WO2005014819A1 - Antigene du cancer de l'oesophage et utilisation de celui-ci - Google Patents

Antigene du cancer de l'oesophage et utilisation de celui-ci Download PDF

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WO2005014819A1
WO2005014819A1 PCT/JP2004/011736 JP2004011736W WO2005014819A1 WO 2005014819 A1 WO2005014819 A1 WO 2005014819A1 JP 2004011736 W JP2004011736 W JP 2004011736W WO 2005014819 A1 WO2005014819 A1 WO 2005014819A1
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cancer
protein
pro
peptide
dna
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PCT/JP2004/011736
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English (en)
Japanese (ja)
Inventor
Tetsuya Nakatsura
Yoshihiro Yoshitake
Yusuke Nakamura
Yasuharu Nishimura
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Kumamoto Technology & Industry Foundation
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Priority to JP2005513040A priority Critical patent/JP4557886B2/ja
Publication of WO2005014819A1 publication Critical patent/WO2005014819A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3046—Stomach, Intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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

  • the present invention relates to a novel human esophageal cancer antigen useful for the diagnosis and immunotherapy of various cancers including esophageal cancer, and uses thereof. More specifically, the present invention relates to a protein specific to human cancer, a partial peptide thereof, a DNA encoding the same, an antibody having the above protein as an epitope, a killer T cell, a cancer vaccine, a cancer diagnostic probe, and a cancer diagnostic. Drugs Prevention and treatment of cancer. Background art
  • Immunotherapy has long been expected as a treatment for cancer, and various attempts have been made, but it has not yet been shown to have a sufficient antitumor effect.
  • radiation therapy and surgery are mainly used as treatments, but the locational burden on the patient is immeasurable due to its location. Therefore, it is desired to establish a treatment method with as few side effects and invasiveness as possible.
  • T cells play an important role in tumor rejection in vivo, and cytotoxic T cells Efforts are being focused on isolating T cell-recognizing tumor antigens that can induce (CTL: Cytotoxic T Lymphocyte) and determining MHC class I-restricted epitopes.
  • CTL Cytotoxic T Lymphocyte
  • An object of the present invention is to provide a human esophageal cancer antigen applicable to diagnosis and treatment of various cancers and tumors, a gene encoding the same, an anticancer vaccine using the same, an antibody, and a CTL that specifically reacts with the above antigen. (Cytotoxic T cells).
  • a second object of the present invention is to provide an effective antigen-specific immunotherapy using the above antigen or cytotoxic T cell.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, using a cDNA microarray array method, a protein excellent in the effect of immunotherapy and many peptides constituting the protein have been demonstrated. O successful isolation of the antigen, which led to the completion of the present invention o
  • any one of the following proteins (A) and (B) is provided.
  • a peptide comprising a part of the above-described protein of the present invention and having immunity-inducing activity.
  • the peptide of the present invention is preferably a peptide capable of activating a cytotoxic T cell recognizing a cancer antigen protein.
  • a peptide comprising an amino acid sequence according to any one of the following (1) to (10).
  • a mammalian DNA encoding the above-described protein of the present invention.
  • the mammal is a human or a mouse.
  • an antibody against the above-described protein or peptide of the present invention there is provided an antibody against the above-described protein or peptide of the present invention.
  • a cytotoxic T cell induced by in vitro stimulation using the above-described protein or peptide of the present invention there is provided a cancer vaccine comprising the above-described protein or peptide of the present invention.
  • a cancer vaccine comprising the above-described DNA of the present invention or a recombinant virus or bacterium containing the DNA.
  • the cancer vaccine of the present invention preferably further comprises an adjuvant.
  • a cancer diagnostic probe comprising the above-described DNA of the present invention.
  • a cancer diagnostic agent comprising the above-described probe for cancer diagnosis and / or antibody of the present invention.
  • a prophylactic / therapeutic agent for cancer comprising the above-mentioned protein, peptide, antibody, and Z or cytotoxic T cell of the present invention.
  • the cancer is esophageal cancer, brain tumor, malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, head and neck cancer, kidney cancer, prostate cancer, lung cancer, thyroid cancer, breast cancer, gastric cancer, colon cancer, Teng cancer , Biliary tract cancer, It is liver cancer, gallbladder cancer, testicular cancer, uterine cancer, ovarian cancer, or sarcoma.
  • nucleic acid capable of suppressing the expression of the above-described protein of the present invention by an RNAi phenomenon.
  • the nucleic acid is siRNA, shRNA or an expression vector thereof.
  • an RNA having the sequence of SEQ ID NO: 17 or an expression vector capable of expressing the same is provided.
  • an antitumor agent comprising the nucleic acid or RNA described above or an expression vector capable of expressing the same.
  • FIG. 1 is a graph showing the relative ratio of PP-RP expression between the expression levels of cancerous and non-cancerous parts in 26 cases of esophageal cancer patients.
  • FIG. 2 is a graph showing relative values of the amount expressed in other organs when the expression amount of PP-RP in normal organs is set to 1.
  • a of FIG. 2 shows the relative ratio of PP-RP gene expression in the cancerous / non-cancerous part of 26 esophageal cancer patients.
  • B in Figure 2. 2 shows the expression of the PP-RP gene in various normal organs.
  • FIG. 3 shows the results of Northern plot analysis on 12 normal cells and an esophageal cancer cell line.
  • FIG. 4 shows the results of analyzing the expression of PP-RP mRNA in various cancer cell lines by RT-PCR.
  • FIG. 5 shows the results of immunostaining of normal and esophageal cancer tissues using goat polyclonal anti-RBQ-l (Santa Cruz).
  • FIG. 6 shows the results of observing changes in cells in which the PP-RP gene was introduced into the NIH3T3 cell line. 1 [1113 C3 cell line? ? When -1? Is transfected, a pile-up image of the cells is observed.
  • FIG. 7 shows the state of the injection site of a mouse two weeks after injection of cells obtained by transfecting the NI H3T3 cell line with PP-RP into a nude mouse. Three mice were injected with 10 6 cells. Cells transfected with PP-RP into the NI H3T3 cell line Trout was formed in one mouse.
  • FIG. 8 is a graph showing the results of 51 Cr release assay for each peptide of the CTL strain obtained by inducing with a mixture of 10 kinds of peptide vaccines derived from PP-RP protein.
  • CTLs obtained by induction with a mixture of 10 types of peptide vaccines derived from PP-RP protein specifically recognize peptide 7.
  • FIG. 9 is a graph showing the results of measuring the cytotoxic activity of a CTL strain obtained by inducing with PP-RP peptide 2 against a C1R-A2402 cancer cell line by 51 Cr release assay.
  • CTLs from esophageal cancer patients induced with PP-RP-derived p420-428GYSVPPPGF peptide can peptide-specifically damage cancer cell lines.
  • FIG. 10 is a graph showing the results obtained by measuring the cytotoxic activity of the CTL strain obtained by inducing with PP-RP peptide 2 against TE9 and TE11 by 51 Cr release assay. ? ? One? CTLs of esophageal cancer patients induced by the derived 420-428GYSVPPPGF peptide damage the cancer cell line in a PP-RP antigen-specific manner.
  • FIG. 11 is a graph showing the results of measuring the cytotoxic activity of a CTL strain obtained by inducing with PP-RP peptide 3 against a C 1 R-A2402 cancer cell line by 51 Cr release assay.
  • PP-RP-derived 634-642 AYYGRS VDF peptide-induced CTLs from esophageal cancer patients can peptide-specifically damage cancer cell lines.
  • FIG. 12 is a graph showing the results of measuring the cytotoxicity of CTL lines obtained by inducing with PP-RP peptide 3 against TE9 and TE11 by 51 Cr release assay.
  • PP-RP-derived p634-642 AYYGRS VDF peptide-induced CTLs of esophageal cancer patients damage PP-RP antigen-specific cancer cell lines.
  • Figure 13 A is PP- RP Bae CTL lines obtained by induced peptide 3, C 1 R- A 2402, and SK- the He p 1 cancer cell line 51 Cr release cytotoxicity against It is a graph which shows the result measured by Suatsushi.
  • FIG. 13B is a graph showing the results obtained by measuring the cytotoxic activity of CTL lines obtained by inducing with PP-RP peptide 3 on TE13 by 51 Cr release assay.
  • Figure 13 C is PP- RP peptide CTL lines obtained by induced 3, the TE 9, TE 1 1, TE 13 Oyo cytotoxic activity against Pi SK_He 1, the results of measurement by 51 Cr release mediation Si It is a graph shown.
  • TE 1 1 HLA—A24,? ? Expression of -1? ++
  • SK-He1 HLA—A24, PP—RP expression—
  • CTLs of esophageal cancer patients induced by PP-RP-derived p634-642 AYYGRSVDF peptide specifically recognize the peptide and damage cancer cell lines expressing HLA-A24-restricted PP-RP.
  • FIG. 14A shows the results of measuring the cytotoxic activity of the CTL strain obtained by inducing PP-RP peptide 9 against C1R-A2402 and SK-Hep1 cancer cell lines using 51 Cr release assay.
  • FIG. Figure 148? 1 is a graph showing the results obtained by measuring the cytotoxic activity of a CTL strain obtained by inducing -1 peptide 9 against TE13 by 51 Cr release assay.
  • Fig. 14 ⁇ Graph showing the results of CTL lines obtained by induced in one P peptide 9, the TE 9, TE 11, TE 1 3 Oyo Pi SK- cytotoxic activity to the He p 1, was determined by 51 Cr release mediation Si It is.
  • TE11 HLA-A24, PP—RP expression + +
  • SK-He 1 HLA_A24, PP— expression of RP
  • PP RP-derived p379-388VFVPVPPPP L-peptide-induced CTL in esophageal cancer patients specifically recognizes the peptide
  • HLA A24-restricted PP— Damages cancer cell lines expressing RP.
  • FIG. 15A is a graph showing the results obtained by measuring the cytotoxic activity of the CTL lines obtained by inducing with PP-RP peptide 10 on C1R-A2402 and SK-He1 cancer cell lines by using 51 Cr release assay. It is.
  • FIG. 15B is a graph showing the results obtained by measuring the cytotoxic activity against TE13 of the CTL strain obtained by inducing with PP-RP peptide 10, using 51 Cr release assay.
  • Figure 1 5 C is the CTL lines obtained by induced in PP- RP peptide 10, a TE 9, TE 1 1, TE 1 3 and cytotoxic activity against SK_He p 1, was measured with a 5 r release mediation Si It is a graph which shows a result.
  • TE11 Expression of HLA-A24, PP-RP ++
  • SK-He 1 HLA_A24, PP—RP expression—
  • FIG. 16 shows the results of measuring the cell growth rate when PP-RP was knocked down by RNAi in the esophageal cancer cell line TE13 overexpressing PP-RP.
  • FIG. 17 is a survival curve showing that PP-RP is a prognostic factor.
  • the protein collected from the esophageal cancer of the present invention is any of the following (A) or (B):
  • A a protein having the amino acid sequence of SEQ ID NO: 1 (hereinafter also referred to as “PP-RP”);
  • B a protein having an amino acid sequence of SEQ ID NO: 1 including substitution, deletion, insertion, Z or addition of one or several amino acids, and having immunity-inducing activity;
  • protein having immunity-inducing activity refers to a protein having an activity of inducing an immune response such as antibody production and cellular immunity.
  • PP— RP proliferation potential related protein
  • RBBP 6 retinoblastoma binding protein 6
  • P 1 792 amino acid residue Is a split mutant a This PP-RP is a nucleoprotein that has a domain that binds directly to the Rb protein and can also bind to DNA.
  • the protein was hardly expressed in normal human organs other than placenta and testis by Northern blotting.
  • the PP-RP gene was transformed when expressed in NIH / 3T3 cells. Moreover, when these cells are transplanted into nude mice, they survive and form tumors, which may be related to canceration.
  • PP-RP which is an esophageal cancer antigen of the present invention
  • PP-RP can be detected, for example, from a cancer cell collected from an esophageal cancer patient by cDNA microarray analysis as described in the Examples below.
  • P 2 P—R A protein having an amino acid sequence similar to that of the human protein PP-RP also exists in mice. This protein was named P 2 P—R.
  • This P 2 P_R is a nuclear protein that binds to p53 and the Rb protein, and its expression level increases in the M phase.
  • the cDNA microarray method according to the present invention is, as is known, for example, mRNA is separately prepared from a tissue extracted from a cancer patient into a cancerous part and a non-cancerous part, and a fluorescently labeled cDNA is prepared therefrom. On a slide glass on which at least about 50%, preferably at least 60%, and particularly preferably at least about 70% of the genes are spotted, hybridized, and the signal was acquired with a scanner to obtain the gene. This is a method for analyzing offspring expression.
  • One or several amino acids in the amino acid sequence of SEQ ID NO: 1 including substitution, deletion, insertion and / or addition of one or several amino acids ''
  • the range is not particularly limited, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, and particularly preferably 1 to 3 means.
  • the method for obtaining and producing the protein of the present invention is not particularly limited, and may be any of a naturally-derived protein, a chemically synthesized protein, and a recombinant protein produced by a genetic recombination technique. Recombinant proteins are preferred in that they can be produced in large quantities with relatively easy operation.
  • the protein of the present invention can be obtained according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t_butyloxycarbonyl method). Can be synthesized.
  • the protein of the present invention can also be synthesized using various commercially available peptide synthesizers.
  • a DNA having a nucleotide sequence encoding the protein for example, the nucleotide sequence of SEQ ID NO: 2 or a mutant or homologue thereof is obtained and preferably used.
  • the protein of the present invention can be produced.
  • the expression vector is preferably any vector that can replicate autonomously in the host cell or can be integrated into the chromosome of the host cell, and contains a promoter at a position capable of expressing the gene of the present invention. Is used.
  • a transformant having a gene encoding the protein 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 expression vector may be introduced into the host by a known method appropriate for each host.
  • the transformant having the gene of the present invention prepared as described above is cultured, the protein of the present invention is produced and accumulated in a culture, and the protein of the present invention is collected from the culture. Thus, the recombinant protein can be isolated.
  • the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, and the like which can be utilized by the microorganisms.
  • a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.
  • the culture conditions may be the same as those usually used for culturing the microorganism.
  • the protein of the present invention may be isolated and purified by a conventional protein isolation and purification method.
  • a protein having an amino acid sequence containing substitution, deletion, insertion and Z or addition of one or several amino acids is a DNA encoding the amino acid sequence of SEQ ID NO: 1.
  • Those skilled in the art can appropriately produce or obtain the sequence based on information on the nucleotide sequence described in SEQ ID NO: 2 showing one example of the sequence.
  • mutant gene having a base sequence encoding a protein having an amino acid sequence including substitution, deletion, insertion, and Z or addition of one or several amino acids is included. It can also be made by any method known to those skilled in the art, such as, chemical synthesis, genetic engineering techniques or mutagenesis. Specifically, mutant DNAs can be obtained by using DNAs having the nucleotide sequence of SEQ ID NO: 2 and introducing mutations into these DNAs.
  • the method can be carried out using a method in which a DNA having the base sequence of SEQ ID NO: 2 is brought into contact with a drug as a mutagen, a method of irradiating ultraviolet rays, or a genetic engineering technique.
  • Site-directed mutagenesis method which is one of genetic engineering technique is useful because it is a method capable of introducing a specific mutation into a specific position, Molecular Cloning:.
  • the present invention further relates to a peptide comprising a part of the above-mentioned protein of the present invention and having immunity-inducing activity.
  • the peptide of the present invention is preferably one that can activate cytotoxic T cells that recognize a cancer antigen protein. Specific examples of such peptides include those having any one of the following amino acid sequences.
  • the peptide of the present invention can be synthesized according to a method used in ordinary peptide chemistry.
  • the commonly used synthesis methods are, for example, Peptide Synthesis, Interscience, New ⁇ , 1966; The Proteins. Vol 2, Academic Press inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; 1985; Pharmaceutical Development, Vol. 14 Peptide Synthesis, Hirokawa Shoten, 1991, etc., and International Publication W099 / 67288.
  • it can be synthesized according to a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butynoleoxycarbonyl method).
  • the peptides of the present invention can also be synthesized using various types of commercially available peptide synthesizers.
  • the peptide of the present invention has a binding motif for HLA-A24. Selection of such a peptide having a binding motif for HLA-A24 can be performed by, for example, (J. Immunol.,
  • the binding affinities of various peptides with HLA antigens can be determined using software available on the Internet, such as those described in Parker KC, J. Immunol., 152, 1994. It can also be calculated in silico. For example, it can be measured as described in (J. Immunol., 152. 1994; Int. J Cancer., 80, 1999. Nukaya,).
  • the DNA of the present invention is a DNA encoding the protein of the present invention described in (1) above, and is preferably any one of the following DNAs (a), (b) and (c).
  • hybridize under stringent conditions refers to the use of DNA as a probe and colony hybridization, plaque hybridization, Southern blot hybridization, or the like. Means the nucleotide sequence of the obtained DNA, for example, using a filter on which DNA or a fragment of the DNA derived from a colony or plaque is immobilized, in the presence of 0.7 to 1.OM NaCl at 65 ° C. After hybridization with C, wash the filter with 0.1 to 2 XSSC solution (1 XSSC solution is 15 OmM sodium chloride and 15 nxM sodium citrate) under 65 conditions. And the like which can be identified by the above method. Hybridization can be carried out in accordance with the method described in, for example, "Molecular Development, Second Edition".
  • DNA that hybridizes under stringent conditions examples include DN ⁇ having a certain degree of homology with the nucleotide sequence of DN ⁇ used as a probe.
  • the method for obtaining DNA of the present invention is not particularly limited. Appropriate probes and primers are prepared based on the amino acid sequence and base sequence information shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing in this specification, and cDNAs for humans or the like are prepared using them.
  • the DNA of the present invention can be isolated by screening the library.
  • the cDNA library is preferably produced from a cell, organ or tissue expressing the DNA of the present invention.
  • DNA having the nucleotide sequence shown in SEQ ID NO: 2 can also be obtained by the PCR method.
  • PCR is performed using a pair of primers designed to amplify the nucleotide sequence described in SEQ ID NO: 2.
  • PCR reaction conditions can be appropriately set. For example, the reaction process consisting of 30 seconds at 94 ° C (denaturation), 30 seconds to 1 minute at 55 ° C (annealing), and 2 minutes at 72 (extension) is defined as one cycle. And a reaction at 72 ° C. for 7 minutes.
  • the amplified DNA fragment can be cloned into an appropriate vector that can be amplified in a host such as E. coli.
  • the present invention relates to an antibody that recognizes part or all of the protein or peptide of the present invention as an epitope (antigen), and a cytotoxicity (killer) induced by in vitro stimulation using the protein or peptide. ) Also related to T cells (CTL). In general, CTL shows stronger antitumor activity than antibodies.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, and its production can be performed by a conventional method.
  • a polyclonal antibody can be obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood from the mammal, and separating and purifying the antibody from the collected blood.
  • mammals such as mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, and birds can be immunized.
  • Methods of immunization are known to those of skill in the art, for example, administering the antigen, for example, 2-3 times at 7 to 30 day intervals.
  • the dose may be, for example, about 0.05 to 2 mg per antigen.
  • the administration route is also 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 by dissolving it in an appropriate buffer, for example, 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 100 ⁇ g of the antigen.
  • blood is collected from the immunized mammal, and the blood is collected, for example, by centrifugation, precipitation using ammonium sulfate or polyethylene dalicol, gel filtration chromatography, ion exchange chromatography. Separation and purification by a conventional method such as chromatography such as affinity chromatography can provide a polyclonal antibody recognizing the protein of the present invention as a polyclonal antiserum.
  • a monoclonal antibody can be obtained by preparing a hybridoma.
  • a hybridoma can be obtained by cell fusion between an antibody-producing cell and a Myeoma cell line.
  • the hybridoma producing the monoclonal antibody of the present invention can be obtained by the following cell fusion method.
  • Antibody-producing cells include spleen cells, lymph node cells, and B phosphorus from immunized animals. Use a ball or the like.
  • the antigen the protein of the present invention or its partial peptide is used. Mice, rats, and the like can be used as immunized animals, and administration of the antigen to these animals is performed by a conventional method.
  • a suspension of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant
  • the protein or peptide of the present invention which is an antigen
  • spleen cells are obtained as antibody-producing cells from the immunized animal, and these are fused with myeloma cells by a known method to produce a hybridoma.
  • myeloma cell lines used for cell fusion include P3X63Ag8, P3U1 strain, and Sp2 / 0 strain in mice.
  • a fusion promoter such as polyethylene glycol or Sendai virus is used.
  • hypoxanthine 'aminopterin' thymidine (HAT) medium is used according to a conventional method.
  • the hybridoma obtained by cell fusion is cloned by a limiting dilution method or the like. If necessary, screening can be performed by enzyme immunoassay using the protein of the present invention to obtain a cell line that produces a monoclonal antibody that specifically recognizes the protein of the present invention.
  • the hybridoma is cultured by a conventional cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites. Just fine. Purification of the monoclonal antibody from the culture supernatant or ascites can be performed 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 include F (ab ') 2 fragments, Fab' fragments and the like.
  • labeled antibodies of the above-mentioned antibodies are also within the scope of the present invention. That is, the antibody of the present invention prepared as described above can be labeled and used.
  • the types of antibody labeling and labeling methods are known to those skilled in the art. For example, horseradish peroxidase or Is an enzyme label such as alkaline phosphatase, a fluorescent label such as FITC (fluorescein isothiosinate) or TRITC (teloramethylrhodamine B isothiosinate), or a coloring substance such as colloidal metal latex Labeling, affinity labeling such as biotin, and isotope labeling such as 125 I.
  • Analysis or measurement of the protein or peptide of the present invention (that is, a cancer antigen) using the labeled antibody of the present invention can be performed by an enzyme antibody method, an immunohistochemical staining method, an immunoplot method, a direct fluorescent antibody method or an indirect fluorescent antibody method.
  • the method can be performed according to a method known to those skilled in the art.
  • the present invention also relates to an activated T cell induced by in vitro stimulation with the protein or pepsid of the present invention.
  • an activated T cell induced by in vitro stimulation with the protein or pepsid of the present invention.
  • peripheral blood lymphocytes or tumor-infiltrating lymphocytes are stimulated in vitro with the protein or peptide of the present invention
  • tumor-reactive activated T cells are induced, and the activated ⁇ cells are effectively used for adoptive immunotherapy. be able to.
  • the protein or peptide of the present invention can be expressed in vitro or in vitro in dendritic cells, which are powerful antigen-presenting cells, to induce immunity by administering the antigen-expressing dendritic cells.
  • the DNA, protein and peptide of the present invention can induce T cells that can damage cancer cell lines in an antigen-specific manner, they can be expected as therapeutic or preventive agents for cancer.
  • bacteria of BCG bacteria transformed with the DNA of the present invention into an appropriate vector and transformed with the recombinant DNA, or viruses such as vaccinia virus with the DNA of the present invention integrated in the genome are used for treatment of human cancer. .
  • viruses such as vaccinia virus with the DNA of the present invention integrated in the genome, are used for treatment of human cancer. .
  • the dose and method of administration of the cancer vaccine are the same as those for normal vaccination and BCG lectin.
  • the DNA of the present invention (as it is or in the form of a plasmid DNA incorporated into an expression vector), a recombinant virus or a recombinant bacterium containing the DNA as it is or dispersed in an adjuvant can be used as a cancer vaccine containing humans. It can be administered to animals. Similarly, the peptide of the present invention is dispersed with adjuvant. Can be administered as a cancer vaccine.
  • adjuvant examples include incomplete Freund's adjuvant, BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum adjuvant, and silica adjuvant. It is preferable to use Freund's incomplete adjuvant (FIA) because of its performance.
  • incomplete Freund's adjuvant BCG, trehalose dimycolate (TDM), lipopolysaccharide (LPS), alum adjuvant, and silica adjuvant.
  • TDM trehalose dimycolate
  • LPS lipopolysaccharide
  • alum adjuvant alum adjuvant
  • silica adjuvant silica adjuvant
  • the DNA of the present invention can be used as a diagnostic probe by extracting DNAs of various human cancers and examining their homology.
  • the probe and the antibody can be used as a cancer diagnostic agent.
  • the present invention relates to a cancer diagnostic probe containing all or a part of the antisense chain of DNA or RNA encoding the protein of the present invention.
  • the present invention further relates to a cancer diagnostic comprising the above-mentioned probe for cancer diagnosis or an antibody against the protein of the present invention.
  • the cancer diagnostic probe of the present invention is all or part of the antisense strand of DNA (cDNA) or RNA encoding the protein of the present invention, and has a length sufficient to be a probe (at least 20 bases or more). Those with are preferred.
  • cancer can be diagnosed by detecting mRNA of the protein (cancer antigen) of the present invention obtained from a specimen using the antisense strand.
  • Samples used for detection include, but are not limited to, genomic DNA, RNA or cDNA obtained from a biopsy of a subject's cells, for example, blood, urine, saliva, tissue, etc. Not something.
  • a sample amplified by PCR or the like may be used.
  • the type of cancer referred to in the present invention is not particularly limited, and specific examples include esophageal cancer, brain tumor, malignant melanoma, chronic myeloid leukemia, acute myeloid leukemia, lymphoma, head and neck cancer, kidney cancer, prostate cancer, and lung cancer. , Thyroid, breast, gastric, colon, knee, biliary tract, liver, gallbladder, testicular, uterine, ovarian, or sarcoma, but are not limited to these. It is not something to be done.
  • IgG antibodies are produced against a wide variety of proteins.
  • the antibody that specifically binds to the protein or a peptide that is a part thereof of the present invention include immunospecific antibodies such as a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single-chain antibody, and a humanized antibody. These can be specifically prepared. These can be prepared by a conventional method using the above-mentioned protein such as the esophageal cancer antigen PP-RP or a part thereof as an antigen, and are used for diagnosis of esophageal cancer using them. be able to.
  • the protein or peptide of the present invention can also induce cancer cell-specific cytotoxic T cells as T cell epitopes, and is therefore useful as an agent for preventing or treating human cancer.
  • the antibody of the present invention is useful as a diagnostic agent for cancer.
  • the protein, peptide or antibody of the present invention may be used as an injectable solution as it is, or together with a pharmaceutically acceptable carrier, and Z or a diluent, and, if necessary, the following adjuvants. It may be administered by administration, or may be administered by percutaneous absorption through mucous membranes by spraying or the like.
  • the carrier for example, human serum albumin and the like can be mentioned.
  • the diluent include PBS, distilled water and the like.
  • the dose of the protein, peptide or antibody of the present invention per adult can be, for example, from 0.01 mg to 100 mg per dose, but is limited to this range. is not.
  • the form of the preparation is not particularly limited, and may be freeze-dried or granulated with excipients such as sugar.
  • adjuvants for enhancing cytotoxic T cell-inducing activity examples include cell components such as BCG bacteria, ISC0M described in nature, vol. 344, p873 (1990), J. Immunol, vol. 148, pl438 (1992), such as saponin QS-21, ribosome, and aluminum hydroxide.
  • immunostimulants such as lentinan, schizophyllan, and picibanil are used as adjuvants. You can also.
  • cytokines that enhance the proliferation and differentiation of T cells such as IL-2, IL_4, IL-12, IL-11, IL-6, and TNF, can also be used as adjuvants.
  • the antigen peptide is added in vitro to cells collected from a patient or cells having the same HLA pap type, and the antigen is presented.
  • Toxic T cells can also be induced.
  • cytotoxic T cells can be induced in the test tube and then returned to the blood vessel of the patient.
  • Such treatment by cell transfer has already been performed as a cancer treatment method, and is a method well known to those skilled in the art.
  • the antigen be a recognition antigen for cytotoxic T cells (killer T cells / CTL).
  • the antigen of the present invention increased killer T cell-inducing activity in the mouth of HLA-A24, which is common in Japanese.
  • injection of the antigen of the present invention into the body induces and activates CTL, and as a result, an antitumor effect can be expected.
  • activated T cells When stimulated with the antigen of the present invention, activated T cells are induced in vitro, and the activated T cells can be effectively used for adoptive immunotherapy by injecting the activated T cells into the body.
  • the present invention further relates to a nucleic acid capable of suppressing the expression of the protein of the present invention by the RNAi phenomenon.
  • a nucleic acid capable of suppressing the expression of the protein of the present invention by the RNAi phenomenon.
  • examples of such a nucleic acid include si RNA, sh RNA and their expression vectors, and specific examples include RNA having the sequence of SEQ ID NO: 17 and an expression vector capable of expressing the same.
  • These nucleic acids (such as RNA or its expression vector) can suppress the growth rate of esophageal cancer cells overexpressing the protein (PP-RP) of the present invention, as shown in the following Examples. It is useful as an antitumor agent.
  • the administration method of the antitumor agent of the present invention is not particularly limited, and oral administration, parenteral administration (for example, Examples include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, topical administration to the affected area, and dermal administration), and direct administration to the affected area.
  • parenteral administration for example, Examples include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, topical administration to the affected area, and dermal administration
  • direct administration to the affected area for example, Examples include intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, topical administration to the affected area, and dermal administration.
  • a pharmaceutically acceptable additive can be added as necessary.
  • pharmaceutically acceptable excipients include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles. Diluents, carriers, excipients and Z or pharmaceutical adjuvants, but are not limited thereto.
  • the dose of the nucleic acid as an active ingredient of the antitumor agent of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the severity of the disease, the age, weight, sex, or history of the patient. it can.
  • the dose of the nucleic acid as an active ingredient is not particularly limited, but it is, for example, about 0.1 ng to about 10 Omg Zkg, preferably about 1 ng to about 1 Omg at a time.
  • the antigen of the present invention, its production method, and effects will be described with reference to examples, but the present invention is not limited to these examples.
  • Example 1 Screening of PP-RP by cDNA microarray method
  • Cancer tissues were collected from 26 esophageal cancer patients with informed consent and consent. The collected cancer tissues were stored at 180 ° C. The cancer tissue sample was collected by a laser-microphone beam microdissection system to collect only the cancerous part or non-cancerous part, prepare mRNA, and then create fluorescently labeled cDNA. The gene was analyzed by placing it on a slide glass on which 23040 genes, 70% of all genes, were spotted, hybridizing, capturing signals with a scanner, and analyzing gene expression. As a result, in 21 out of 26 esophageal cancer patients, the relative ratio of the expression level of the cancerous part to the non-cancerous part was 5 times or more in the expression of the PP-RP gene. In addition, the gene, PP-RP, which is hardly expressed outside the normal placenta, Identified. ( Figure 1, Figure 2) Example 2: Northern blot analysis
  • the Northern plot method was performed by the following method. That is, in order to confirm the expression of the PP-RP gene in normal tissues, a human tissue poly (A) + RNA blot (human 12-lane MTN blot, Clontech) was used. In addition, membranes were prepared by hand using total RA of normal esophagus, normal testis, normal placenta, and cancer cell lines.
  • PCR products obtained with the primers of 5'-TGCTGTTGTGATTCCCTGCTG-3, (SEQ ID NO: 13) and Antisense 5, -AGGAAACTGAGGAGAAAACTG-3, (SEQ ID NO: 14) were hybridized as PP-RP probes to these membranes. .
  • Fig. 5 shows the results. As can be seen from the results in Fig. 5, in normal tissues, expression was observed in the testis and placenta, but not in spleen, lymph nodes, brain, kidney, lung, liver, and esophagus tissue (A ⁇ ! Expression is not observed in normal esophageal tissue, but expression is observed in esophageal cancer (S-V). Its localization was around the chromosome (W, X).
  • Example 5 Gene transfer into NIH3T3 cells
  • a vector with a 3056 bp to 4942 bp portion (ORF 92 bp to 4942 bp) of the PP-RP gene was purchased from Takara Holding Co., Ltd. This and the neo-resistance gene expression vector pCI-neo were simultaneously transfected using Ribofectamine TM 2000. Cells were selected by Neo and cloned by limiting dilution. The cells were inoculated once subcutaneously into nude mice to verify whether a bulge was formed.
  • HLA-A24 accounts for 60% of the total Japanese population.
  • a 9- or 10-mer peptide derived from PP-RP having a binding motif was synthesized.
  • Select peptides common to human PP-RP and mouse P2P-R predicted to bind to both HLA-A24 and Kd using HLA-peptide binding prediction from the PP-RP sequence, 9 or 1
  • An automatic peptide synthesizer converts the following 10 types of peptides consisting of 0 amino acids (PSSM8, manufactured by Shimadzu Corporation) by the Fmoc (9-fluorenylmethyloxycarbonyl) method. The synthesized peptides were converted into pure peptides using high performance liquid chromatography.
  • dendritic cells derived from peripheral blood monocytes as antigen-presenting cells, and using T cell lines obtained by stimulating CD8-positive T cells with synthetic PP-RP peptide, their peptide specificity and cancer cell injury Activity was measured.
  • the target cells were C1R-A2402 cells with or without peptide addition, TE11 (HLA-A24, expression of PP-RP ++), TE13 (expression of HLA-A24, PP-RP ++), TE9 (HLA-A24).
  • PP-RP expression ++ A33, PP-RP expression ++), SK-Hepl (HLA-A24, PP-RP expression-), and PP-RP siRNA (small interfering RNA) expression vector pSilencer TM siRNA expression vector (Ambion (registered) (Trademark)
  • a cell line called TE13 shPP-RP in which the expression of PP-RP was suppressed and a cell line called TE13 shGFP were prepared as a control, and a CTL line induced by the above method was added as an effector to the cell line.
  • a mixture of the above 10 peptides was used.
  • Example 9 The procedure was performed in the same manner as in Example 7 except that peptide 2 was used as the antigen peptide. The test results are shown in FIGS. 9 and 10.
  • Example 9 The procedure was performed in the same manner as in Example 7 except that peptide 2 was used as the antigen peptide. The test results are shown in FIGS. 9 and 10.
  • Example 10 The procedure was performed in the same manner as in Example 7, except that peptide 3 was used as the antigen peptide. The test results are shown in Fig. 11 to Fig. 13 A, B and C.
  • Example 10 The procedure was performed in the same manner as in Example 7, except that peptide 3 was used as the antigen peptide. The test results are shown in Fig. 11 to Fig. 13 A, B and C.
  • Example 10
  • Example 11 The procedure was performed in the same manner as in Example 7 except that peptide 9 was used as the antigen peptide. The test results are shown in Figures 14A, B, and C.
  • Example 11 The procedure was performed in the same manner as in Example 7 except that peptide 9 was used as the antigen peptide. The test results are shown in Figures 14A, B, and C.
  • Example 11 The procedure was performed in the same manner as in Example 7 except that peptide 9 was used as the antigen peptide. The test results are shown in Figures 14A, B, and C.
  • Example 11 Example 11:
  • Example 12 Growth rate
  • PP-RP was knocked down using RAi5, -GAACAGCACUCCUGGAAUC-3 '(SEQ ID NO: 17) from an esophageal cancer cell line TE13 which highly expresses PP-RP.
  • Western blotting confirmed that the expression level of PP-RP protein was reduced in TE13-shPP-RP cells in which PP-RP was knocked down compared to TE13 cells.
  • the cells were 1x10 s / plate at 1 ° /. When cultured in serum, the cell growth rate was significantly (p-slow) in TE13-shPP-RP cells compared to TE13 (Fig. 16).
  • Example 13 PP-RP expression level and prognosis
  • R0 indicates no cancer remains
  • R2 indicates gross cancer remains
  • R1 indicates conditions other than R2.
  • the lifetime was examined. The results are shown in Table 1 below.
  • Table 1 Rl clinicopathologic features of 15 patients with esophageal cancer Gender Age 7 ⁇ in cancer tissue, mouth a Survival time b T M histopathology Residual tumor d
  • Gl, G2 and G3 indicate that the degree of SCC differentiation is sufficient, moderate or weak, respectively.
  • the antigen protein and antigen peptide of the present invention can be used as an excellent anticancer vaccine with few side effects such as self-damage.
  • antibodies can be used as diagnostics.
  • Killer T cells stimulated and activated by the antigen of the present invention can be used as anticancer agents.
  • the antigenic protein of the present invention may be related to the malignancy of esophageal cancer. Further, it can be used for the treatment of esophageal cancer and the like which highly express the antigen protein of the present invention.

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Abstract

L'invention concerne un antigène du cancer de l'oesophage humain utile pour diagnostiquer et traiter divers cancers tels que le cancer de l'oesophage et des tumeurs de l'oesophage ; un gène codant pour cet antigène ; un vaccin anticancéreux utilisant celui-ci, etc. Elle concerne notamment un antigène comprenant une protéine qui comporte la séquence d'acides aminés représentée par SEQ ID NO :1 ; un peptide comprenant une partie de cette protéine et qui possède une activité ayant un effet immunitaire ; un vaccin anticancéreux contenant le peptide ; la séquence de bases représentée par SEQ ID NO :2 ou une séquence complémentaire à celle-ci, et l'ADN contenant tout ou partie de cette séquence ; et un vaccin anticancéreux contenant cet ADN. La protéine antigénique décrite permet d'établir un lien entre le degré de malignité d'un cancer de l'oesophage et l'ARNi inhibant l'expression de celle-ci (par exemple 5'-CAACAGCACUCCUGGAAUC-3' (SEQ ID NO :17), et peut servir à traiter un cancer de l'oesophage ou une affection analogue dans laquelle cette protéine est surexprimée.
PCT/JP2004/011736 2003-08-11 2004-08-10 Antigene du cancer de l'oesophage et utilisation de celui-ci WO2005014819A1 (fr)

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RU2612090C1 (ru) * 2016-03-31 2017-03-02 Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации Способ лечения местнораспространённого нерезектабельного рака пищевода

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FR2836687A1 (fr) * 2002-03-04 2003-09-05 Gene Signal Genes impliques dans la regulation de l'angiogenese, preparations pharmaceutiques les contenant et leurs applications

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FR2836687A1 (fr) * 2002-03-04 2003-09-05 Gene Signal Genes impliques dans la regulation de l'angiogenese, preparations pharmaceutiques les contenant et leurs applications

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2612090C1 (ru) * 2016-03-31 2017-03-02 Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации Способ лечения местнораспространённого нерезектабельного рака пищевода

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