WO2012141201A1 - Peptide pour l'immunothérapie du cancer et procédé d'utilisation de celui-ci - Google Patents

Peptide pour l'immunothérapie du cancer et procédé d'utilisation de celui-ci Download PDF

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WO2012141201A1
WO2012141201A1 PCT/JP2012/059878 JP2012059878W WO2012141201A1 WO 2012141201 A1 WO2012141201 A1 WO 2012141201A1 JP 2012059878 W JP2012059878 W JP 2012059878W WO 2012141201 A1 WO2012141201 A1 WO 2012141201A1
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peptide
cells
cancer
protein
kidney cancer
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PCT/JP2012/059878
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Japanese (ja)
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秀紀 川嶋
亜衣子 大林
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公立大学法人大阪市立大学
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Priority to JP2013509939A priority Critical patent/JPWO2012141201A1/ja
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/868—Vaccine for a specifically defined cancer kidney
    • 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
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/56—Kidney

Definitions

  • the present invention relates to a peptide capable of inducing kidney cancer-specific cytotoxic lymphocytes useful for immunotherapy of kidney cancer and use of the peptide.
  • antigen protein specific to kidney cancer, partial peptide thereof, DNA encoding them, antibody having antigen protein as epitope, inducible immune cell, kidney cancer vaccine, kidney cancer diagnostic agent, kidney cancer onset The present invention also relates to a predisposition evaluation method and a method for searching for a target gene useful for immunotherapy such as kidney cancer.
  • cytotoxic T cells CTL: Isolation of a T cell-recognizing tumor antigen capable of inducing Cytotoxic T Lymphocyte
  • identification of an MHC class I-restricted epitope are required.
  • CTL cytotoxic T cells
  • a cDNA expression cloning method using cytotoxic T cells has been performed as an isolation of many tumor antigens, it is necessary to establish a tumor cell line and establish cytotoxic T cells. Isolation of tumor antigens from other carcinomas was difficult.
  • Immunotherapy using peptide vaccines includes SART and MAGE peptide vaccine therapy for lung cancer, HLA-A24SART peptide vaccine therapy for spleen cancer, and WT1 for most solid cancers such as hematological malignancies, digestive organs, chest, urinary organs, and genitals
  • SART and MAGE peptide vaccine therapy for lung cancer
  • HLA-A24SART peptide vaccine therapy for spleen cancer
  • WT1 for most solid cancers
  • solid cancers such as hematological malignancies, digestive organs, chest, urinary organs, and genitals
  • Non-Patent Documents 1 to 6 disclose CA9 peptide vaccine therapy.
  • specific cytotoxic T cells can be induced by vaccination, it has a very limited clinical effect, and only 2 of the 23 patients have seen partial reduction in cancer.
  • Non-Patent Document 4 discloses mutant VHL peptide vaccine therapy
  • Non-Patent Document 5 discloses WT1 peptide vaccine therapy.
  • Non-Patent Document 6 discloses the induction of cytotoxic T cells by HIFPH3 peptide. However, although specific cytotoxic T cell induction has been demonstrated, clinical studies are now on.
  • Patent Document 1 mainly relates to esophageal cancer, but discloses an antigen of esophageal cancer using a cDNA microarray and discloses vaccine therapy using a peptide derived from the antigen amino acid sequence.
  • Patent documents 2 to 6 are also related to kidney cancer or vaccine. However, in the prior art, no peptide useful for immunotherapy of kidney cancer has been found, and no vaccine or early diagnosis method has been obtained.
  • the present invention finds a peptide useful for immunotherapy of kidney cancer, provides an effective treatment method and early diagnosis method, and further provides a method for searching a target gene useful for immunotherapy of kidney cancer and the like.
  • the task is to do.
  • the peptide of the present invention comprises the amino acid sequence of any one of (1) to (6) below, and is characterized by having immunity-inducing activity.
  • AYPMPFITTI (1) AYCETHYNQL (2) FLVQSSDFKV (3) ILFVQYFHRV (4) KLTLKNKFV (5) ELYHEQCFV (6)
  • amino acid sequence of any one of (1) to (6) above it may be a peptide consisting of an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acids, and having immunity-inducing activity.
  • the protein of the present invention contains any of the peptides described above, and is characterized in that it can activate cytotoxic T cells that recognize the renal cancer antigen protein.
  • the cell of the present invention is a helper T cell, cytotoxic T cell, or pulsed in vitro characterized by being induced by in vitro stimulation using any of the peptides described above or a mixture thereof An antigen-presenting cell, a dendritic cell, or an immune cell population containing these.
  • kidney cancer vaccine of the present invention is characterized by including any of the peptides described above.
  • the antibody of the present invention is characterized in that it can cause an antigen-antibody reaction with any of the peptides described above.
  • the renal cancer diagnostic agent of the present invention is characterized by comprising the above-described antibody.
  • the method for evaluating a predisposition to developing kidney cancer according to the present invention is a method for evaluating whether or not a predisposition to develop kidney cancer is present, and the expression level of the peptide-related gene according to claim 1 is determined in a biological sample derived from a subject. And a step of comparing the expression level with a normal control level and evaluating that the predisposition for developing kidney cancer is high if the expression level is higher than a predetermined threshold value.
  • the method for searching for a target gene of the present invention is a method for searching for a target gene useful for immunotherapy, which comprises a step of collecting serum from a cancer patient for which cytokine therapy was effective, and a SEREX method using the serum as a probe. Screening the cDNA expression library.
  • an effective immunotherapy for kidney cancer and a method for evaluating the predisposition to kidney cancer can be obtained, which contributes to the treatment and prevention of kidney cancer.
  • Photo showing target gene analysis results shows HLA-A * 2402 restriction and antigen-specific cytotoxic activity in a peptide based on the sequence (1) derived from gene 36-6-1, and has high cytotoxic activity against renal cancer cell TUHR-10TKB.
  • Graph (b) shows HLA-A * 2402 restriction and antigen-specific cytotoxic activity in the peptide based on the sequence (2) derived from gene 113-3-1, and is a high cell for renal cancer cell TUHR-10TKB Graph showing injury activity Photograph showing target gene expression in carcinomas other than kidney cancer (A) is a graph showing HLA-A * 0201 restriction and antigen-specific cytotoxic activity in a peptide based on sequence (3) derived from gene 36-6-1, and high cytotoxic activity against renal cancer cell A498.
  • (B) shows HLA-A * 0201 restriction and antigen-specific cytotoxic activity in a peptide based on the sequence (4) derived from gene 36-6-1, and high cells against renal cancer cells A498 and TUHR-10TKB
  • Graph showing injury activity (A) is a graph showing HLA-A * 0201 restriction and antigen-specific cytotoxic activity in a peptide based on the sequence (5) derived from gene 113-3-1 and high cytotoxic activity against renal cancer cell A498.
  • (B) shows HLA-A * 0201 restriction and antigen-specific cytotoxic activity in a peptide based on the sequence (6) derived from gene 113-3-1, and has high cells against renal cancer cells A498 and TUHR-10TKB.
  • Graph showing injury activity is a graph showing HLA-A * 0201 restriction and antigen-specific cytotoxic activity in a peptide based on the sequence (5) derived from gene 113-3-1 and high cytotoxic activity against renal cancer cell A498.
  • the present inventor searched for an antigen protein that reacts with the serum of a patient who succeeded in cytokine therapy. Then, two types of antigen genes (36-6-1, 113-3-1) were identified, and six types of peptides that induce specific cytotoxic T cells having higher activity than these antigens were found. The invention has been completed.
  • the present invention is the first analysis using serum from patients with advanced renal cancer who have been effective for cytokine therapy.
  • the peptides and proteins according to the present invention are as follows.
  • the peptide of the present invention consists of any one of the following amino acid sequences (1) to (6) and has immunity-inducing activity.
  • AYPMPFITTI (1) AYCETHYNQL (2) FLVQSSDFKV (3) ILFVQYFHRV (4) KLTLKNKFV (5) ELYHEQCFV (6)
  • the protein of the present invention comprises any one of the amino acid sequences (1) to (6) above and has immunity-inducing activity.
  • amino acid substitutions may be included in any one of the amino acid sequences (1) to (6) above.
  • those capable of activating cytotoxic T cells that recognize kidney cancer antigen protein are preferred.
  • the above (1) is a peptide A24 / 10mer190 derived from the amino acid sequence of sugar chain binding protein galectin 9 (36-6-1) (Non-patent Document 7) involved in cellular immunity.
  • Galectin 9 is remarkably expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues. Although the gene sequence is known, this peptide is not known and its use is not known.
  • PINCH is peptide A24 / 10mer238 derived from the amino acid sequence of adapter protein PINCH (113-3-1) (Non-patent Document 8) relating to signaling of cell adhesion.
  • PINCH is also highly expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues, and the gene sequence is known, but this peptide is not known and its use is not known.
  • (3) and (4) above are peptides 10m-103 and 10m-117 derived from the amino acid sequence of sugar chain binding protein galectin 9 (36-6-1) (Non-patent Document 7) involved in cellular immunity. It is. Galectin 9 is remarkably expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues. Although the gene sequence is known, this peptide is not known and its use is not known.
  • peptides 9m-284 and 9m-29 derived from the amino acid sequence of adapter protein PINCH (113-3-1) (Non-Patent Document 8) relating to cell adhesion signaling.
  • PINCH is also highly expressed in clear cell carcinoma, which accounts for the majority of kidney cancer, compared to normal tissues, and the gene sequence is known, but this peptide is not known and its use is not known.
  • kidney cancer antigen proteins can be detected from, for example, cancer cells collected from kidney cancer patients by cDNA microarray analysis.
  • mRNA is prepared by dividing a tissue extracted from a subject into a cancerous part and a non-cancerous part, and then fluorescence-labeled cDNA is prepared. This is a method of analyzing the gene expression by placing the signal on a glass slide and hybridizing, and then capturing the signal with a scanner.
  • the method for producing the renal cancer antigen protein is not particularly limited, and may be a naturally derived protein, a chemically synthesized protein, or a recombinant protein prepared by a gene recombination technique.
  • Recombinant proteins are preferred in that they can be produced in large quantities by a relatively easy operation.
  • a naturally-derived protein it can be isolated from cells or tissues expressing the protein by appropriately combining methods for protein isolation and purification.
  • a chemically synthesized protein When a chemically synthesized protein is obtained, it can be synthesized according to a chemical synthesis method such as the Fmoc method or the tBoc method. It can also be synthesized using various commercially available peptide synthesizers.
  • To produce it as a recombinant protein it can be produced by obtaining a DNA having a base sequence encoding the protein or a mutant or homologue thereof and introducing it into a suitable expression system.
  • 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 a vector containing a promoter at a position where the gene can be expressed is used.
  • the transformant which has the gene which codes a protein can be produced by introduce
  • 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 conventionally known standard method according to each host.
  • a recombinant protein can be isolated by culturing a transformant having a gene, producing and accumulating a desired protein in the culture, and collecting the protein from the culture.
  • the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism.
  • a natural medium or a synthetic medium may be used as long as the medium can efficiently be cultured.
  • the culture conditions may be the conditions usually used for culturing microorganisms.
  • a conventionally known protein isolation and purification method may be used.
  • a protein having an amino acid sequence containing substitution, deletion, insertion and / or addition of one or several amino acids is a DNA sequence encoding the amino acid sequence. Based on this information, it can be produced by a conventionally known method. That is, a mutant gene having a base sequence encoding a protein having a desired amino acid sequence can also be prepared by chemical synthesis, genetic engineering techniques, mutagenesis, and the like. For example, it can be carried out by using a method of bringing a drug that acts as a mutagen into contact with DNA before displacement, a method of irradiating ultraviolet rays, or a genetic engineering technique.
  • Peptide synthesis can be performed according to methods used in normal peptide chemistry. For example, it can be synthesized according to a chemical synthesis method such as the Fmoc method or the tBoc method. Moreover, the peptide of this invention can also be synthesize
  • the peptides according to the invention have a binding motif for HLA-A * 2402 or HLA-A * 0201. Such selection of peptides having binding motifs for HLA-A * 2402 or HLA-A * 0201 can be performed based on conventionally known methods including calculation of binding affinity between various peptides and HLA antigens. it can.
  • the DNA according to the present invention is as follows.
  • the DNA of the present invention is a DNA encoding the protein or peptide described above, and is preferably the DNA described in any one of (a), (b) and (c) below.
  • A DNA having the base sequence according to any one of (1) to (6) above.
  • B DNA that hybridizes with the DNA of (a) above under stringent conditions and encodes a protein or peptide having immunity-inducing activity.
  • C DNA encoding a protein or peptide having a partial sequence of the DNA of (a) or (b) and having immunity-inducing activity.
  • Hybridization under stringent conditions means a DNA base sequence obtained by using DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. Examples thereof include DNA that can be identified by a conventionally known method and has a certain degree of homology with the base sequence of DNA used as a probe.
  • the method for obtaining DNA is not particularly limited, and appropriate probes and primers are prepared based on the sequence information described in any of (1) to (6) above, and cDNA libraries such as humans are screened using them. Can be isolated. It can also be obtained by the PCR method. Using a human chromosomal DNA or cDNA library as a template, PCR is performed using a pair of primers designed to amplify the desired base sequence, and then amplified. DNA fragments can be cloned into an appropriate vector that can be amplified in a host such as E. coli.
  • the antibodies and cytotoxic cells according to the present invention are as follows.
  • the present invention also relates to an antibody that recognizes part or all of the protein or peptide of the present invention as an epitope, and cytotoxic T cells induced by in vitro stimulation using the protein or peptide of the present invention.
  • cytotoxic T cells show stronger antitumor activity than antibodies.
  • the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, a fragment thereof, or a labeled antibody of the antibody.
  • the production can be performed by a conventionally known method.
  • a polyclonal antibody is obtained by immunizing a mammal with the protein of the present invention as an antigen, collecting blood, and separating and purifying the antibody from the collected blood. Examples of mammals include mice, hamsters, guinea pigs, chickens, rats, rabbits, dogs, goats, sheep, cows, and the like, for example, about 0.05 to 2 mg of antigen 2 to 3 times at intervals of 7 to 30 days. Administer.
  • Antigen is dissolved in an appropriate buffer containing adjuvant such as complete Freund's adjuvant or aluminum hydroxide, and the administration route includes subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. Is mentioned.
  • adjuvant such as complete Freund's adjuvant or aluminum hydroxide
  • the administration route includes subcutaneous administration, intradermal administration, intraperitoneal administration, intravenous administration, intramuscular administration, etc. Is mentioned.
  • chromatography such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, etc.
  • a polyclonal antibody that recognizes the protein of the present invention can be obtained.
  • Monoclonal antibodies can be obtained by preparing hybridomas.
  • a hybridoma can be obtained by cell fusion between an antibody-producing cell and a myeloma cell line.
  • antibody-producing cells spleen cells, lymph node cells, B lymphocytes and the like from immunized animals are used.
  • antigen the protein or peptide of the present invention is used.
  • immunized animal mice, rats and the like are used, and the animal is immunized by administering a suspension of an adjuvant and an antigen protein or peptide several times into the vein, subcutaneous, intradermal or intraperitoneal cavity of the animal.
  • spleen cells are obtained as antibody-producing cells from the immunized animal, and this and myeloma cells are fused by a conventionally known method to prepare a hybridoma.
  • myeloma cell strains used for cell fusion include P3X63Ag8, P3U1 strain, Sp2 / 0 strain and the like in mice.
  • fusion promoters such as polyethylene glycol and Sendai virus are used, and hypoxanthine / aminopterin / thymidine medium or the like is used for selection of hybridomas after cell fusion.
  • Hybridomas obtained by cell fusion are cloned by limiting dilution or the like.
  • a cell line producing a monoclonal antibody that specifically recognizes a desired protein By screening by enzyme immunoassay, a cell line producing a monoclonal antibody that specifically recognizes a desired protein can be obtained.
  • the hybridoma In order to produce a target monoclonal antibody from a hybridoma, the hybridoma is cultured by a cell culture method or ascites formation method, and the monoclonal antibody is purified from the culture supernatant or ascites.
  • ammonium sulfate fractionation, gel filtration, ion exchange chromatography, affinity chromatography, etc. are used in appropriate combination.
  • the antibody of the present invention can be used after being labeled.
  • an enzyme label a fluorescent label, a label with a coloring substance, an affinity label, an isotope label, and the like can be given.
  • protein or peptide analysis using the labeled antibody of the present invention include enzyme antibody method, immunohistochemical staining method, immunoblot method, direct fluorescent antibody method, indirect fluorescent antibody method and the like.
  • the present invention also relates to activated T cells induced by in vitro stimulation using the protein or pepsid of the present invention.
  • activated T cells induced by in vitro stimulation using 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 these activated T cells are effectively used for immunotherapy.
  • the protein or peptide of the present invention can be expressed in vivo or in vitro in dendritic cells which are strong antigen-presenting cells, and immunity induction can be performed by administration of the antigen-expressing dendritic cells.
  • the vaccine according to the present invention is as follows. Since the protein, peptide, and DNA of the present invention can induce T cells that can specifically damage kidney cancer cell lines, they can be used as therapeutic or preventive agents for kidney cancer.
  • BCG bacteria transformed with recombinant DNA by incorporating the DNA of the present invention into an appropriate vector, or viruses such as vaccinia virus integrated with the DNA of the present invention in the genome are used as vaccines.
  • adjuvants include Freund's incomplete adjuvant, BCG, trehalose dimycolate, lipopolysaccharide, alum adjuvant, silica adjuvant and the like.
  • the probe and diagnostic agent for diagnosing kidney cancer according to the present invention are as follows.
  • the DNA of the present invention can be used as a diagnostic probe by taking out the DNA of kidney cancer and examining its homology, and can also be used as a diagnostic agent for kidney cancer using this probe or the above-mentioned antibody. it can.
  • As the diagnostic probe a probe which is the whole or a part of the antisense strand of DNA or RNA encoding the protein of the present invention and has a length sufficient to be established as a probe is preferable.
  • diagnosis can be performed by detecting mRNA of kidney cancer antigen obtained from a specimen using an antisense strand.
  • Examples of the sample used for detection include cells such as kidneys of a subject, genomic DNA that can be obtained from a biopsy of blood, urine, saliva, tissue, and RNA.
  • Immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies that specifically bind to the proteins and peptides of the present invention can be used for the diagnosis of kidney cancer.
  • the protein, peptide, or antibody of the present invention can be administered as it is, or together with a pharmaceutically acceptable carrier, diluent or adjuvant, by injection or percutaneous absorption.
  • the renal cancer preventive and therapeutic agents according to the present invention are as follows. Since the protein or peptide of the present invention can induce a renal cancer cell-specific cytotoxic T cell as a T cell epitope, it is useful as a preventive or therapeutic agent for kidney cancer.
  • auxiliary agents for enhancing the inducing activity of cytotoxic T cells include saponin-based QS-21, liposome, aluminum hydroxide and the like.
  • immunostimulants such as lentinan, schizophyllan, and picibanil can be used as adjuvants.
  • cytokines that enhance proliferation and differentiation of T cells such as IL-2, IL-4, IL-12, IL-1, IL-6, TNF, and IFN can also be used as adjuvants.
  • antigen peptides are added to cells collected from patients with kidney cancer or cells having the same HLA paprotype in vitro and presented to the antigen, then administered into the patient's blood vessels and effectively cytotoxic in the patient's body.
  • T cells can also be induced.
  • cytotoxic T cells can be induced in a test tube by adding an antigen peptide to the patient's peripheral blood lymphocytes and cultured in a test tube, and then returned to the patient's blood vessel.
  • the antigen is a recognition antigen for cytotoxic T cells.
  • the antigen of the present invention increases in vitro killer T cell-inducing activity in HLA-A * 2402, which is common in Japanese, and in HLA-A * 0201, which is common in Westerners.
  • cytotoxic T cells are induced and activated and used for antitumor.
  • activated T cells are induced in vitro when stimulated with the antigen of the present invention, it can be effectively used for immunotherapy by injecting activated T cells into the body.
  • RNAi according to the present invention is as follows.
  • examples of the nucleic acid capable of suppressing the expression of the protein of the present invention by the RNAi phenomenon include siRNA, shRNA, or expression vectors thereof.
  • the antitumor agent of the present invention can be mixed with a pharmaceutically acceptable additive as necessary.
  • Pharmaceutically acceptable additives include antioxidants, preservatives, colorants, flavors, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, Examples include carriers, excipients, and pharmaceutical adjuvants.
  • peptide of this invention may provide as a peptide derivative.
  • Such derivatives include modifications to promote synthesis and purification, modifications to promote physical and chemical stabilization, stability and instability to metabolism in vivo, and activation modifications such as conditioning.
  • Other modifications in peptide derivatives include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, Phosphatidylinositol covalent bond, cross-linking, cyclization, disulfide bond, demethylation, cross-linking covalent bond formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodine , Methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, race
  • the peptide derivative is generated as a side chain of a residue or an N-terminal group or a C-terminal group as long as the activity of the peptide of the present invention is not destroyed and the composition containing the peptide is not toxic. It can be prepared as a functional group.
  • derivatives containing polyethylene glycol side chains that extend the residue of peptides in body fluids, aliphatic esters of carboxyl groups, amides of carboxyl groups by reacting with ammonia or amines, liberation of amino acid residues formed with acyl moieties examples thereof include N-acyl derivatives of amino groups and O-acyl derivatives of free hydroxyl groups formed with acyl moieties.
  • the peptide may be provided as a pharmacologically acceptable salt.
  • This salt includes both a carboxyl group salt and an amino acid addition salt of a polypeptide.
  • the salt of the carboxyl group include inorganic salts such as sodium, calcium, ammonium, iron, and zinc, and salts with organic bases formed using amines such as triethanolamine, arginine, lysine, piperidine, and procaine. It is done.
  • acid addition salts include salts with mineral acids such as hydrochloric acid and sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid.
  • the method for evaluating the predisposition to developing kidney cancer according to the present invention is as follows.
  • the biological sample include kidney cells and blood.
  • Conventionally known conventional methods can be used as a method for collecting and preparing the same and a method for measuring and evaluating the expression level of peptide-related genes.
  • SEREX serological analysis of cancer antigens by The recombinant cDNA expression cloning method is a method in which mRNA is directly extracted from a cancer tissue extracted from a cancer patient, and a cancer antigen is searched from the prepared cDNA expression library using the serum of the cancer patient. It is a comprehensive analysis method different from microarrays, and many genes are registered as SEREX antigens in the database, but most are not yet evaluated, and there are very few reports on kidney cancer.
  • NC membrane Nitrocellulose (NC) membrane was immersed in E. coli phage lysate, and proteins expressed in E. coli and phage were adsorbed on the NC membrane.
  • the NC membrane was soaked in kidney cancer patient serum diluted 5-fold with TBST, and antibodies cross-reacting with E. coli and phage were removed from the serum and used for screening by the SEREX method.
  • RNA (OCUU1) established from a patient's kidney cancer cells (clear cell carcinoma) as a cell culture strain, and after mRNA was isolated, a cDNA library was cloned into ⁇ ZAP.
  • ZAP-cDNAgenesynthesis kit Gigapack III gold cloning kit, manufactured by Stratagene.
  • a cDNA library was seeded so that there would be 19000 plaques per 10 ⁇ 14 cm NZY plate. After culturing at 42 ° C. for 4.5 hours, an NC membrane containing IPTG was placed on the plate and further cultured at 37 ° C. for 3.5 hours, and protein expression was carried out by phage plaques. The plate was cooled overnight at 4 ° C., the NC membrane was peeled off from the plate, and a plaque lift was taken. After washing with TBST, blocking was performed for 1 hour, and reaction was performed at room temperature for 2 hours with the above-treated patient serum diluted 100 times as a primary antibody.
  • phages were seeded on an NZY plate having a diameter of 10 cm so that the number of phages per plate was 500, and screening was performed in the same manner.
  • a single clone was obtained by the third screening, and in-vivo excision was performed from the phage, and the gene was cloned into a plasmid (pBluescript) and sequenced.
  • plaques were screened (121 plates in the primary screening), and 43 positive clones including duplicates were obtained. They encoded 15 genes including chromatin modulators, ubiquitin-related, tumor suppressor-related, mitosis-related, and adhesion-related proteins. Among them, one gene protein that reacts only with the serum of IFN treatment effective cases was obtained, and two genes whose expression was remarkably increased in renal cancer tissues compared with normal kidney tissues were obtained.
  • kidney cancer tissue and normal kidney tissue were compared, and the expression in each tissue was examined by RT-PCR method.
  • two types of genes whose expression was remarkably increased in kidney cancer tissues of all clinical specimens as compared with normal kidney tissues were obtained.
  • FIG. 1 shows RT-PCR of the gene.
  • Gene 36-6-1 is the same as the gene of sugar chain binding protein galectin 9 (Non-patent Document 7) involved in cellular immunity, and the other gene 113-3-1 is an adapter protein related to cell adhesion signaling. It was found to be identical to the PINCH gene (Non-Patent Document 8). In any case, the sequence of the gene is known, but this peptide is not known, and the use is unknown.
  • PCR primers the following sequences (3) and (4) were used for 36-6-1 FW and RV, respectively, denaturation 94 ° C., 30 seconds, annealing 65 ° C., 30 seconds, polymerization 72 ° C. , 3 minutes, 30 cycles (TaKaRa, RNA PCR kit).
  • FIG. 1 data of a specific cultured cell line of kidney cancer is shown as a reference.
  • the gene expression is selected and the expression is lost by continuing to culture under certain conditions.
  • 36-6-1 is such a protein or gene
  • 113-3-1 is considered to be a protein or gene that remains relatively indefinitely despite continuation of culture.
  • FIG. 1 shows the results of only 5 cases in which the expression of genes in the normal part and the kidney cancer part are compared. However, even if the number of cases is increased to 815, 36-6-1 and 113-3 -1 gave similar results.
  • cytokine therapy 36-6-1 was screened using the serum of a rare case of multiple bone metastases of kidney cancer that completely disappeared with interferon ⁇ . It was obtained. 113-3-1 is derived from a patient whose sera used for cloning had lung cancer metastasis clearly reduced by interferon ⁇ .
  • Peptides were synthesized based on these two genes 36-6-1 (galectin 9) and 113-3-1 (PINCH), respectively. Induced from normal human peripheral blood mononuclear cells (PBMC) using peptide A24 / 10mer190 (sequence (1)) derived from 36-6-1 and peptide A24 / 10mer238 (sequence (2)) derived from 113-3-1 The cytotoxic T cells thus obtained were examined for HLA-A * 2402 restriction and antigen-specific cytotoxic activity. HLA-A * 2402 is the most common type of HLA in Japanese. As a result, as shown in FIGS.
  • the conventional peptide that induces cytotoxic T cells is derived from a gene screened by a method different from the peptide of the present invention, and induces cytotoxic T cells by stimulating lymphocytes in kidney cancer patient blood. As shown, the peptide of the present invention was able to induce highly active cytotoxic T cells from normal human lymphocytes.
  • 36-6-1 is a protein or gene that loses its expression when it is selected for gene expression by continuing to be cultured under certain conditions.
  • 113-3-1 is considered to be a relatively long-lasting protein and gene despite continued culture.
  • 36-6-1 is specific to kidney cancer, but 113-3-1 is also expressed in other cancer cells (PC3, DU145, LNCaP is prostate cancer, T24 is bladder Cancer, NEC8 is testicular tumor, HeLa is cervical cancer, OCUB1 and MCF7 are breast cancer cell lines).
  • cytotoxic T cells were induced as follows.
  • PBMCs were isolated from the blood of healthy individuals with HLA-A * 2402 / A * 0201 using Ficoll-Paque PLUS (GE Healthcare Bio-Science AB) (responder cells).
  • JTK-LCL cells RB1873
  • B-LCLs B-cells transformed and immortalized by EB virus
  • PBMCs were separated from HLA-A * 2402 donors, dispensed at 3 ⁇ 10 6 / ml / well in 24-well plates, added with peptides at a concentration of 5 ⁇ M, and cultured for 5 days.
  • JTK-LCL cells were collected, the cells were counted and adjusted to a density of 0.8 ⁇ 10 6 / ml, and then the peptide was added at a concentration of 3 ⁇ M (peptide pulse).
  • 500 ⁇ L of JTK-LCL cells irradiated so as not to proliferate were layered on the above-described responder cells and co-cultured. From 2 days later, IL2 was prepared to a final concentration of 20 IU / mL every 2 days.
  • CD8 + T cells in the responder cells were 90% Concentrated to the same extent and stimulated similarly using JTK-LCL cells.
  • the cytotoxic activity of CD8 + T cells against target cells was measured by 51Cr releasing assay.
  • tumor cells (5 types) were collected from the plate, 51Cr was added as 5 ⁇ 10 6 cells / ml each, cultured in an incubator, 51Cr was taken up into the tumor cells, and a sample was prepared. Effector cells are induced with CTL recovered, 2 ⁇ 10 6 cells / ml using the cell concentration in the liquid was made a dilution series microplate 96 well (2 ⁇ 10 6 cells / ml, the 3/10 And 1/10). After adding 100 ⁇ l of 51Cr-labeled target cells to effector cells and co-culturing for 4 hours, the radioactivity released from the target cells damaged by CTL attack was determined.
  • the cytotoxic activity against TUHR-10TKB by each cytotoxic T cell was high even at a low E: T ratio.
  • Both cytotoxic T cells derived from normal human peripheral blood lymphocytes by the two peptides of the present invention have cancer cell cytotoxicity of over 60% at a low E: T ratio (10: 1).
  • the peptide is remarkably highly effective. This indicates that these antigens have high antigenicity as kidney cancer antigens and are useful immunotherapy targets.
  • the two peptides of the present invention can be used in combination.
  • kidney cancer cells since the two types of genes identified in the present invention are highly expressed in kidney cancer cells as shown in FIG. 1, detection of kidney cancer cells in the circulating blood using the RT-PCR method is performed. It is also applicable to diagnosis.
  • FIGS. 4 (a) and (b) show HLA-A * 0201 restriction in cytotoxic T cells induced with peptide (3) and peptide (4) based on galectin 9 (36-6-1). 5 shows antigen-specific cytotoxic activity, and FIGS. 5 (a) and (b) show cytotoxicity T cells induced using peptide (5) and peptide (6) based on PINCH (113-3-1). It shows HLA-A * 0201 restriction and antigen-specific cytotoxic activity.
  • kidney cancer cell line A498 expressing each antigen was transformed into HLA- by the induced cytotoxic T cells. It becomes clear that A * 0201 restriction is impaired, and as shown in FIGS. 4 (b) and 5 (b), the antigens having the sequences (4) and (6) are expressed. It was found that kidney cancer cell lines A498 and TUHR-10TKB are impaired by HLA-A * 0201 restriction by induced cytotoxic T cells.
  • the antigenic protein, peptide of the present invention, or DNA encoding them can be used as an excellent kidney cancer vaccine with few side effects such as self-injury.
  • antibodies can be used as diagnostic agents, cytotoxic T cells stimulated and activated by antigens can be used as anticancer agents, and RNAi that suppresses the expression of the antigen of the present invention can enhance the antigen of the present invention. It can be used to treat developing kidney cancer.
  • the search method of the present invention is useful for searching for target genes useful for cancer immunotherapy. Therefore, it contributes to the treatment and early detection of cancer such as kidney and is industrially useful.

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Abstract

L'invention concerne un peptide utile pour l'immunothérapie du cancer du foie, et une méthode de traitement efficace ainsi qu'un procédé de diagnostic précoce. Le peptide comprend une séquence d'acides aminés comprise dans les séquences (1)-(6) suivantes et qui possède une activité induisant une immunité. AYPMPFITTI (1) AYCETHYNQL (2) FLVQSSDFKV (3) ILFVQYFHRV (4) KLTLKNKFV (5) ELYHEQCFV (6).
PCT/JP2012/059878 2011-04-11 2012-04-11 Peptide pour l'immunothérapie du cancer et procédé d'utilisation de celui-ci WO2012141201A1 (fr)

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JP2014065680A (ja) * 2012-09-26 2014-04-17 Osaka City Univ 癌免疫療法のためのペプチド及びその利用

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Publication number Priority date Publication date Assignee Title
JP2014065680A (ja) * 2012-09-26 2014-04-17 Osaka City Univ 癌免疫療法のためのペプチド及びその利用

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