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WO2007018198A1 - Peptide antigène dérivé du hsp105 pour une utilisation chez des patients positifs à l’hal-a2 et produit pharmaceutique comprenant l’antigène - Google Patents

Peptide antigène dérivé du hsp105 pour une utilisation chez des patients positifs à l’hal-a2 et produit pharmaceutique comprenant l’antigène Download PDF

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Publication number
WO2007018198A1
WO2007018198A1 PCT/JP2006/315630 JP2006315630W WO2007018198A1 WO 2007018198 A1 WO2007018198 A1 WO 2007018198A1 JP 2006315630 W JP2006315630 W JP 2006315630W WO 2007018198 A1 WO2007018198 A1 WO 2007018198A1
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Prior art keywords
cells
peptide
cancer
killer
hsp105
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PCT/JP2006/315630
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English (en)
Japanese (ja)
Inventor
Yasuharu Nishimura
Tetsuya Nakatsura
Hiroyuki Komori
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Kumamoto University
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Priority to JP2007529587A priority Critical patent/JPWO2007018198A1/ja
Publication of WO2007018198A1 publication Critical patent/WO2007018198A1/fr

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • HSP105-derived cancer rejection antigen peptide for HLA-A2 positive persons and pharmaceuticals including the same
  • the present invention includes esophageal cancer, breast cancer, thyroid cancer, colon cancer, spleen cancer, malignant melanoma, melanoma, osteosarcoma, pheochromocytoma, head and neck cancer, uterine cancer, ovarian cancer, etc.
  • the present invention relates to a novel peptide effective as a vaccine against cancer that highly expresses heat shock protein 05 (HSP105), and a medicament for treating and preventing tumors containing the peptide.
  • HSP105 heat shock protein 05
  • Immunotherapy has long been expected as a cancer treatment, and various attempts have been made, but it has not yet shown a sufficient antitumor effect.
  • immunotherapy of cancer has been centered on non-specific immunotherapy, but in recent years it has become clear that T cells play an important role in tumor rejection in vivo, and killer T cells (cells) Efforts to isolate tumor antigens that can recognize toxic T cells and induce killer T cells and to identify tumor antigen peptides that bind to HLA class I molecules and activate killer T cells. Yes.
  • HLA class I molecules are expressed on the surface of all nucleated cells of the body. Proteins produced in the cytoplasm and nucleus are bound to peptides produced by degradation in the cell, and the cell surface Expressed in On the surface of normal cells, peptides derived from normal self proteins are bound to HLA class I molecules, which are not identified and destroyed by T cells of the immune system. On the other hand, cancer cells are in the process of becoming cancerous and may be expressed in a large amount of protein with little or no expression in normal cells. Peptides produced by degrading proteins specifically expressed in cancer cells in the cytoplasm bound to HLA class I molecules and expressed on the surface of cancer cells are recognized by killer T cells. Only destroy cancer cells.
  • cancer-specific antigens and peptides are administered to individuals.
  • cancer cells that do not harm normal cells can be destroyed to suppress cancer growth.
  • This is called cancer immunotherapy using a cancer-specific antigen.
  • HLA class II molecules are mainly expressed on the surface of antigen-presenting cells, and bind to peptides derived from cancer-specific antigens that are produced by the antigen-presenting cells taken from outside the cells and decomposed inside the cells. It is expressed on the cell surface. Recognizing this, helper T cells are activated and produce various site force-ins that activate other immunocompetent cells, thereby inducing or enhancing an immune response against the tumor.
  • HSP105 Since HSP105 is highly expressed only in various cancers and testis such as human large intestine, spleen, esophagus and breast cancer, it can be said to be a suitable target for antitumor immunotherapy.
  • the inventors already have HSP105 By binding to HLA-A24 with a peptide derived from humans, we identified a number of human killer T cells that were recognized and activated by human 'killer T cells in peripheral blood mononuclear cells (PBM C) of cancer patients. In addition, it has been proved in animal experiments using mice that immunotherapy using these is effective, and a patent application has already been filed! (WO2004Z020624 (PCT / JP03 / 11049)). Furthermore, since HSP105 is highly expressed only in the testis in normal organs, it has been confirmed in mouse experiments that no adverse events such as autoimmunity occur even when immunotherapy targeting HSP105 is performed. And then.
  • HLA-A24 With only peptides presented to killer T cells by HLA-A24, only about 60% of Japanese cancer patients are subject to peptide vaccine administration, and about 40% of Japanese patients are positive for HLA-A2. By identifying the peptides presented to more killer T cells, when combined, about 85% of Japanese cancer patients can be targeted for peptide vaccine administration.
  • HLA-A2 can be applied to many Western white cancer patients because of the high frequency of positives in Western whites. Therefore, the identification of peptides presented to killer T cells by HLA-A2 is an important issue.
  • the present invention can target about 40% of various cancer patients who highly express HSP105 in Japanese by identifying HSP105-derived peptides presented to killer T cells by HLA-A2. Therefore, we decided to solve the problem of providing means to enable immunotherapy.
  • HSP105 is highly expressed only in various cancers and testis such as human large intestine, spleen, esophagus, and mammary gland, and can be said to be a suitable target for antitumor immunotherapy.
  • HSP105-derived peptides that bind to HLA-A24 that can induce human killer T cells by stimulating peripheral blood mononuclear cells in cancer patients.
  • an epitopic peptide that can induce human killer T cells by binding to HLA-A2 derived from HSP105 was newly identified.
  • a peptide having the ability to induce killer T cells which also has an amino acid sequence in which one or two amino acids are substituted or appended in the amino acid sequence shown in SEQ ID NO: 1.
  • a medicament for treating and / or preventing tumor comprising at least one peptide according to (1).
  • a drug for inducing antigen-presenting cells having high inducibility of tumor-reactive T cells comprising the peptide according to (1).
  • a peptide having the ability to induce killer T cells which also has an amino acid sequence in which one or two amino acids are substituted or appended in the amino acid sequence shown in SEQ ID NO: 1.
  • the peptide of the present invention is any of the following peptides.
  • a peptide having the ability to induce killer T cells wherein one or two amino acids are substituted or added in the amino acid sequence shown in SEQ ID NO: 1.
  • the peptide having the ability to induce killer T cells referred to in the present specification means a peptide that activates killer ⁇ cells that injure a cancer expressing the peptide.
  • HSP105 is a variety of cancers such as esophageal cancer, breast cancer, thyroid cancer, colon cancer, knee cancer, malignant melanoma, melanoma, osteosarcoma, pheochromocytoma, head and neck cancer, uterine cancer, and ovarian cancer. It is a good target for anti-tumor immunotherapy because it is highly expressed only in normal cancer and normal testis.
  • ⁇ SP105 is a high-molecular-weight heat shock protein belonging to the HSP105 / 110 family, and has a 10 5 ⁇ force with HSP105a.
  • 105a is a 105kDa heat shock protein that is induced by various stresses.
  • 105 ⁇ is a protein having a molecular weight smaller than that of 105 ⁇ , wherein 105 a mRNA is produced by alternative splicing.
  • the method for obtaining the peptide of the present invention is not particularly limited, and may be either a chemically synthesized protein or a recombinant protein prepared by a gene recombination technique.
  • a chemically synthesized peptide for example, according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarboxyl method) or the tBoc method (tbutyloxycarbon method).
  • Inventive peptides can be synthesized.
  • the peptides of the present invention can be synthesized using various commercially available peptide synthesizers.
  • DNA having a base sequence encoding the peptide or a mutant or homologue thereof is obtained and introduced into an appropriate expression system.
  • Inventive peptides can be produced.
  • the expression vector is preferably a force capable of autonomous replication in a host cell, or a position capable of expressing a gene encoding a peptide as long as it can be inserted into the host cell chromosome.
  • the one containing a promoter is used.
  • a transformant having a gene encoding the peptide of the present invention can be prepared by introducing the above expression vector into a host.
  • the host may be any of bacteria, yeast, animal cells, and insect cells, and the introduction of the expression vector into the host may be performed by a known method according to each host.
  • the transformant produced as described above is cultured, the peptide of the present invention is produced and accumulated in the culture, and the peptide of the present invention is collected from the culture, thereby recombination.
  • the peptide can be isolated.
  • the transformant is a prokaryote such as Escherichia coli or a eukaryote such as yeast
  • the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the microorganism. Any medium can be used as long as the medium can efficiently culture the transformant.
  • the culture conditions may be those usually used for culturing the microorganism.
  • the usual peptide isolation and purification methods may be used.
  • a peptide having an amino acid sequence ability in which one or two amino acids are substituted or added is a DNA sequence encoding the amino acid sequence shown in SEQ ID NO: 1.
  • Those skilled in the art can appropriately produce or obtain the base sequence information. That is, in the amino acid sequence shown in SEQ ID NO: 1, a gene encoding a peptide having an ability to induce killer T cells is also obtained by chemical synthesis, genetic engineering, which has an amino acid sequence ability in which one or two amino acids are substituted or added. It can also be made by any method known to those skilled in the art, such as techniques or mutagenesis.
  • site-directed mutagenesis which is one of genetic methods, is useful because it can introduce a specific mutation at a specific position.
  • Molecular Cloning A laboratory Mannual, 2 ⁇ Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplements 1-38, John Wiley & Sons (1987-1997) and the like.
  • the peptide of the present invention described above can induce immunity against cancer, as shown in Examples described later. Therefore, according to the present invention, an immunity-inducing agent for cancer comprising the peptide of the present invention is provided.
  • the immunity-inducing agent for cancer of the present invention can induce helper T cells, killer T cells, or an immune cell population containing these by using in vitro or in vivo, preferably in vitro, and thereby cancer. Immunity against can be conferred.
  • the present invention also relates to an antibody that recognizes part or all of the peptide of the present invention as an epitope (antigen), and killer T cells induced by in vitro stimulation using the protein or peptide.
  • killer T cells are stronger than antibodies Shows tumor activity.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and can be produced by a conventional method.
  • a polyclonal antibody is obtained by immunizing a mammal or a bird using the peptide of the present invention as an antigen, collecting blood from the mammal or bird, and separating and purifying the antibody from the collected blood.
  • mammals such as mice, mice, mussels, guinea pigs, chickens, rats, rabbits, dogs, goats, hidges, and rabbits can be immunized. Methods of immunization are known to those skilled in the art.
  • antigens may be administered 2 to 3 times, for example, at intervals of 7 to 30 days. The dose can be about 0.05 to 2 mg of antigen per dose, for example.
  • the administration route is not particularly limited, and subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, and the like can be appropriately selected.
  • the antigen can be used after being dissolved in an appropriate buffer containing a commonly used adjuvant such as complete Freund's adjuvant or aluminum hydroxide.
  • booster immunization can be performed using, for example, 100 g to 1000 g of antigen.
  • Mammalian or avian immunized immunized one to two months after the last administration blood is also collected, and the blood is subjected to, for example, centrifugation, precipitation using ammonium sulfate or polyethylene glycol, gel filtration chromatography. Separation and purification by a conventional method such as chromatography such as ion exchange chromatography, affinity chromatography, etc., yields a purified polyclonal antibody that recognizes the peptide of the present invention.
  • a monoclonal antibody can be obtained by preparing a hyperidoma.
  • hypridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line.
  • a hyperidoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
  • spleen cells As antibody-producing cells, spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used.
  • the peptide of the present invention is used as the antigen.
  • Mice, rats, and the like can be used as immunized animals, and antigens are administered to these animals by conventional methods.
  • a book that is an adjuvant and antigen such as complete Freund's adjuvant or incomplete Freund's adjuvant
  • the animal is immunized by administering a suspension or emulsion with the peptide of the invention several times, such as intravenously, subcutaneously, intradermally or intraperitoneally.
  • spleen cells are obtained from the immunized animal as antibody-producing cells, and this and myeloma cells are fused with a known method (G. Kohler et al., ⁇ ature, 256 495 (1975)) to produce a hyperidoma. Can be produced.
  • Examples of myeloma cell lines used for cell fusion include the P3X63Ag8, P3U1 and Sp2Z0 strains in mice.
  • a fusion promoter such as polyethylene glycol or Sendai virus is used, and hypoxanthine 'aminopterin' thymidine (HAT) medium is used in accordance with a conventional method for selection of the hyperidoma after cell fusion.
  • Hyperidoma obtained by cell fusion is cloned by the limiting dilution method or the like.
  • a cell line producing a monoclonal antibody that specifically recognizes the peptide of the present invention can be obtained by screening by an enzyme immunoassay using the peptide of the present invention.
  • the hyperidoma is cultured by a normal cell culture method or ascites formation method, and then the supernatant is obtained from the culture supernatant or ascites.
  • Purify monoclonal antibodies can be purified from the culture supernatant or ascites by a conventional method. For example, ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography and the like can be used in appropriate combination.
  • antibody fragments are also within the scope of the present invention.
  • antibody fragments include F (ab ′) 2 fragments, Fab ′ fragments, and the like.
  • the present invention also relates to killer T cells induced by in vitro stimulation using the peptides of the present invention.
  • killer T cells induced by in vitro stimulation using the peptides of the present invention.
  • peripheral blood lymphocytes or tumor-infiltrating lymphocytes are stimulated in vitro with the peptide of the present invention, tumor-reactive activated T cells are induced, and these activated T cells can be effectively used for adoptive immunotherapy.
  • the peptide of the present invention can be used for immunotherapy by expressing the antigen-expressing rod cells in vivo or in vitro after being expressed in rod cells that are strong antigen-presenting cells.
  • the peptide of the present invention and an immunostimulant are used to effect a killer by in vitro stimulation.
  • One T cell can be induced.
  • the immunostimulant used here include cell growth factor and cytodynamic force-in.
  • the tumor By transferring the killer sputum cells obtained as described above into the body, the tumor can be suppressed, and cancer can be prevented and / or treated.
  • a killer sputum cell capable of suppressing tumor growth can be produced as described above. Therefore, according to the present invention, a cell culture medium containing the peptide of the present invention is provided. This cell culture medium is rich in killer sputum cells that can suppress tumor growth. Furthermore, according to the present invention, there is also provided a cell culture kit for producing killer sputum cells, comprising the cell culture solution and the cell culture container.
  • the peptide of the present invention can induce cancer cell-specific killer cells, it can be expected as an agent for treating or preventing cancer.
  • a BCG bacterium transformed with this recombinant DNA containing the gene encoding the peptide of the present invention into an appropriate vector, or a vaccinia virus integrated with the DNA encoding the peptide of the present invention in the genome Such viruses can be effectively used as a live vaccine for the treatment and prevention of human cancer.
  • the dose and method of administration of cancer vaccine are the same as those for normal vaccination and BCG vaccine.
  • DNA encoding the peptide of the present invention (as it is or in the form of plasmid DNA incorporated into an expression vector), a recombinant virus or a recombinant cell containing the DNA, is suspended as it is or in an adjuvant. In this state, it can be administered as a cancer vaccine to mammals including humans. Similarly, the peptide of the present invention can be administered as a cancer vaccine in a suspended state with adjuvant.
  • adjuvants examples include Freund's incomplete adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), myoban adjuvant, silica adjuvant, and the like. It is preferable to use Freund's incomplete adjuvant (IFA) because of its induction ability.
  • IFA Freund's incomplete adjuvant
  • the type of cancer referred to herein is not particularly limited, and specific examples include esophageal cancer, breast cancer, thyroid cancer, colon cancer, knee cancer, malignant melanoma (melanoma), malignant lymphoma, osteosarcoma, Pheochromocytoma , Head and neck cancer, uterine cancer, ovarian cancer, brain tumor, chronic myeloid leukemia, acute myeloid leukemia, kidney cancer, prostate cancer, lung cancer, stomach cancer, liver cancer, gallbladder cancer, testicular cancer, thyroid cancer, bladder cancer or sarcoma, etc. Is mentioned.
  • the peptide of the present invention can induce cancer cell-specific killer T cells as T cell epitopes, it is useful as a prophylactic / therapeutic agent for human cancer.
  • the antibody of the present invention is also useful as a prophylactic / therapeutic agent for human cancer as long as it can inhibit the activity of HSP105, which is a cancer antigen.
  • the peptide or antibody of the present invention is administered as an injection, as it is, together with a pharmaceutically acceptable carrier and Z or diluent, and if necessary, the following adjuvants are added. It can also be administered by transdermal absorption from the mucous membrane by a method such as spraying.
  • the carrier mentioned here is, for example, human serum albumin, and examples of the diluent include PBS, distilled water and the like.
  • the dose is not limited to this range, and can be administered so that the dose of the peptide or antibody of the present invention per adult is, for example, in the range of O.Olmg to 100mg per dose.
  • the form of the preparation is not particularly limited, and it may be freeze-dried or granulated by adding an excipient such as sugar! /.
  • Examples of adjuvants that can be added to the drug of the present invention to enhance tumor-reactive T cell-inducing activity include muramyl dipeptide (MDP) and other bacterial cell components such as BCG bacteria, Nature, vol. 344, p873 (1990), saponin-based QS-21 described in J. Immunol. Vol. 148, pl438 (1992), ribosome, aluminum hydroxide and the like.
  • MDP muramyl dipeptide
  • immunostimulants such as lentinan, schizophyllan and picibanil can also be used as adjuvants.
  • Ceramides such as CpG and lipopolysaccharide (LP S) that bind to ToU-like receptors and activate the innate immune system can also be used as adjuvants.
  • the antigen peptide is added in vitro to a cell collected from a patient or from another person's (mouth) cell that shares some HLA alleles. It is also possible to induce killer T cells effectively in the patient's body. In addition, by adding the peptide to the patient's peripheral blood lymphocytes and culturing them in a test tube, it is possible to achieve a killer in the test tube. One T cell can be induced and then returned to the patient's blood vessel. Such treatment by cell transfer has already been carried out as a cancer treatment method and is well known among those skilled in the art.
  • killer sputum cells By injecting the peptide of the present invention into the body, killer sputum cells are induced, and as a result, an antitumor effect can be expected.
  • activated sputum cells when stimulated lymphocytes in vitro with the peptide of the present invention, activated sputum cells are induced, and the activated sputum cells can be injected into the affected area to be effectively used for adoptive immunotherapy. it can.
  • HSP1 05 peptide presented to human 'killer sputum cells by binding to HLA-A2 examined this time
  • the amino acid sequence of human HSP105 was searched using the BIMAS system, and one kind of peptide that was assumed to have a relatively high binding affinity with HLA-A2 was selected.
  • HSP105 169-177 peptide induces killer T cells in humans
  • PBMC peripheral blood mononuclear cells
  • PBMC strength separation of CD8 + and CD14 + cell fractions and induction of killer T cells The method previously reported from isolated PBMC (Monji, M et al. Clin Cancer Resl0, 6047-6057, 2 004) was used to induce killer T cells. First, using MACS, CD8 positive cells in PBMC And CD14 positive cell fractions were separated, and CD14 positive cell fractions were separated from GM-CSF (100 ng / ml) and IL-4.
  • Immature rod cells were induced by co-culture with (20 ng / ml). Further, on day 5 of culture, TNF-a (20 ng / ml) was added to mature the rod cells, and on day 7, HSP105 169-177 peptide was added (10 M) and CD8 positive. Co-cultured with cell fraction. Antigen stimulation with rod-like cells derived from the autologous CD14-positive cell fraction was repeated 3 to 4 times per week to induce peptide-specific killer T cells. The medium during induction was changed by half every 2 days, and IL-2 was added at a concentration of 10 U / ml.
  • the ELISPOT method was used to examine whether killer T cells that specifically react with HSP105 and produce IFN- ⁇ exist among the induced killer T cells. When a killer T cell (effector) reacts with a target cell (target) to produce IFN- ⁇ , it is detected as a red spot. IFN- ⁇ was detected using ELISPOT Human IFN- ⁇ ELISPOT set (BD).
  • BD ELISPOT Human IFN- ⁇ ELISPOT set
  • HLA-A2-positive mature rods not loaded with peptide As target cells, HLA-A2-positive mature rods not loaded with peptide, mature rods loaded with HSP105 A2-7 peptide, HLA-A2-positive but not expressing HSP105 and human hepatoma cell line HepG2 HLA105 high expression HLA-A2 positive human colon cancer cell line SW620, HSP105 high expression ability HLA-A2 human melanoma cell line 888mel, HSP105 high expression and HLA-A2 The expressing human melanoma cell line 526mel cells was used. First, an anti-human IFN- ⁇ antibody was coated on an ELISPOT plate (BD Bioscience) for 18 hours. Thereafter, blocking was performed with 10% FCS / RPMI for 2 hours.
  • ELISPOT plate BD Bioscience
  • Effector cells (100 L / well) and target cells (100 L / well) were mixed and cultured at 37 ° C. for 22 hours.
  • the effector Z target ratio (E / T ratio) was 5: 1. Thereafter, the plate was washed with sterilized water, reacted with a piotinylated anti-human IFN- ⁇ antibody for 2 hours and further with streptavidin-HRP for 1 hour, and an IFN- ⁇ positive spot was detected in the substrate solution. Spot counting was performed using automatic analysis software of MINERVA TECH.
  • killer T cells induced with the HSP105 169-177 peptide were compared to mature rodent cells loaded with the HSP105 A2-7 peptide compared to HLA-A2 positive mature rod cells loaded with no peptide.
  • HepG2 which is HLA-A2 positive and does not express HSP105 very much, it also expresses HSP105 more highly than SW620 which is HLA-A2 positive and highly expresses HSP105.
  • 888mel which does not express HLA-A2
  • more T cells respond strongly to 526mel cells that express HSP105 and express HLA-A2, and the number of spots and the area of the spot The sum increased significantly (Figure 1). That is, HSP105-specific killer T cells could be induced using the HSP105 169-177 peptide.
  • Cytotoxic activity of induced killer T cells targets HLA-A2 positive and does not express HSP105 very much V, human hepatoma cell line HepG2, and HLA-A2 positive and human colon cancer cell line S W620 that highly expresses HSP105 As a cytotoxicity test.
  • the cytotoxic activity of killer T cells was evaluated by a cytotoxicity test using Terascan VP. First, target cells were fluorescently labeled with a strong lucein AM staining solution at 37 ° C for 30 minutes. These cells were co-cultured with killer T cells on a Coster 96-well half-area plate, and the degree of cytotoxicity was measured by detecting fluorescent cells over time.
  • HSP105-derived peptide that can induce killer T cells by binding to HLA-A24 expresses a mouse H-2K d molecule whose binding peptide structure is identical to HLA-A24
  • the effectiveness has been confirmed by animal experiments using BALB / c mice.
  • the peptide presented to killer T cells by HLA-A24 is about 60% of Japanese cancer patients and cannot be the subject of force vaccine administration.
  • This time by identifying peptides presented to killer T cells by HLA-A2, when combined, approximately 85% of Japanese cancer patients will be subject to S vaccine administration. If the effectiveness of exploratory medicine using the HSP105 peptide presented to killer T cells by HLA-A2 can be demonstrated, the possibility of clinical application also to Western Caucasian cancer patients will increase.
  • FIG. 1 shows the results of ELISPOT analysis.
  • CD8 + T cells from cancer patients Killer T cells induced by stimulation with rod 14 cells derived from CD 14 positive cells loaded with 9-177 peptide are compared to mature rod cells that are HLA-A2 positive and not loaded with peptide (upper left) H SP105 In contrast to mature rodent cells loaded with A2-7 peptide (upper right) and HLA-A2 positive, HSP105 is less expressed !, compared to HepG2 (middle left), HLA-A2 positive and HSP105 Higher expression of HSP105 and HLA-A2 compared to 888mel (lower left), which does not express HLA-A2 5 26mel (lower right) more T cells responded strongly, and the sum of the number of spots and the area of the spots clearly showed a significant increase. Based on this, it was determined that the HSP105 169-177 peptide is an epitope peptide capable of inducing killer T cells specific for HSP105.
  • FIG. 2 shows the results of cytotoxicity test.
  • Killer T cells obtained by sorting CD8 positive T cells from peripheral blood of HLA-A2 positive human colon cancer patients and stimulating with CD14 positive cell-derived rod-shaped cells loaded with HSP105 169-177 peptide, A cytotoxicity test was conducted to determine whether or not HSP105-expressing cells are cytotoxic.
  • As the target cells HLA-A2 positive and HSP105 was not expressed so much! Human liver cancer cell line HepG2 and HLA-A2 positive and human colon cancer cell line SW620 highly expressing HSP105 were used.
  • killer T cells induced with HSP105 169-177 peptide are HLA-A2 positive and do not express HSP105 very much! Compared with HepG2, SWA which is HLA-A2 positive and highly expressed HSP105 Clearly showed strong cytotoxic activity.

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Abstract

L’invention concerne un moyen permettant une immunothérapie, efficace dans 40 % des types variés de patients cancéreux japonais qui expriment un niveau élevé de HSP105, en identifiant une peptide dérivée de HSP105 pouvant se lier à l’HLA-A2, étant ainsi reconnue par un lymphocyte T tueur humain. Une peptide sélectionnée à partir des peptides suivantes (1) et (2) : (1) une peptide comprenant la séquence d’acides aminés décrite dans la SEQ ID NO 1 ; et (2) une peptide ayant un ou deux résidus d’acide aminé substitués ou ajoutés dans la séquence d’acide aminé décrite dans la SEQ ID NO:1 et capable d’induire un lymphocyte T tueur.
PCT/JP2006/315630 2005-08-09 2006-08-08 Peptide antigène dérivé du hsp105 pour une utilisation chez des patients positifs à l’hal-a2 et produit pharmaceutique comprenant l’antigène WO2007018198A1 (fr)

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JP2010159257A (ja) * 2002-08-30 2010-07-22 Kumamoto Technology & Industry Foundation 癌抗原及びその利用
JP2013047230A (ja) * 2005-08-09 2013-03-07 Medeinetto:Kk Ｈｌａ−ａ２陽性者用ｈｓｐ１０５由来癌拒絶抗原ペプチド及びこれを含む医薬
JP2016509839A (ja) * 2013-03-01 2016-04-04 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
JP2016509840A (ja) * 2013-03-01 2016-04-04 アメリカ合衆国 末梢血から腫瘍反応性ｔ細胞の濃縮された集団を作製する方法
WO2023224096A1 (fr) * 2022-05-18 2023-11-23 国立研究開発法人国立がん研究センター Vaccin contre le cancer utilisant un cocktail d'antigènes courants du cancer, un agent thérapeutique des cellules tcr/car-t, procédé de diagnostic compagnon et procédé de diagnostic du risque d'apparition du cancer par détection de cellules cancéreuses circulant dans le sang

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US9404925B2 (en) 2002-08-30 2016-08-02 Medinet Co., Ltd. Cancer antigen and use thereof
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JP2013047230A (ja) * 2005-08-09 2013-03-07 Medeinetto:Kk Ｈｌａ−ａ２陽性者用ｈｓｐ１０５由来癌拒絶抗原ペプチド及びこれを含む医薬
JP2021019634A (ja) * 2013-03-01 2021-02-18 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
JP2016509840A (ja) * 2013-03-01 2016-04-04 アメリカ合衆国 末梢血から腫瘍反応性ｔ細胞の濃縮された集団を作製する方法
US9844569B2 (en) 2013-03-01 2017-12-19 The United State Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing enriched populations of tumor reactive T cells from peripheral blood
JP2018148898A (ja) * 2013-03-01 2018-09-27 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
US10716809B2 (en) 2013-03-01 2020-07-21 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing enriched populations of tumor reactive T cells from peripheral blood
JP2016509839A (ja) * 2013-03-01 2016-04-04 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
JP2022058791A (ja) * 2013-03-01 2022-04-12 アメリカ合衆国 末梢血から腫瘍反応性ｔ細胞の濃縮された集団を作製する方法
JP7181917B2 (ja) 2013-03-01 2022-12-01 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
JP2023018043A (ja) * 2013-03-01 2023-02-07 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
JP7252383B2 (ja) 2013-03-01 2023-04-04 アメリカ合衆国 末梢血から腫瘍反応性ｔ細胞の濃縮された集団を作製する方法
US11679128B2 (en) 2013-03-01 2023-06-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Methods of producing enriched populations of tumor reactive T cells from peripheral blood
JP7653962B2 (ja) 2013-03-01 2025-03-31 アメリカ合衆国 濃縮された腫瘍反応性ｔ細胞集団を腫瘍から作製する方法
WO2023224096A1 (fr) * 2022-05-18 2023-11-23 国立研究開発法人国立がん研究センター Vaccin contre le cancer utilisant un cocktail d'antigènes courants du cancer, un agent thérapeutique des cellules tcr/car-t, procédé de diagnostic compagnon et procédé de diagnostic du risque d'apparition du cancer par détection de cellules cancéreuses circulant dans le sang

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