胞；
优选地，所述癌症为表达如第一方面所述的多肽的癌症；
进一步优选地，所述癌症选自肺癌，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，白血病和脑肿瘤。
第十五方面，本发明提供了一种在患者中治疗或预防癌症的药物组合物，其包括如第一方面所述的多肽，以及药学上可接受的载体。
第十六方面，本发明提供了如第一方面所述的多肽在制备抗体中的用途，所述抗体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，白血病和脑肿瘤。
第十七方面，本发明提供了如第一方面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，白血病和脑肿瘤。
第十八方面，本发明提供了如第一方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，白血病和脑肿瘤。
第十九方面，本发明提供了如第一方面所述的多肽或者如第二方面所述的核酸的检测试剂在制备用于在患者中诊断癌症的套件中的用途；
优选地，所述癌症为表达如第一方面所述的多肽的癌症；
进一步优选地，所述癌症选自肺癌，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，白血病和脑肿瘤。
第二十方面，本发明提供了一种在患者中诊断癌症的套件，其包括如第一方面所述的多肽或者如第二方面所述的核酸的检测试剂。
第二十一方面，本发明提供了一种治疗方法，其包括：
向患者施用有效量的如第一方面所述的多肽、如第六方面所述的抗原呈递细胞、如第八方面所述的免疫效应细胞、如第十方面所述的靶向性免疫细胞群、如第十一方面所述的缀合物、如第十二方面所述的抗体、如第十四方面所述的疫苗或如第十五方面所述的药物组合物。
第二十二方面，本发明提供了一种诊断方法，其包括：
检测患者来源的生物样品是否携带如第一方面所述的多肽；
基于所述生物样品是否携带所述多肽的事实，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，白血病和脑肿瘤。
有益效果
本发明的肿瘤抗原多肽TLISSLMPI由CACNA1H基因的肿瘤特异性突变所产生，其在未发生该突变的人体正常组织内不存在，只存在于发生该突变的的患者的肿瘤组织中；由于其只存在于患者肿瘤组织，而不存在于正常组织，因此其特异性较高，引起的免疫反应的特异性也较高，其可以引起针对肿瘤的特异性免疫反应；当将其用作肿瘤疫苗时，比其他肿瘤多肽疫苗更为安全，并且副作用小，很少引起严重的免疫反应，又因其结构简单、易于人工合成，因而适于产业化生产。
上述多肽的可变形式由于具有与HLA-A0201的增强的结合力，而与T细胞之间 的特异性不改变，因此，这些可变形式与多肽TLISSLMPI一样，均具有相同的激活特异性T免疫能力。因此，多肽TLISSLMPI或其可变形式可以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。多肽TLISSLMPI或其可变形式可以采用多肽+佐剂，或多肽负载的DC疫苗，或多肽特异性DC-CTL，DC-CIK疫苗等方式，用于肿瘤的预防及治疗；所述肿瘤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。
此外，由于本发明的多肽仅存在于肿瘤组织中，而且可以通过质谱的方式检测到血清中是否存在该游离多肽，因此，其可以作为肿瘤标志物应用于肿瘤的诊断。
附图说明
图1显示流式细胞术检测本发明多肽与T2亲和力的结果图。
图2显示免疫细胞特异性杀伤呈递本发明多肽的靶细胞的结果图。
图3显示使用本发明多肽免疫治疗后的肿瘤生长抑制效果和小鼠生存率；其中，图3A显示使用佐剂、佐剂+野生型多肽(TLISSLRPI，SEQ ID NO：1)、佐剂+突变型多肽TLISSLMPI或其可变形式多肽治疗后的肿瘤生长抑制效果，图3B显示使用佐剂、佐剂+野生型多肽、佐剂+突变型多肽TLISSLMPI或其可变形式多肽治疗后的小鼠生存率。
图4显示使用本发明多肽免疫治疗后的肿瘤生长抑制效果和小鼠生存率；其中，图4A显示使用DC-负载野生型多肽、DC-负载突变型多肽(TLISSLMPI)或其可变形式多肽治疗后的肿瘤生长抑制效果，图4B显示使用DC-负载野生型多肽、DC-负载突变型多肽TLISSLMPI或其可变形式多肽治疗后的小鼠生存率。
图5显示使用本发明多肽免疫治疗后的肿瘤生长抑制效果和小鼠生存率；其中，图5A显示使用携带野生型多肽(TLISSLRPI)、突变型多肽(TLISSLMPI)或其可变 形式多肽的慢病毒载体感染的DC细胞治疗后的肿瘤生长抑制效果，图5B显示使用携带野生型多肽(TLISSLRPI)、突变型多肽(TLISSLMPI)或其可变形式多肽的慢病毒载体感染的DC细胞治疗后的小鼠生存率。
图6显示使用本发明多肽免疫治疗后的肿瘤生长抑制效果和小鼠生存率；其中，图6A显示使用DC-负载野生型多肽(TLISSLRPI)+CTL、DC-负载突变型多肽(TLISSLMPI)或其可变形式多肽+CTL治疗后的肿瘤生长抑制效果，图6B显示使用DC-负载野生型多肽(TLISSLRPI)+CTL、DC-负载突变型多肽(TLISSLMPI)或其可变形式多肽+CTL治疗后的小鼠生存率。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1本发明的突变型多肽的亲和力预测
通过以下程序预测多肽的亲和力：
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)对多肽进行亲和力预测。预测结果用IC50分值表示，IC50小于500表示该多肽有亲和力，IC50小于50表示该多肽具有高亲和力。
通过标准固相合成法，获得野生型多肽TLISSLRPI(SEQ ID NO：1)、本发明的突变型多肽TLISSLMPI及其5种可变形式多肽(也属于本发明)，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。采用基于肿瘤DNA和RNA测序的突变多肽结合能力预测软件预测野生型多肽以及本发明的几种突变型多肽与HLA-A0201的亲和力，预测得分如以下表1所示。
表1、多肽与HLA-A0201的亲和力预测结果
待预测多肽序列 IC50(nM) 野生型多肽序列 IC50(nM)
TLISSLMPI 3.28 TLISSLRPI 3.39
TLISSLMPL 3.18 - -
TLISSLMPV 2.86 - -
TMISSLMPI 2.67 - -
TMISSLMPL 2.49 - -
TMISSLMPV 2.24 - -
由表1可知，经计算机软件预测，本发明的突变型多肽的IC50得分均低于50nM，说明本发明的突变型多肽与HLA-A0201具有高亲和力。
实施例2本发明突变型多肽的T2亲和力验证
按照实施例1中所记载的方式，获得野生型多肽TLISSLRPI、本发明多肽TLISSLMPI及其5种可变形式多肽；取2×105个T2细胞(一种淋巴细胞，肿瘤细胞系，表达HLA-A0201；T2细胞是内源性抗原提呈途径中必需的抗原多肽转运蛋白(TAP)缺陷的细胞株，为HLA-A2阳性的T、B淋巴细胞杂交瘤细胞，可用于研究多肽与HLA-A2的亲和力，T细胞与MHC-I分子的相互作用；ATCC货号：CRL-1992TM)，用500μl IMDM无血清培养基重悬于24孔板中，分别加入10μg/ml上述多肽，并加入人类β2微球蛋白(最终浓度，3μg/ml)，在培养箱(37℃，5％CO2)中培养过夜。每个组设2个重复孔；没有加多肽的T2细胞被用作背景对照(即，空白)，加入CMV多肽(NLVPMVATV)的组用作阳性对照。
将细胞培养物以200g离心5分钟收集细胞。所收集的细胞用PBS洗涤两次后，将细胞直接用抗HLA-A 0201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(MFI)。荧光指数(FI)用下列公式计算：
FI＝[MFI样品-MFI背景]/MFI背景；
其中，MFI背景代表不含肽的值；FI＞1.5表明该肽具有用于HLA-A0201分子的高亲和性，1.0＜FI＜1.5表明该肽具有对HLA-A0201分子中等亲和力，以及0.5＜FI＜1.0表明该肽为HLA-A0201分子低亲和力。
上述各多肽与HLA-A 0201的亲和力检测结果如以下表2所示。
表2、多肽与HLA-A0201亲和力的检测结果
样本 加入多肽浓度 平均荧光强度 FI
TLISSLRPI 100μM 672.3 1.99
TLISSLMPI 100μM 682.7 2.04
TLISSLMPL 100μM 692.4 2.08
TLISSLMPV 100μM 713.6 2.18
TMISSLMPI 100μM 701.3 2.12
TMISSLMPL 100μM 763.9 2.40
TMISSLMPV 100μM 776.5 2.46
空白 0μM 224.6 0
CMV 100μM 656.7 1.92
由表2可知，经过亲和力验证，空白组的FI为0，作为阳性对照的CMV多肽的FI为1.92，两个均正常；而野生型多肽和本发明突变型多肽的FI均大于1.5，进一步证明野生型多肽和本发明突变型多肽都是高亲和力的。
实施例3本发明突变型多肽体外刺激扩增CD8+T细胞
取HLA-A0201亚型阳性的志愿者的PBMC细胞，2×107个PBMC细胞，用贴壁法分离单核细胞(贴3h)，以及CD8磁珠的方法分离得到CD8+的T细胞。采用GM-CSF(1000U/ml)、IL-4(1000U/ml)、诱导贴壁单核细胞为未成熟DC，再用IFN-γ(100U/ml)、CD40L(100U/ml)、本发明突变型多肽TLISSLMPI或其5种可变形式多 肽的任一种诱导贴壁细胞为多肽特异性成熟DC细胞。将负载多肽的成熟DC细胞辐照，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(IL-21、IL-2和IL-7的终浓度分别为30ng/ml、5ng/ml和10ng/ml)，第10天取共培养的细胞进行计数，并进行后续的ELISPOTs以及LDH检测。计数结果如以下表3所示。
表3、培养后细胞计数结果
  培养前孔内细胞总数 培养后孔内细胞总数
TLISSLMPI 2.5×10^6 1.32×10^7
TLISSLMPL 2.5×10^6 1.37×10^7
TLISSLMPV 2.5×10^6 1.63×10^7
TMISSLMPI 2.5×10^6 1.46×10^7
TMISSLMPL 2.5×10^6 1.76×10^7
TMISSLMPV 2.5×10^6 1.87×10^7
由表3可知，培养10天后，细胞有明显增殖，总的细胞数目扩增倍数在5-7倍之间，说明本发明多肽的加入可明显刺激CD8+T细胞的扩增。
实施例4ELISPOTs方法验证本发明突变型多肽激活CD8+T细胞免疫反应
本实施例采用ELISPOTs检测试剂盒(货号：3420-4AST-10，MABTECH)，验证本发明多肽激活CD8+T细胞的免疫反应。
ELISPOTs检测方法原理：CD8+T细胞能够特异性识别HLA-A0201和多肽的复合物，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多肽的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-γ干扰素。而CD8+T细胞被激活分泌的IFN-γ干扰素可以被ELISPOTs 板子上的抗体所捕获，最终识别IFN-γ的抗体可以通过偶联在抗体上的酶，降解底物显色，最终产生斑点。斑点的数目代表了被激活分泌IFN-γ干扰素的细胞数目。
将实验例3中培养后的细胞分别与负载本发明突变型多肽TLISSLMPI和野生型多肽TLISSLRPI的T2细胞加入到ELISPOTs板中进行培养，20小时后进行ELISPOTs检测(见试剂盒说明书)。最后，对ELISPOTs检测产生的斑点进行计数。
测试多肽具有免疫原性的要求如下：斑点数(测试多肽)/斑点数(无关多肽)＞2；即，测试多肽引起的斑点数超过无关多肽斑点数目的两倍以上，表明测试多肽具有免疫原性。
上述各多肽的ELISPOTs检测结果见如下表4。
表4、多肽刺激特异性CD8+T细胞分泌IFN-γ干扰素
Figure PCTCN2016100978-appb-000001
Figure PCTCN2016100978-appb-000002
由表4结果可知，本发明多肽及其可变形式具有免疫原性，可以特异性地激活CD8+T细胞免疫反应。
实施例5LDH释放实验证明CD8+T细胞对呈递本发明多肽的靶细胞的特异性 杀伤活性
将实验例3中培养的细胞与负载本发明的突变型多肽或野生型多肽或未负载多肽的T2细胞进行共培养，实验中设置最大释放孔，体积校正孔，培养基对照孔，自发释放孔，不同效靶比(T细胞与T2细胞的数目比)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，计算靶细胞杀伤T2的杀伤活性。检测结果见图2和如下表5。
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％
表5、T细胞特异性识别并杀伤呈递实验多肽的靶细胞
组别 效靶比(1∶1) 效靶比(10∶1)
T细胞(TLISSLMPI)+T2(TLISSLRPI) 2.23％ 4.23％
T细胞(TLISSLMPI)+T2(TLISSLMPI) 7.96％ 38.89％
T细胞(TLISSLMPL)+T2(TLISSLRPI) 3.12％ 5.71％
T细胞(TLISSLMPL)+T2(TLISSLMPI) 8.23％ 39.66％
 T细胞(TLISSLMPV)+T2(TLISSLRPI) 2.57％ 4.73％
T细胞(TLISSLMPV)+T2(TLISSLMPI) 8.56％ 43.82％
T细胞(TMISSLMPI)+T2(TLISSLRPI) 2.85％ 5.13％
T细胞(TMISSLMPI)+T2(TLISSLMPI) 8.33％ 41.16％
T细胞(TMISSLMPL)+T2(TLISSLRPI) 3.13％ 5.83％
T细胞(TMISSLMPL)+T2(TLISSLMPI) 9.12％ 45.23％
T细胞(TMISSLMPV)+T2(TLISSLRPI) 3.51％ 6.31％
T细胞(TMISSLMPV)+T2(TLISSLMPI) 9.32％ 47.35％
由图2和表5结果可知，在效靶比为1∶1或1∶10时，本发明的突变型多肽TLISSLMPI及其可变形式多肽激活的T细胞，能够杀伤呈递了突变型多肽的T2细胞，而不能杀伤呈递野生型多肽的T2细胞，这进一步验证了本发明的突变型多肽能特异性杀伤呈递突变型多肽TLISSLMPI的靶细胞。
实施例6构建并包装突变型多肽TLISSLMPI及其可变形式多肽的重组慢病毒
合成野生型多肽TLISSLRPI对应的DNA序列“ACGCTGATATCATCACTCAGGCCCATT”(SEQ ID NO：8)，合成突变型多肽TLISSLMPI对应的DNA序列“ACGCTGATATCATCACTCATGCCCATT”(SEQ ID NO：9)，合成其可变形式多肽TLISSLMPL的DNA序列“ACGCTGATATCATCACTCATGCCCCTG”(SEQ ID NO：10)，合成其可变形式多肽TLISSLMPV的DNA序列“ACGCTGATATCATCACTCATGCCCGTC”(SEQ ID NO：11)，合成其可变形式多肽TMISSLMPI的DNA序列“ACGATGATATCATCACTCATGCCCATT”(SEQ ID NO：12)，合成其可变形式多肽TMISSLMPL的DNA序列“ACGATGATATCATCACTCATGCCCCTG”(SEQ ID  NO：13)，合成其可变形式多肽TMISSLMPV的DNA序列“ACGATGATATCATCACTCATGCCCGTC”(SEQ ID NO：14)；并分别构建野生型多肽TLISSLRPI、突变型多肽TLISSLMPI及其可变形式多肽的慢病毒载体pHBLV-Puro，分别命名为pHBLV-Puro-TLISSLRPI、pHBLV-Puro-TLISSLMPI、pHBLV-Puro-TLISSLMPL、pHBLV-Puro-TLISSLMPV、pHBLV-Puro-TMISSLMPI、pHBLV-Puro-TMISSLMPL和pHBLV-Puro-TMISSLMPV。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，并包装出野生型多肽TLISSLRPI、突变型多肽TLISSLMPI及其可变形式多肽的慢病毒。
实施例7表达突变型多肽TLISSLMPI的人源肺癌细胞系的建立
人非小细胞肺腺癌细胞系NCI-H2087购买于ATCC，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100U/ml链霉素的DMEM培养基中，在37℃，5％CO2的孵箱中培养。将实施例6所包装的TLISSLMPI慢病毒转染H2087细胞系，并采用Puromycin抗生素，持续筛选存活的H2087细胞系，最终建立表达TLISSLMPI多肽的H2087细胞系，并命名为H2087-TLISSLMPI细胞系。
实施例8NOD 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。选取4周后的小鼠准备接种人肺癌细胞系模型。
实施例9H2087-TLISSLMPI的皮下移植瘤模型的建立
将实施例7所建立的人非小细胞肺腺癌细胞系H2087-TLISSLMPI，培养于含10％胎牛血清、100U/mL青霉素和100U/mL链霉素的DMEM培养基中，并在37℃，5％CO2的孵箱中培养。收集H2087-TLISSLMPI肿瘤细胞，1500rpm离心5min，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NOD/SCID小鼠或免疫重建后的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和短径b，并计算肿瘤的大小＝a*b*b/2，每天称量小鼠体重和肿瘤并记录；7天后，小鼠皮下可摸到约米粒大小肿瘤，此时对H2087-TLISSLMPI皮下瘤模型的NOD/SCID小鼠进行DC-CTL疫苗的治疗。对免疫重建4周的H2087-TLISSLMPI皮下瘤模型NOD/SCID小鼠分别进行多肽+完全弗氏佐剂，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10多肽疫苗治疗方案
将免疫重建4周的H2087-TLISSLMPI皮下瘤模型NOD/SCID小鼠随机分为8组：佐剂+野生型多肽组、佐剂组、佐剂+TLISSLMPI多肽组以及佐剂+其5种可变形式多肽组，每组各6只。上述多肽首次免疫剂量为100μg/只小鼠。多肽用PBS重悬后，与150μl/只小鼠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。每天观察荷瘤鼠的一般特征，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(a)和最短径(b)。肿瘤体积计算为：1/2×长×宽2。结果见图3。结果显示，相对于单纯佐剂组和野生型多肽组，TLISSLMPI或其可变形式+弗氏佐剂均可以有效的抑制肿瘤的生长，并延长小鼠的生存期。生存期计算公式：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％。
实施例11DC多肽疫苗的制备以及使用该疫苗的治疗方案
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，收集PBMC细胞，按2～3×106个/ml重悬于RPMI 1640培养基中，37℃孵育2h，贴壁细胞即为DC，采用1000U/ml GM-CSF，1000U/ml IL-4，100U/ml IFN-γ，100U/ml CD40L，诱导贴壁细胞为成熟DC细胞；收获成熟DC后，分别加入野生型多肽，突变型多肽TLISSLMPI及其5种可变形式多肽(浓度为10μg/ml)，共同孵育4h后用生理盐水洗涤3次。用生理盐水将负载多肽的DC调整为(4.0±0.5)×107个/ml，用于后续实验。
将荷瘤小鼠随机分为7组：DC-负载野生型多肽组、DC-负载突变型多肽TLISSLMPI以及DC-负载其5种可变形式多肽，每组各6只。制备DC-负载野生型多肽，DC-负载TLISSLMPI多肽及DC-负载其5种可变形式多肽的细胞悬液。对荷瘤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各细胞/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4，图4结果显示，相对于野生型多肽负载的DC疫苗组，TLISSLMPI或其可变形式多肽负载的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12多肽基因重组慢病毒感染DC疫苗的制备及使用该疫苗的治疗方案
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，收集PBMC细胞，37℃孵育2h，洗去未贴壁细胞，经重组人粒细胞-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，更换半量的培养基及调整细胞密度为1*106个/ml；加入含有适量的野生型多肽TLISSLRPI，突变型多肽TLISSLMPI或其5种可变形式多肽的重组慢病毒液(如实施例6所构建)。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。16h后并将DC细胞调整为(4.0±0.5)x107个/ml，用于后续实验。
将荷瘤小鼠随机分为7组：野生型多肽-DC组、TLISSLMPI多肽-DC组以及其5种可变形式多肽-DC组，每组各6只。制备DC-负载野生型多肽，DC-负载TLISSLMPI多肽以及DC-负载其5种可变形式多肽的细胞悬液。对免疫重建的荷瘤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个细胞/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＝1/2x长x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5，图5结果显示，相对于野生型多肽组，突变型多肽TLISSLMPI或其5种可变形式多肽基因的重组慢病毒感染的DC疫苗，具有明显的肿瘤抑制效果，并且延长小鼠的生存期，但是野生型多肽对该肿瘤没有反应。
实施例13多肽特异性CTL疫苗的制备以及使用该疫苗在体内的治疗方案
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，收集PBMC细胞，按2～3×106个/ml重悬于RPMI 1640培养基中，37℃孵育2h，吸取未贴壁细胞即是外周血淋巴细胞(peripheral blood lymphocyte，PBL)。
所收集的PBL经过磁珠分选获得CD8+T，将CD8+T与负载野生型多肽的DC，负载突变型多肽TLISSLMPI及其五种可变形式多肽的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周再加入负载TLISSLMPI多肽的DC及负载其5种可变形式多肽的DC、负载野生型多肽的DC和500IU/ml IL-2进行第二轮刺激。第3周进行同样处理。如此，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)。PI＝扩增后细胞数/接种细胞数。第3次刺激后第7天(即，培养第21天)收获 细胞，即细胞毒性T淋巴细胞(cytotoxic T lymphocytes，CTL)。将细胞用生理盐水重悬，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个细胞。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6所示；图6结果显示，相对于野生型多肽组，突变型多肽TLISSLMPI或其5种可变形式多肽激活的DC-CTL疫苗，具有明显的肿瘤抑制效果，并且延长小鼠的生存期。
申请人声明，本发明通过上述实施例来说明本发明的产品及其用途和使用效果，但本发明并不局限于此，所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (24)

  1. 一种分离的多肽或其免疫学活性片段，所述多肽选自：
    (a)由SEQ ID NO：2所示氨基酸序列组成的多肽；
    (b)由SEQ ID NO：2所示氨基酸序列经过一个或多个氨基酸残基的取代、添加和/或缺失而形成的，且具有细胞毒性T淋巴细胞诱导能力的多肽；以及
    (c)(a)多肽或(b)多肽的变体或衍生物，其具有细胞毒性T淋巴细胞诱导能力。
  2. 如权利要求1所述的多肽，其特征在于，所述多肽可被CD8+T细胞识别。
  3. 如权利要求1所述的多肽，其特征在于，所述一个或多个氨基酸的取代、添加和/或缺失为如SEQ ID NO：2所示氨基酸序列的第2位和/或第9位氨基酸的取代；
    优选地，所述一个或多个氨基酸的取代、添加或缺失为如SEQ ID NO：2所示氨基酸序列的第2位氨基酸取代为M和/或第9位氨基酸取代为L或V；
    进一步优选地，所述(b)多肽具有如SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示的氨基酸序列。
  4. 一种分离的核酸，其编码如权利要求1-3任一项所述的多肽。
  5. 一种核酸构建体，其包含权利要求4所述的核酸，以及与之可操作连接、可指导多肽在表达宿主中生产的一个或多个控制序列。
  6. 一种表达载体，其包含权利要求5所述的核酸构建体。
  7. 一种宿主细胞，其中转化或转染了如权利要求5所述的核酸构建体或如权利要求6所述的表达载体。
  8. 一种抗原呈递细胞，其在细胞表面呈递如权利要求1-3任一项所述的多肽。
  9. 产生如权利要求8所述的抗原呈递细胞的方法，其包括：将如权利要求1-3任一项所述的多肽与具有抗原呈递能力的细胞接触的步骤，或者包括：将如权利要求4 所述的核酸、或者如权利要求5所述的核酸构建体、或者如权利要求6所述的表达载体导入具有抗原呈递能力的细胞中表达的步骤；
    优选地，所述具有抗原呈递能力的细胞为树突细胞。
  10. 一种免疫效应细胞，其可识别如权利要求1-3任一项所述的多肽或者识别在细胞表面呈递如权利要求1-3任一项所述的多肽的抗原呈递细胞。
  11. 产生如权利要求10所述的免疫效应细胞的方法，其包括：将如权利要求8所述的抗原呈递细胞与具有免疫效应能力的细胞接触的步骤；
    优选地，所述具有免疫效应能力的细胞为T细胞，优选为CD8+T细胞。
  12. 一种靶向性免疫细胞群，其由抗原呈递细胞与淋巴细胞混合并共培养而形成。
  13. 一种缀合物，其包含如权利要求1-3任一项所述的多肽和一种抗癌药。
  14. 一种抗体，其特征在于，所述抗体特异性识别如权利要求1-3任一项所述的多肽。
  15. 一种制备抗体的方法，其特征在于，包括：
    利用如权利要求1-3任一项所述的多肽对动物进行免疫接种；
    采集经免疫接种的动物血清；以及
    从所述血清中纯化出目的抗体。
  16. 一种在患者中治疗或预防癌症的疫苗，其包括如权利要求1-3任一项所述的多肽，或包括如权利要求4所述的核酸，或包括如权利要求5所述的核酸构建体，或包括如权利要求6所述的表达载体，或包括如权利要求8所述的抗原呈递细胞，或包括如权利要求10所述的免疫效应细胞；
    优选地，所述癌症为表达如权利要求1-3任一项所述的多肽的癌症；
    进一步优选地，所述癌症选自肺癌，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，白血病和脑肿瘤。
  17. 一种在患者中治疗或预防癌症的药物组合物，其包括如权利要求1-3所述的多肽，以及药学上可接受的载体。
  18. 如权利要求1-3任一项所述的多肽在制备抗体中的用途，所述抗体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，白血病和脑肿瘤。
  19. 如权利要求1-3任一项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，白血病和脑肿瘤。
  20. 如权利要求1-3任一项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，白血病和脑肿瘤。
  21. 如权利要求1-3任一项所述的多肽或者如权利要求4所述的核酸的检测试剂在制备用于在患者中诊断癌症的套件中的用途；
    优选地，所述癌症为表达如权利要求1-3任一项所述的多肽的癌症；
    进一步优选地，所述癌症选自肺癌，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，白血病和脑肿瘤。
  22. 一种在患者中诊断癌症的套件，其包括如权利要求1-3任一项所述的多肽或 者如权利要求4所述的核酸的检测试剂。
  23. 一种治疗方法，其特征在于，包括：
    向患者施用有效量的如权利要求1-3任一项所述的多肽、如权利要求8所述的抗原呈递细胞、如权利要求10所述的免疫效应细胞、如权利要求12所述的靶向性免疫细胞群、如权利要求13所述的缀合物、如权利要求14所述的抗体、如权利要求16所述的疫苗或如权利要求17所述的药物组合物。
  24. 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如权利要求1-3任一项所述的多肽；
    基于所述生物样品是否携带所述多肽的事实，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，白血病和脑肿瘤。
PCT/CN2016/100978 2016-09-30 2016-09-30 Cacna1h衍生的肿瘤抗原多肽及其应用 WO2018058489A1 (zh)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1521268A (zh) * 2003-01-27 2004-08-18 北京诺赛基因组研究中心有限公司 检测儿童失神癫痫主效基因cacna1h突变基因的方法及cacna1h突变基因
CN103957923A (zh) * 2011-09-12 2014-07-30 Tau治疗有限公司 用于抑制包括癌干细胞在内的干细胞的增殖、发育或分化的hs.459642单基因簇产物的拮抗剂

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102659921A (zh) * 2012-01-19 2012-09-12 中国人民解放军南京军区福州总医院四七六医院 Neuritin蛋白的HLA-A*0201限制性CTL表位及其应用
CN103570818B (zh) * 2012-07-27 2016-06-29 北京智飞绿竹生物制药有限公司 肿瘤抗原性多肽及其作为肿瘤疫苗的用途
CN104830779A (zh) * 2015-05-05 2015-08-12 杨光华 基于cea抗原的dc细胞、靶向性免疫细胞群及其制备方法和用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1521268A (zh) * 2003-01-27 2004-08-18 北京诺赛基因组研究中心有限公司 检测儿童失神癫痫主效基因cacna1h突变基因的方法及cacna1h突变基因
CN103957923A (zh) * 2011-09-12 2014-07-30 Tau治疗有限公司 用于抑制包括癌干细胞在内的干细胞的增殖、发育或分化的hs.459642单基因簇产物的拮抗剂

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