WO2015049985A1 - Medicinal composition for fgf4 gene-amplified tumor - Google Patents

Medicinal composition for fgf4 gene-amplified tumor Download PDF

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WO2015049985A1
WO2015049985A1 PCT/JP2014/074530 JP2014074530W WO2015049985A1 WO 2015049985 A1 WO2015049985 A1 WO 2015049985A1 JP 2014074530 W JP2014074530 W JP 2014074530W WO 2015049985 A1 WO2015049985 A1 WO 2015049985A1
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fgf4
gene
pharmaceutical composition
tumor
amplified
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PCT/JP2014/074530
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French (fr)
Japanese (ja)
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西尾和人
荒尾徳三
松本和子
福島雅夫
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住友ベークライト株式会社
学校法人近畿大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates to a pharmaceutical composition of an FGF4 gene-amplified tumor, a method for predicting the effect of the pharmaceutical composition, a kit used therefor, the use of the pharmaceutical composition, and a method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor. .
  • Patent Document 1 predicts whether therapeutic administration of an epidermal growth factor receptor (EGFR) inhibitor is effective or ineffective is an increase in EGFR gene copy number, tyrosine kinase receptor type receptor (HER2) copy. Disclose what is possible by detecting an increase in number.
  • EGFR epidermal growth factor receptor
  • HER2 tyrosine kinase receptor type receptor
  • Patent Document 2 discloses that it is possible to predict the therapeutic effect of sorafenib, which is a multi-target kinase inhibitor and a cancer molecular target therapeutic agent, by detecting an increase in the copy number of the FGF3 gene.
  • Patent Document 3 discloses that an increase in the copy number of the ⁇ -actinin-4 gene may indicate the presence of cancer.
  • JP 2013-5800 A JP 2012-249633 A Japanese Patent Application Laid-Open No. 2009-10073
  • the present disclosure provides a pharmaceutical composition of an FGF4 gene amplified tumor. Moreover, this indication provides the effect prediction method of this pharmaceutical composition in one or some embodiment.
  • the present disclosure is a pharmaceutical composition for improving, suppressing progression, and / or treating an FGF4 gene-amplified tumor, comprising an anti-FGF4 antibody, an FGF receptor inhibitor, an FGF4 siRNA, And a pharmaceutical composition containing as an active ingredient at least one selected from the group consisting of FGF4 shRNA.
  • the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and the target FGF4 gene is amplified. If the effectiveness of therapeutic administration of the pharmaceutical composition according to the present invention is predicted, the subject is classified, or the therapeutic administration of the pharmaceutical composition according to the present disclosure is effective when the subject FGF4 gene is not amplified. It relates to a method comprising classifying said object if gender is not predicted.
  • the present disclosure detects the gene amplification level of the FGF4 gene in the tumor tissue sample or tumor cell sample of the subject, and the FGF4 protein when the subject FGF4 gene is amplified.
  • the present invention relates to a method for improving, suppressing the progression of and / or treating FGF4 gene-amplified tumor, comprising administering to the subject a pharmaceutical composition comprising a substance that suppresses the expression of or inhibits the function thereof as an active ingredient.
  • FIG. 1 is a graph showing an example of the results of measuring the FGF4 gene copy number of 194 esophageal cancer specimens.
  • the horizontal axis is the sample sample and is arranged in order of copy number.
  • the vertical axis represents the measured copy number.
  • FIG. 2 shows an example of the results of measuring the tumor size of a cell line (WiDr / FGF4) over time when FGF4 gene is introduced into colon cancer cell line WiDr and FGF4 protein is overexpressed. It is a graph.
  • an EGFP gene was introduced (WiDr / EGFP).
  • FIG. 3 is a graph showing an example of the results of measuring the tumor size of a cell line (A549 / FGF4) over time when the FGF4 gene was introduced into the lung cancer cell line A549 and the FGF4 protein was overexpressed. It is.
  • an EGFP gene was introduced (A549 / EGFP).
  • FIG. 4 shows the results of measuring the tumor size of the cell line (KYSE-270 / FGF4) over time when the FGF4 gene was introduced into the esophageal cancer cell line KYSE-270 and the FGF4 protein was overexpressed. It is a graph which shows an example.
  • an EGFP gene was introduced (KYSE-270 / EGFP).
  • FIG. 5 is a graph showing an example of the results of confirming the effect of anti-FGF4 antibody on FGF4 overexpressing esophageal cancer cell line KYSE-270 / FGF4 transplanted into mice.
  • goat polyclonal IgG was used instead of anti-FGF4 antibody.
  • the vertical axis represents the tumor size (mm 3 )
  • the horizontal axis represents the culture time (days).
  • the vertical axis represents the mouse body weight (g)
  • the horizontal axis represents the culture time (days).
  • FIG. 6 is a graph showing an example of the results of confirming the effect of an FGFR inhibitor on the FGF4-overexpressing lung cancer cell line A549 / FGF4 transplanted into mice.
  • the control is an example in which no FGFR inhibitor is administered.
  • the vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days).
  • the present disclosure indicates that some tumors and cancers have an amplified copy number of the FGF4 gene, and suppress or inhibit the function of the FGF4 protein in such an FGF4 gene-amplified tumor. Based on the finding that a pharmaceutical composition containing a substance as an active ingredient has an antitumor effect. The present disclosure is also based on the finding that, in one or more embodiments, amplification of the FGF4 gene copy number occurs in about 40% of esophageal cancers.
  • the present disclosure is a pharmaceutical composition for improving, suppressing progression and / or treating FGF4 gene-amplified tumor, and contains a substance that suppresses the expression of FGF4 protein or inhibits its function as an active ingredient.
  • the present invention relates to a pharmaceutical composition.
  • the tumor that is the target of the pharmaceutical composition according to the present disclosure is a tumor in which the copy number of FGF4 (Fibroblast growth factor 4) gene is amplified (hereinafter also referred to as “FGF4 gene amplified tumor”). It is.
  • FGF4 gene amplified tumors can be present in various cancers. For example, about 40% of esophageal cancers are FGF4 gene amplified tumors. In addition, for example, there is a report that FGF4 gene amplification is observed in tumor samples of about 10% of bladder cancer and about 35% of head and neck squamous cell carcinoma.
  • the pharmaceutical composition according to the present disclosure can exert an antitumor effect on an FGF4 gene-amplified tumor.
  • the pharmaceutical composition according to the present disclosure can be used for improvement, suppression of progression, and / or treatment of an individual having an FGF4 gene-amplified tumor.
  • the FGF4 gene amplified tumor is esophageal cancer.
  • the effectiveness of the pharmaceutical composition according to the present disclosure can be determined by examining the presence or absence of amplification of the copy number of the FGF4 gene in the subject tumor. Therefore, in another aspect, the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and the pharmaceutical composition according to the present disclosure when the target FGF4 gene is amplified Classifying the subject as the efficacy of therapeutic administration of the product is predicted, or the efficacy of therapeutic administration of the pharmaceutical composition according to the present disclosure is not expected when the subject's FGF4 gene is not amplified And a method including classifying the object (hereinafter, also referred to as “classification method according to the present disclosure”). Examples of the classification method according to the present disclosure include humans and mammals other than humans in one or a plurality of embodiments.
  • the presence or absence of amplification of the copy number of the FGF4 gene a conventionally performed method or a method developed in the future can be used in one or a plurality of embodiments.
  • the presence or absence of amplification of the copy number of the FGF4 gene can be performed by fluorescent fluorescence in situ hybridization (FISH), a next-generation sequencer, a DNA chip, or real-time quantitative PCR.
  • FISH fluorescent fluorescence in situ hybridization
  • the fact that the copy number of the FGF4 gene is increased means that the copy number of the FGF4 gene of the target tumor is larger than the copy number of the control gene of normal cells.
  • Amplification of gene copy number by fluorescence in situ hybridization can be detected / measured in one or more embodiments as follows.
  • FISH fluorescence in situ hybridization
  • a fragment DNA (probe) of a specific gene is hybridized with a chromosome or nuclear DNA using a fluorescent substance, and the increase / decrease (deletion or amplification) of the gene and the position (mapping) in a staining pair are examined. It is. Therefore, cells can be stained with a probe specific to the sequence of the target gene, and the increase or decrease in copy number can be measured according to the number of signals.
  • FGF3 / FGF4 amplification and multiple lung metastasesin responders to sorafenib in hepatocellular carcinoma. ”
  • amplification of gene copy number by a next-generation sequencer can be detected / measured as follows. Unlike conventional Sanger methods, next-generation sequencers can process tens of millions to hundreds of millions of DNA fragments in parallel. Therefore, by increasing and decreasing the copy number of the target gene by sequencing the DNA from the tumor sample and the control sample and comparing the coverage of the target gene. Also, “PLoS One. 2013; 8 (5): e64271. Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation Sequencing” and “Clin Cancer Res. 2013 May 15; 19 (10): 2668-76. Relapsed classic E-cadherin (CDH1) -mutated invasive lobular breast cancer shows a high frequency of HER2 (ERBB2) gene mutations. ”
  • amplification of gene copy number by a DNA chip can be detected / measured as follows. That is, CGH (comparative genomic hybridization) analysis is performed using a slide glass (DNA chip) in which a large number of DNA fragments are arrayed. Tumor samples and control samples labeled with different fluorescent dyes are hybridized with DNA fragments on the DNA chip. Based on the intensity ratio of fluorescent signals derived from each sample, the copy number of tumor DNA in the region corresponding to the arrayed DNA fragments is quantified. Also, “http://www.cghtmd.jp/CGHDatabase/about/about#high.jsp” and “Hepatology. 2013 Apr; 57 (4): 1407-15. FGF3 / FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma. ”
  • Amplification of gene copy number by real-time quantitative PCR can be detected / measured as follows. That is, using a sample DNA as a template, a target gene site is amplified by a PCR reaction using primers specific to the target gene. The amplification reaction is detected in real time, the number of PCR cycles for amplification up to a certain amount is compared between the control sample and the tumor sample, and the copy number is calculated according to the difference. Also, “http://tools.invitrogen.com/content/sfs/manuals/cms#062368.pdf” and “Hepatology. 2013 Apr; 57 (4): 1407-15. FGF3 / FGF4 amplification and multiple lung metastases in Refer to "responders to sorafenib in hepatocellular carcinoma.”
  • the active ingredient of the pharmaceutical composition according to the present disclosure is a substance that suppresses the expression of FGF4 protein or a substance that inhibits the function.
  • the active ingredient of the pharmaceutical composition according to the present disclosure includes at least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA.
  • the anti-FGF4 antibody has neutralizing activity or high neutralizing activity in one or a plurality of embodiments.
  • the anti-FGF4 antibody is a monoclonal antibody in one or more embodiments. Further, in one or a plurality of embodiments, the anti-FGF4 antibody may be in the form of an antibody fragment or may be modified with a sugar chain.
  • FGF receptor is a receptor for FGF4 protein. Therefore, the FGF receptor inhibitor can be used as an active ingredient of the pharmaceutical composition according to the present disclosure.
  • the FGF receptor inhibitor conventionally known and future developed ones can be used.
  • FGF4 gene siRNA small interfering RNA
  • shRNA small hairpin
  • a person skilled in the art can design and manufacture appropriate FGF4 gene siRNA and shRNA according to standard methods.
  • the “pharmaceutical composition” may be a dosage form suitable for an administration form by applying a well-known formulation technique in one or a plurality of embodiments.
  • the dosage form include, but are not limited to, oral administration in a dosage form such as a tablet, capsule, granule, powder, pill, troche, syrup, and liquid.
  • parenteral administration in dosage forms such as injections, liquids, aerosols, suppositories, patches, lotions, liniments, ointments, eye drops and the like can be mentioned.
  • These preparations can be produced by known methods using additives such as, but not limited to, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
  • the pharmaceutical composition according to the present disclosure does not contain other active ingredients having a therapeutic effect, or further contains one or a plurality of active ingredients.
  • excipient examples include, but are not limited to, starch such as starch, potato starch, and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like.
  • coating agent examples include, but are not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, paraffin, and the like.
  • binder include, but are not limited to, polyvinyl pyrrolidone, macrogol and the same compound as the excipient.
  • disintegrant examples include, but are not limited to, compounds similar to the excipients and chemically modified starch and celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone.
  • stabilizer examples include, but are not limited to, paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenol, cresol Mention may be made of such phenols; thimerosal; dehydroacetic acid; and sorbic acid.
  • flavoring agent examples include, but are not limited to, sweeteners, acidulants, and fragrances that are commonly used.
  • the solvent is not limited to these, but ethanol, phenol, chlorocresol, purified water, distilled water and the like can be used, and a surfactant or an emulsifier can also be used as necessary.
  • a surfactant or an emulsifier include, but are not limited to, polysorbate 80, polyoxyl 40 stearate, lauromacrogol, and the like.
  • the method of using the pharmaceutical composition according to the present disclosure may vary depending on symptoms, age, administration method, and the like.
  • the method of use is not limited to these, but intermittently or continuously, orally, transdermally, submucosally, subcutaneously, intramuscularly, vascularly, so that the concentration of the active ingredient in the body is between 100 nM and 1 mM. It can be administered internally, intracerebrally, or intraperitoneally.
  • the lower limit is 0.01 mg (preferably 0.1 mg) and the upper limit is 2000 mg as converted into the active ingredient per day for a subject (adult if human). (Preferably 500 mg, more preferably 100 mg) may be divided into 1 or several times and administered according to symptoms.
  • the lower limit is 0.001 mg (preferably 0.01 mg) and the upper limit is 500 mg (preferably 50 mg) per day for a subject (adult if human). Is divided into one or several times and administered according to symptoms.
  • the present disclosure relates to at least one use selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA for producing a pharmaceutical composition according to the present disclosure.
  • the present disclosure relates to a method for improving, suppressing progression, and / or treating an FGF4 gene-amplified tumor, comprising administering a pharmaceutical composition according to the present disclosure to a subject in need.
  • the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and suppresses the expression of the FGF4 protein when the target FGF4 gene is amplified.
  • the present invention relates to a method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor, comprising administering to the subject a pharmaceutical composition containing a substance that inhibits function as an active ingredient.
  • the present disclosure provides at least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA in the above-described improvement, suppression of progression, and / or treatment of an FGF4 gene-amplified tumor.
  • Use, or use of a pharmaceutical composition according to the present disclosure are examples of a pharmaceutical composition according to the present disclosure.
  • the present disclosure provides a reagent for detecting the FGF4 gene amplification level by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR, And a reagent for detecting the FGF4 gene amplification level and a nucleotide that hybridizes to the FGF4 gene by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR Relates to a kit comprising:
  • a pharmaceutical composition for improvement, suppression of progression and / or treatment of FGF4 gene-amplified tumor which is selected from the group consisting of anti-FGF4 antibody, FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA.
  • a pharmaceutical composition containing at least one of these as an active ingredient [2] The pharmaceutical composition according to [1], wherein the FGF4 gene-amplified tumor is esophageal cancer. [3] The pharmaceutical composition according to [1] or [2], wherein the anti-FGF4 antibody is a neutralizing antibody having specificity for FGF4.
  • [4] detecting the gene amplification level of the FGF4 gene in the tumor tissue sample or tumor cell sample of the subject; and Classifying the subject when the efficacy of therapeutic administration of the pharmaceutical composition according to any one of [1] to [3] is predicted when the subject's FGF4 gene is amplified, or Categorizing the subject as not predicting the effectiveness of therapeutic administration of the pharmaceutical composition of any of [1] to [3] when the FGF4 gene is not amplified.
  • [5] The method according to [4], wherein the FGF4 gene amplification level is detected by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR.
  • FISH fluorescence in situ hybridization
  • [6] The method according to [4] or [5], wherein the tumor tissue or tumor cell is collected from the subject's esophagus.
  • a reagent for detecting the FGF4 gene amplification level and the FGF4 gene are hybridized by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR.
  • FISH fluorescence in situ hybridization
  • a method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor comprising administering the pharmaceutical composition according to any one of [1] to [3] to a subject in need.
  • [11] Detecting the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and effectively inhibiting the function or inhibiting the function of the FGF4 protein when the target FGF4 gene is amplified
  • a method for improving, suppressing progression and / or treating FGF4 gene-amplified tumor comprising administering a pharmaceutical composition as a component to the subject.
  • the ID of the primer used was HS01235235 # cn (Forward: 5'-GAG CAG CAA GGG CAA GCT CTA-3 ': SEQ ID No.1, Reverse: 5'-ACC TTC ATG GTG GGC GAC A-3': SEQ ID No.2).
  • TERT locus was used as an internal control (Forward: 5′-GCA CCG TCA AGGCTG AGA AC-3 ′: SEQ ID No. 3, Reverse: 5′-ATG GTG GTG AAG ACG CCA GT-3 ′: SEQ ID No .Four).
  • Human Genomic DNA (Clontech) was used as a normal control. The result is shown in FIG.
  • FIG. 1 is a graph showing an example of the results of measuring the FGF4 gene copy number of 194 esophageal cancer specimens.
  • the horizontal axis is the sample sample and is arranged in order of copy number.
  • the vertical axis represents the measured copy number.
  • the copy number of the FGF4 gene was 5 or more in about 40% of the esophageal cancer specimens.
  • the CGH method was performed using the trade name: Oncoscan, and FISH used a probe obtained from a human genomic clone library (GSP Laboratory, Kawasaki, Japan).
  • the FGF4 gene was introduced into the colon cancer cell line WiDr, the lung cancer cell line A549, and the esophageal cancer cell line KYSE-270 to prepare cell lines that overexpress the FGF4 protein.
  • the prepared FGF4 overexpressing strains are referred to as WiDr / FGF4, A549 / FGF4, and KYSE-270 / FGF4, respectively.
  • cell lines WiDr / EGFP, A549 / EGFP, and KYSE-270 / EGFP in which the EGFP gene was introduced and the EGFP protein was overexpressed were prepared as these controls. (Evaluation of tumorigenicity)
  • the prepared FGF4 overexpression strain was examined for tumorigenicity.
  • FIG. 2 is a graph showing an example of the results of measuring the tumor size of a cell line (WiDr / FGF4) over time when FGF4 gene is introduced into a colon cancer cell line WiDr and overexpressed in culture.
  • FIG. 3 is a graph showing an example of the results of measuring the tumor size of the cell line (A549 / FGF4) over time when the FGF4 gene was introduced into the lung cancer cell line A549 and overexpressed.
  • FIG. 4 shows an example of the results of measuring the tumor size of a cell line (KYSE-270 / FGF4) over time when the FGF4 gene was introduced into the cell line KYSE-270 of esophageal cancer and overexpressed. It is a graph to show. In any case, significant enhancement of tumor-forming ability was observed with respect to the control into which the EGFP gene was introduced. Moreover, these results show that the FGF4 gene can exert a function (oncogenic activity) as an oncogene.
  • a tumor-bearing mouse model in which the FGF4-overexpressing esophageal cancer cell line KYSE-270 / FGF4 was transplanted was prepared. Specifically, a mouse model was prepared by transplanting KYSE-270 / FGF4 cells subcutaneously into nude mice.
  • mice were divided into 2 groups of 4 mice, goat polyclonal IgG was administered to the control group, and anti-FGF4 antibody was administered intraperitoneally to the other group (5 mg / kg, 2 times / week, 2 weeks). . Tumor diameter and body weight were measured every few days.
  • FIG. 5 is a graph showing an example of the result of confirming the effect of the anti-FGF4 antibody on the tumor of KYSE-270 / FGF4 transplanted into a mouse.
  • the vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days).
  • the vertical axis represents the mouse body weight (g), and the horizontal axis represents the culture time (days).
  • T / C 0.7
  • FIG. 6 is a graph showing an example of the results of confirming the effect of an FGFR inhibitor on the FGF4-overexpressing lung cancer cell line A549 / FGF4 transplanted into mice.
  • the vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days).
  • the FGFR inhibitor showed significant antitumor activity.

Abstract

In one or more embodiments, provided is a medicinal composition for FGF4 (fibroblast growth factor 4) gene-amplified tumor. In one embodiment, the present invention relates to a medicinal composition for improving FGF4 gene-amplified tumor, inhibiting the progression of the same, and/or treating the same, said medicinal composition comprising as an active ingredient at least one member selected from the group consisting of anti-FGF4 antibody, FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA.

Description

ＦＧＦ４遺伝子増幅腫瘍の医薬組成物Pharmaceutical composition of FGF4 gene amplified tumor
　本開示は、ＦＧＦ４遺伝子増幅腫瘍の医薬組成物、該医薬組成物の効果予測方法、それに用いるキット、該医薬組成物の使用、並びに、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制及び／又は治療方法に関する。 The present disclosure relates to a pharmaceutical composition of an FGF4 gene-amplified tumor, a method for predicting the effect of the pharmaceutical composition, a kit used therefor, the use of the pharmaceutical composition, and a method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor. .
　特許文献１は、上皮成長因子受容体（ＥＧＦＲ）阻害薬の治療的投与が有効であるか有効でないかの予測が、ＥＧＦＲ遺伝子のコピー数の増加、チロシンキナーゼ受容体型受容体（ＨＥＲ２）のコピー数の増加を検出することで可能になることを開示する。 Patent Document 1 predicts whether therapeutic administration of an epidermal growth factor receptor (EGFR) inhibitor is effective or ineffective is an increase in EGFR gene copy number, tyrosine kinase receptor type receptor (HER2) copy. Disclose what is possible by detecting an increase in number.
　特許文献２は、多標的キナーゼ阻害剤であって癌分子標的治療薬であるソラフェニブの治療効果の予測が、ＦＧＦ３遺伝子のコピー数の増加を検出することで可能になることを開示する。 Patent Document 2 discloses that it is possible to predict the therapeutic effect of sorafenib, which is a multi-target kinase inhibitor and a cancer molecular target therapeutic agent, by detecting an increase in the copy number of the FGF3 gene.
　特許文献３は、α－アクチニン－４遺伝子のコピー数の増加が、癌の存在を示しうることを開示する。 Patent Document 3 discloses that an increase in the copy number of the α-actinin-4 gene may indicate the presence of cancer.
特開２０１３－５８００号公報JP 2013-5800 A 特開２０１２－２４９６３３号公報JP 2012-249633 A 特開２００９－１００７３７号公報Japanese Patent Application Laid-Open No. 2009-10073
　本開示は、一又は複数の実施形態において、ＦＧＦ４遺伝子増幅腫瘍の医薬組成物を提供する。また、本開示は、一又は複数の実施形態において、該医薬組成物の効果予測方法を提供する。 In one or a plurality of embodiments, the present disclosure provides a pharmaceutical composition of an FGF4 gene amplified tumor. Moreover, this indication provides the effect prediction method of this pharmaceutical composition in one or some embodiment.
　本開示は、一又は複数の実施形態において、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療のための医薬組成物であって、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４　ｓｉＲＮＡ、及びＦＧＦ４　ｓｈＲＮＡからなる群から選択される少なくとも１つを有効成分として含有する医薬組成物に関する。 In one or a plurality of embodiments, the present disclosure is a pharmaceutical composition for improving, suppressing progression, and / or treating an FGF4 gene-amplified tumor, comprising an anti-FGF4 antibody, an FGF receptor inhibitor, an FGF4 siRNA, And a pharmaceutical composition containing as an active ingredient at least one selected from the group consisting of FGF4 shRNA.
　本開示は、その他の一又は複数の実施形態において、対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、対象のＦＧＦ４遺伝子が増幅している場合に本開示にかかる医薬組成物の治療的投与の有効性が予測されると前記対象を分類すること、又は、対象のＦＧＦ４遺伝子が増幅していない場合に本開示にかかる医薬組成物の治療的投与の有効性が予測されないと前記対象を分類することを含む方法に関する。 In one or a plurality of other embodiments, the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and the target FGF4 gene is amplified. If the effectiveness of therapeutic administration of the pharmaceutical composition according to the present invention is predicted, the subject is classified, or the therapeutic administration of the pharmaceutical composition according to the present disclosure is effective when the subject FGF4 gene is not amplified. It relates to a method comprising classifying said object if gender is not predicted.
　本開示は、その他の一又は複数の実施形態において、対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、対象のＦＧＦ４遺伝子が増幅している場合にＦＧＦ４タンパク質の発現を抑制又は機能阻害する物質を有効成分とする医薬組成物を前記対象に投与することを含む、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法に関する。 In one or a plurality of other embodiments of the present disclosure, the present disclosure detects the gene amplification level of the FGF4 gene in the tumor tissue sample or tumor cell sample of the subject, and the FGF4 protein when the subject FGF4 gene is amplified. The present invention relates to a method for improving, suppressing the progression of and / or treating FGF4 gene-amplified tumor, comprising administering to the subject a pharmaceutical composition comprising a substance that suppresses the expression of or inhibits the function thereof as an active ingredient.
図１は、１９４例の食道癌標本のＦＧＦ４遺伝子のコピー数を測定した結果の一例を示すグラフである。横軸が標本サンプルでコピー数の順に配置されている。縦軸が測定されたコピー数である。FIG. 1 is a graph showing an example of the results of measuring the FGF4 gene copy number of 194 esophageal cancer specimens. The horizontal axis is the sample sample and is arranged in order of copy number. The vertical axis represents the measured copy number. 図２は、大腸癌の細胞株ＷｉＤｒにＦＧＦ４遺伝子を導入してＦＧＦ４タンパク質を過剰発現させた培養した場合細胞株（ＷｉＤｒ／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。コントロールは、ＥＧＦＰ遺伝子を導入した（ＷｉＤｒ／ＥＧＦＰ）である。FIG. 2 shows an example of the results of measuring the tumor size of a cell line (WiDr / FGF4) over time when FGF4 gene is introduced into colon cancer cell line WiDr and FGF4 protein is overexpressed. It is a graph. As a control, an EGFP gene was introduced (WiDr / EGFP). 図３は、肺癌の細胞株Ａ５４９にＦＧＦ４遺伝子を導入してＦＧＦ４タンパク質を過剰発現させた培養した場合細胞株（Ａ５４９／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。コントロールは、ＥＧＦＰ遺伝子を導入した（Ａ５４９／ＥＧＦＰ）である。FIG. 3 is a graph showing an example of the results of measuring the tumor size of a cell line (A549 / FGF4) over time when the FGF4 gene was introduced into the lung cancer cell line A549 and the FGF4 protein was overexpressed. It is. As a control, an EGFP gene was introduced (A549 / EGFP). 図４は、食道癌の細胞株ＫＹＳＥ－２７０にＦＧＦ４遺伝子を導入してＦＧＦ４タンパク質を過剰発現させた培養した場合細胞株（ＫＹＳＥ－２７０／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。コントロールは、ＥＧＦＰ遺伝子を導入した（ＫＹＳＥ－２７０／ＥＧＦＰ）である。FIG. 4 shows the results of measuring the tumor size of the cell line (KYSE-270 / FGF4) over time when the FGF4 gene was introduced into the esophageal cancer cell line KYSE-270 and the FGF4 protein was overexpressed. It is a graph which shows an example. As a control, an EGFP gene was introduced (KYSE-270 / EGFP). 図５は、マウスに移植したＦＧＦ４過剰発現食道癌細胞株ＫＹＳＥ－２７０／ＦＧＦ４に対する抗ＦＧＦ４抗体の効果を確認した結果の一例を示すグラフである。コントロールとして、抗ＦＧＦ４抗体に換えてヤギのポリクローナルＩｇＧを使用した。図５左図は、縦軸が腫瘍の大きさ（ｍｍ3）であり、横軸が培養時間（日）である。図５右図は、縦軸がマウスの体重（ｇ）であり、横軸が培養時間（日）である。FIG. 5 is a graph showing an example of the results of confirming the effect of anti-FGF4 antibody on FGF4 overexpressing esophageal cancer cell line KYSE-270 / FGF4 transplanted into mice. As a control, goat polyclonal IgG was used instead of anti-FGF4 antibody. In the left diagram of FIG. 5, the vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days). In the right diagram of FIG. 5, the vertical axis represents the mouse body weight (g), and the horizontal axis represents the culture time (days). 図６は、マウスに移植したＦＧＦ４過剰発現肺癌細胞株Ａ５４９／ＦＧＦ４に対するＦＧＦＲ阻害剤の効果を確認した結果の一例を示すグラフである。コントロールは、ＦＧＦＲ阻害剤を投与しない例である。縦軸が腫瘍の大きさ（ｍｍ3）であり、横軸が培養時間（日）である。FIG. 6 is a graph showing an example of the results of confirming the effect of an FGFR inhibitor on the FGF4-overexpressing lung cancer cell line A549 / FGF4 transplanted into mice. The control is an example in which no FGFR inhibitor is administered. The vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days).
　本開示は、腫瘍や癌の中には、ＦＧＦ４遺伝子のコピー数が増幅しているものがあること、そして、そのようなＦＧＦ４遺伝子増幅腫瘍に対して、ＦＧＦ４タンパク質の発現を抑制又は機能阻害する物質を有効成分とする医薬組成物が抗腫瘍効果を有するという知見に基づく。また、本開示は、一又は複数の実施形態において、食道癌の約４０％においてＦＧＦ４遺伝子のコピー数の増幅が起っているという知見に基づく。 The present disclosure indicates that some tumors and cancers have an amplified copy number of the FGF4 gene, and suppress or inhibit the function of the FGF4 protein in such an FGF4 gene-amplified tumor. Based on the finding that a pharmaceutical composition containing a substance as an active ingredient has an antitumor effect. The present disclosure is also based on the finding that, in one or more embodiments, amplification of the FGF4 gene copy number occurs in about 40% of esophageal cancers.
　したがって、本開示は一態様において、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療のための医薬組成物であって、ＦＧＦ４タンパク質の発現を抑制又は機能阻害する物質を有効成分として含有する医薬組成物に関する。 Therefore, in one aspect, the present disclosure is a pharmaceutical composition for improving, suppressing progression and / or treating FGF4 gene-amplified tumor, and contains a substance that suppresses the expression of FGF4 protein or inhibits its function as an active ingredient. The present invention relates to a pharmaceutical composition.
　［対象の腫瘍］
　本開示にかかる医薬組成物の対象となる腫瘍は、一又は複数の実施形態において、ＦＧＦ４（Fibroblast growth factor 4）遺伝子のコピー数が増幅した腫瘍（以下、「ＦＧＦ４遺伝子増幅腫瘍」ともいう。）である。ＦＧＦ４遺伝子増幅腫瘍は、各種癌に存在しうる。例えば、食道癌の約４０％がＦＧＦ４遺伝子増幅腫瘍である。また、例えば、膀胱癌の約１０％、頭頸部扁平上皮癌の約３５％の腫瘍サンプルでＦＧＦ４遺伝子の増幅が見られるという報告がある。本開示にかかる医薬組成物は、一又は複数の実施形態において、ＦＧＦ４遺伝子増幅腫瘍に対して抗腫瘍効果を発揮しうる。本開示にかかる医薬組成物は、一又は複数の実施形態において、ＦＧＦ４遺伝子増幅腫瘍を有する個体の改善、進行抑制、及び／又は、治療に使用できる。本開示の一又は複数の実施形態において、ＦＧＦ４遺伝子増幅腫瘍は、食道癌である。 [Target tumor]
In one or a plurality of embodiments, the tumor that is the target of the pharmaceutical composition according to the present disclosure is a tumor in which the copy number of FGF4 (Fibroblast growth factor 4) gene is amplified (hereinafter also referred to as “FGF4 gene amplified tumor”). It is. FGF4 gene amplified tumors can be present in various cancers. For example, about 40% of esophageal cancers are FGF4 gene amplified tumors. In addition, for example, there is a report that FGF4 gene amplification is observed in tumor samples of about 10% of bladder cancer and about 35% of head and neck squamous cell carcinoma. In one or a plurality of embodiments, the pharmaceutical composition according to the present disclosure can exert an antitumor effect on an FGF4 gene-amplified tumor. In one or a plurality of embodiments, the pharmaceutical composition according to the present disclosure can be used for improvement, suppression of progression, and / or treatment of an individual having an FGF4 gene-amplified tumor. In one or more embodiments of the present disclosure, the FGF4 gene amplified tumor is esophageal cancer.
　［コピー数の検出］
　したがって、本開示にかかる医薬組成物の有効性は、対象の腫瘍のＦＧＦ４遺伝子のコピー数の増幅の有無を調べることで判定することができる。よって、本開示はその他の態様において、対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、対象のＦＧＦ４遺伝子が増幅している場合に本開示にかかる医薬組成物の治療的投与の有効性が予測されると前記対象を分類すること、又は、対象のＦＧＦ４遺伝子が増幅していない場合に本開示にかかる医薬組成物の治療的投与の有効性が予測されないと前記対象を分類することを含む方法（以下、「本開示にかかる分類方法」ともいう。）に関する。本開示にかかる分類方法の対象としては、一又は複数の実施形態において、ヒト、又は、ヒト以外の哺乳類などが挙げられる。 [Detect number of copies]
Therefore, the effectiveness of the pharmaceutical composition according to the present disclosure can be determined by examining the presence or absence of amplification of the copy number of the FGF4 gene in the subject tumor. Therefore, in another aspect, the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and the pharmaceutical composition according to the present disclosure when the target FGF4 gene is amplified Classifying the subject as the efficacy of therapeutic administration of the product is predicted, or the efficacy of therapeutic administration of the pharmaceutical composition according to the present disclosure is not expected when the subject's FGF4 gene is not amplified And a method including classifying the object (hereinafter, also referred to as “classification method according to the present disclosure”). Examples of the classification method according to the present disclosure include humans and mammals other than humans in one or a plurality of embodiments.
　ＦＧＦ４遺伝子のコピー数の増幅の有無は、一又は複数の実施形態において、従来行われる方法、又は、今後開発される方法が使用できる。ＦＧＦ４遺伝子のコピー数の増幅の有無は、一又は複数の実施形態において、蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、又は、リアルタイム定量ＰＣＲによって行うことができる。本開示の一又は複数の実施形態において、ＦＧＦ４遺伝子のコピー数が増加しているとは、対象腫瘍のＦＧＦ４遺伝子のコピー数が、正常細胞のコントロール遺伝子のコピー数よりも多いことをいう。 For the presence or absence of amplification of the copy number of the FGF4 gene, a conventionally performed method or a method developed in the future can be used in one or a plurality of embodiments. In one or a plurality of embodiments, the presence or absence of amplification of the copy number of the FGF4 gene can be performed by fluorescent fluorescence in situ hybridization (FISH), a next-generation sequencer, a DNA chip, or real-time quantitative PCR. In one or a plurality of embodiments of the present disclosure, the fact that the copy number of the FGF4 gene is increased means that the copy number of the FGF4 gene of the target tumor is larger than the copy number of the control gene of normal cells.
　蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）による遺伝子コピー数の増幅は、一又は複数の実施形態において、以下のようにして検出／測定することができる。
　ＦＩＳＨ法は、蛍光物質を利用して特定遺伝子の断片ＤＮＡ（プローブ）を染色体や核ＤＮＡとハイブリダイズさせ、その遺伝子の増減（欠失や増幅）や染色対中の位置（マッピング）を調べる方法である。したがって、目的とする遺伝子の配列に特異的なプローブで細胞を染色し、シグナルの多寡に応じて、コピー数の増減を測定できる。また、“https://www.yodosha.co.jp/jikkenigaku/keyword/234.html”及び“Hepatology. 2013 Apr;57(4):1407-15. FGF3/FGF4 amplification and multiple lung metastasesin responders to sorafenib in hepatocellular carcinoma.”を参照できる。 Amplification of gene copy number by fluorescence in situ hybridization (FISH) can be detected / measured in one or more embodiments as follows.
In the FISH method, a fragment DNA (probe) of a specific gene is hybridized with a chromosome or nuclear DNA using a fluorescent substance, and the increase / decrease (deletion or amplification) of the gene and the position (mapping) in a staining pair are examined. It is. Therefore, cells can be stained with a probe specific to the sequence of the target gene, and the increase or decrease in copy number can be measured according to the number of signals. Also, “https://www.yodosha.co.jp/jikkenigaku/keyword/234.html” and “Hepatology. 2013 Apr; 57 (4): 1407-15. FGF3 / FGF4 amplification and multiple lung metastasesin responders to sorafenib in hepatocellular carcinoma. ”
　次世代シーケンサーによる遺伝子コピー数の増幅は、一又は複数の実施形態において、以下のようにして検出／測定することができる。
　次世代シーケンサーは従来のサンガー法と違い、数千万から数億のＤＮＡ断片に対して大量並列に処理できる。したがって、腫瘍サンプルとコントロールサンプルからのＤＮＡをシーケンスし、目的遺伝子のカバー率を比較することで、目的遺伝子のコピー数の増減を推定できる。また、“PLoS One. 2013; 8(5): e64271. Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation Sequencing”及び“Clin Cancer Res. 2013 May 15;19(10):2668-76. Relapsed classic E-cadherin (CDH1)-mutated invasive lobular breast cancer shows a high frequency of HER2 (ERBB2) gene mutations.”を参照できる。 In one or a plurality of embodiments, amplification of gene copy number by a next-generation sequencer can be detected / measured as follows.
Unlike conventional Sanger methods, next-generation sequencers can process tens of millions to hundreds of millions of DNA fragments in parallel. Therefore, by increasing and decreasing the copy number of the target gene by sequencing the DNA from the tumor sample and the control sample and comparing the coverage of the target gene. Also, “PLoS One. 2013; 8 (5): e64271. Targeted Sequencing of Cancer-Related Genes in Colorectal Cancer Using Next-Generation Sequencing” and “Clin Cancer Res. 2013 May 15; 19 (10): 2668-76. Relapsed classic E-cadherin (CDH1) -mutated invasive lobular breast cancer shows a high frequency of HER2 (ERBB2) gene mutations. ”
　ＤＮＡチップによる遺伝子コピー数の増幅は、一又は複数の実施形態において、以下のようにして検出／測定することができる。
　すなわち、多数のＤＮＡ断片をアレイ化したスライドグラス（ＤＮＡチップ）を用いてＣＧＨ（comparative genomic hybridization）解析法を行う。それぞれ異なる蛍光色素でラベルした腫瘍サンプルならびにコントロールサンプルを、ＤＮＡチップ上のＤＮＡ断片とハイブリダイズさせる。それぞれのサンプル由来の蛍光シグナルの強度比をもとにアレイ化されたＤＮＡ断片に相当する領域の腫瘍ＤＮＡのコピー数を定量化する。また、“http://www.cghtmd.jp/CGHDatabase/about/about#high.jsp”及び“Hepatology. 2013 Apr;57(4):1407-15. FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma.”を参照できる。 In one or a plurality of embodiments, amplification of gene copy number by a DNA chip can be detected / measured as follows.
That is, CGH (comparative genomic hybridization) analysis is performed using a slide glass (DNA chip) in which a large number of DNA fragments are arrayed. Tumor samples and control samples labeled with different fluorescent dyes are hybridized with DNA fragments on the DNA chip. Based on the intensity ratio of fluorescent signals derived from each sample, the copy number of tumor DNA in the region corresponding to the arrayed DNA fragments is quantified. Also, “http://www.cghtmd.jp/CGHDatabase/about/about#high.jsp” and “Hepatology. 2013 Apr; 57 (4): 1407-15. FGF3 / FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma. ”
　リアルタイム定量ＰＣＲによる遺伝子コピー数の増幅は以下のようにして検出／測定することができる。
　すなわち、サンプルのＤＮＡをテンプレートとし、標的遺伝子に特異的プライマー用いてＰＣＲ反応により標的となる遺伝子の部位を増幅する。増幅反応をリアルタイムに検出し、一定量までの増幅にかかるPCRのサイクル数をコントロールサンプルと腫瘍サンプルで比較して、その差に応じて、コピー数を算出する。また、“http://tools.invitrogen.com/content/sfs/manuals/cms#062368.pdf”及び“Hepatology. 2013 Apr;57(4):1407-15. FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma.”を参照できる。 Amplification of gene copy number by real-time quantitative PCR can be detected / measured as follows.
That is, using a sample DNA as a template, a target gene site is amplified by a PCR reaction using primers specific to the target gene. The amplification reaction is detected in real time, the number of PCR cycles for amplification up to a certain amount is compared between the control sample and the tumor sample, and the copy number is calculated according to the difference. Also, “http://tools.invitrogen.com/content/sfs/manuals/cms#062368.pdf” and “Hepatology. 2013 Apr; 57 (4): 1407-15. FGF3 / FGF4 amplification and multiple lung metastases in Refer to "responders to sorafenib in hepatocellular carcinoma."
　［有効成分］
　本開示にかかる医薬組成物の有効成分は、ＦＧＦ４タンパク質の発現を抑制する物質又は機能阻害する物質である。本開示にかかる医薬組成物の有効成分は、一又は複数の実施形態において、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４ｓｉＲＮＡ、及びＦＧＦ４ｓｈＲＮＡからなる群から選択される少なくとも１つが挙げられる。 [Active ingredients]
The active ingredient of the pharmaceutical composition according to the present disclosure is a substance that suppresses the expression of FGF4 protein or a substance that inhibits the function. In one or a plurality of embodiments, the active ingredient of the pharmaceutical composition according to the present disclosure includes at least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA.
　抗ＦＧＦ４抗体は、医薬組成物の抗腫瘍効果を向上させる点から、一又は複数の実施形態において、中和活性を有し、又は、高い中和活性を有する。抗ＦＧＦ４抗体は、一又は複数の実施形態において、モノクローナル抗体である。また、抗ＦＧＦ４抗体は、一又は複数の実施形態において、抗体断片の形態であってもよいし、糖鎖で修飾されていてもよい。 In view of improving the antitumor effect of the pharmaceutical composition, the anti-FGF4 antibody has neutralizing activity or high neutralizing activity in one or a plurality of embodiments. The anti-FGF4 antibody is a monoclonal antibody in one or more embodiments. Further, in one or a plurality of embodiments, the anti-FGF4 antibody may be in the form of an antibody fragment or may be modified with a sugar chain.
　ＦＧＦ受容体は、ＦＧＦ４タンパク質の受容体である。よって、ＦＧＦ受容体阻害剤は本開示にかかる医薬組成物の有効成分として使用されうる。ＦＧＦ受容体阻害剤としては、従来公知及び今後開発されるものが使用できる。 FGF receptor is a receptor for FGF4 protein. Therefore, the FGF receptor inhibitor can be used as an active ingredient of the pharmaceutical composition according to the present disclosure. As the FGF receptor inhibitor, conventionally known and future developed ones can be used.
　ＦＧＦ４遺伝子のｓｉＲＮＡ（small interfering RNA）やｓｈＲＮＡ（small hairpin）も本開示にかかる医薬組成物の有効成分として使用できる。当業者であれば、定法に従って適切なＦＧＦ４遺伝子のｓｉＲＮＡ及びｓｈＲＮＡを設計及び製造できる。 FGF4 gene siRNA (small interfering RNA) and shRNA (small hairpin) can also be used as an active ingredient of the pharmaceutical composition according to the present disclosure. A person skilled in the art can design and manufacture appropriate FGF4 gene siRNA and shRNA according to standard methods.
　本開示において「医薬組成物」は、一又は複数の実施形態において、周知の製剤技術を適用し、投与形態に適した剤形とすることができる。その投与形態としては、これらに限定されないが、例えば、錠剤、カプセル剤、顆粒剤、散剤、丸剤、トローチ剤、シロップ剤、液剤等の剤形による経口投与が挙げられる。或いは、注射剤、液剤、エアゾール剤、座剤、貼布剤、パップ剤、ローション剤、リニメント剤、軟膏剤、点眼剤等の剤形による非経口投与を挙げることができる。これらの製剤は、これらに限定されないが、賦形剤、滑沢剤、結合剤、崩壊剤、安定化剤、矯味矯臭剤、希釈剤などの添加剤を用いて周知の方法で製造されうる。 In the present disclosure, the “pharmaceutical composition” may be a dosage form suitable for an administration form by applying a well-known formulation technique in one or a plurality of embodiments. Examples of the dosage form include, but are not limited to, oral administration in a dosage form such as a tablet, capsule, granule, powder, pill, troche, syrup, and liquid. Alternatively, parenteral administration in dosage forms such as injections, liquids, aerosols, suppositories, patches, lotions, liniments, ointments, eye drops and the like can be mentioned. These preparations can be produced by known methods using additives such as, but not limited to, excipients, lubricants, binders, disintegrants, stabilizers, flavoring agents, and diluents.
　本開示にかかる医薬組成物は、一又は複数の実施形態において、治療効果を有する他の有効成分を含まず、或いは、さらに１又は複数の有効成分を含有する。 In one or a plurality of embodiments, the pharmaceutical composition according to the present disclosure does not contain other active ingredients having a therapeutic effect, or further contains one or a plurality of active ingredients.
　前記賦形剤としては、これらに限定されないがデンプン、バレイショデンプン、トウモロコシデンプン等のデンプン、乳糖、結晶セルロース、リン酸水素カルシウム等を挙げることができる。前記コーティング剤としては、これらに限定されないが、エチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、セラック、タルク、カルナウバロウ、パラフィン等を挙げることができる。前記結合剤としては、これらに限定されないが、ポリビニルピロリドン、マクロゴール及び前記賦形剤と同様の化合物を挙げることができる。前記崩壊剤としては、これらに限定されないが、前記賦形剤と同様の化合物及びクロスカルメロースナトリウム、カルボキシメチルスターチナトリウム、架橋ポリビニルピロリドンのような化学修飾されたデンプン・セルロース類を挙げることができる。前記安定化剤としては、これらに限定されないが、メチルパラベン、プロピルパラベンのようなパラオキシ安息香酸エステル類；クロロブタノール、ベンジルアルコール、フェニルエチルアルコールのようなアルコール類；塩化ベンザルコニウム；フェノール、クレゾールのようなフェエノール類；チメロサール；デヒドロ酢酸；及びソルビン酸を挙げることができる。前記矯味矯臭剤としては、これらに限定されないが、通常使用される、甘味料、酸味料、香料等を挙げることができる。 Examples of the excipient include, but are not limited to, starch such as starch, potato starch, and corn starch, lactose, crystalline cellulose, calcium hydrogen phosphate, and the like. Examples of the coating agent include, but are not limited to, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, shellac, talc, carnauba wax, paraffin, and the like. Examples of the binder include, but are not limited to, polyvinyl pyrrolidone, macrogol and the same compound as the excipient. Examples of the disintegrant include, but are not limited to, compounds similar to the excipients and chemically modified starch and celluloses such as croscarmellose sodium, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone. . Examples of the stabilizer include, but are not limited to, paraoxybenzoates such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenol, cresol Mention may be made of such phenols; thimerosal; dehydroacetic acid; and sorbic acid. Examples of the flavoring agent include, but are not limited to, sweeteners, acidulants, and fragrances that are commonly used.
　また、液剤の製造には、溶媒として、これらに限定されないが、エタノール、フェノール、クロロクレゾール、精製水、蒸留水等を使用することができ、必要に応じて界面活性剤又は乳化剤等も使用できる。前記界面活性剤又は乳化剤としては、これらに限定されないが、ポリソルベート８０、ステアリン酸ポリオキシル４０、ラウロマクロゴール等を挙げることができる。 In the production of the liquid agent, the solvent is not limited to these, but ethanol, phenol, chlorocresol, purified water, distilled water and the like can be used, and a surfactant or an emulsifier can also be used as necessary. . Examples of the surfactant or emulsifier include, but are not limited to, polysorbate 80, polyoxyl 40 stearate, lauromacrogol, and the like.
　本開示にかかる医薬組成物の使用方法は、症状、年齢、投与方法等により異なりうる。使用方法は、これらに限定されないが、有効成分の体内濃度が１００ｎＭ～１ｍＭの間のいずれかになるように、間欠的若しくは持続的に、経口、経皮、粘膜下、皮下、筋肉内、血管内、脳内、又は腹腔内に投与することができる。限定されない実施形態として、経口投与の場合、対象（ヒトであれば成人）に対して１日あたり、有効成分に換算して、下限として０．０１ｍｇ（好ましくは０．１ｍｇ）、上限として、２０００ｍｇ（好ましくは５００ｍｇ、より好ましくは１００ｍｇ）を１回又は数回に分けて、症状に応じて投与することが挙げられる。限定されない実施形態として、静脈内投与の場合には、対象（ヒトであれば成人）に対して１日当たり、下限として０．００１ｍｇ（好ましくは０．０１ｍｇ）、上限として、５００ｍｇ（好ましくは５０ｍｇ）を１回又は数回に分けて、症状に応じて投与することが挙げられる。 The method of using the pharmaceutical composition according to the present disclosure may vary depending on symptoms, age, administration method, and the like. The method of use is not limited to these, but intermittently or continuously, orally, transdermally, submucosally, subcutaneously, intramuscularly, vascularly, so that the concentration of the active ingredient in the body is between 100 nM and 1 mM. It can be administered internally, intracerebrally, or intraperitoneally. As a non-limiting embodiment, in the case of oral administration, the lower limit is 0.01 mg (preferably 0.1 mg) and the upper limit is 2000 mg as converted into the active ingredient per day for a subject (adult if human). (Preferably 500 mg, more preferably 100 mg) may be divided into 1 or several times and administered according to symptoms. As a non-limiting embodiment, in the case of intravenous administration, the lower limit is 0.001 mg (preferably 0.01 mg) and the upper limit is 500 mg (preferably 50 mg) per day for a subject (adult if human). Is divided into one or several times and administered according to symptoms.
　［方法及び使用］
　本開示は、一態様において、本開示にかかる医薬組成物を製造するための、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４ｓｉＲＮＡ、及びＦＧＦ４ｓｈＲＮＡからなる群から選択される少なくとも１つの使用に関する。また、本開示は、一態様において、本開示にかかる医薬組成物を必要とされる対象に投与することを含む、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法に関する。また、本開示は、一態様において、対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、対象のＦＧＦ４遺伝子が増幅している場合にＦＧＦ４タンパク質の発現を抑制又は機能阻害する物質を有効成分とする医薬組成物を前記対象に投与することを含む、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法に関する。さらに、本開示は、上述したＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法における、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４ｓｉＲＮＡ、及びＦＧＦ４ｓｈＲＮＡからなる群から選択される少なくとも１つの使用、若しくは、本開示にかかる医薬組成物の使用に関する。 [Method and use]
In one aspect, the present disclosure relates to at least one use selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA for producing a pharmaceutical composition according to the present disclosure. In addition, in one aspect, the present disclosure relates to a method for improving, suppressing progression, and / or treating an FGF4 gene-amplified tumor, comprising administering a pharmaceutical composition according to the present disclosure to a subject in need. Further, in one aspect, the present disclosure detects the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and suppresses the expression of the FGF4 protein when the target FGF4 gene is amplified. Alternatively, the present invention relates to a method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor, comprising administering to the subject a pharmaceutical composition containing a substance that inhibits function as an active ingredient. Furthermore, the present disclosure provides at least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA in the above-described improvement, suppression of progression, and / or treatment of an FGF4 gene-amplified tumor. Use, or use of a pharmaceutical composition according to the present disclosure.
　［キット］
　本開示は、その他の態様において、蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によりＦＧＦ４遺伝子増幅レベルの検出を行うための試薬、及び、蛍光in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によりＦＧＦ４遺伝子増幅レベルの検出を行うための試薬及びＦＧＦ４遺伝子にハイブリダイズするヌクレオチドを備えるキットに関する。 [kit]
In another aspect, the present disclosure provides a reagent for detecting the FGF4 gene amplification level by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR, And a reagent for detecting the FGF4 gene amplification level and a nucleotide that hybridizes to the FGF4 gene by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR Relates to a kit comprising:
　本開示は、さらに以下の一又は複数の実施形態に関する。
　［１］　ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療のための医薬組成物であって、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４　ｓｉＲＮＡ、及びＦＧＦ４　ｓｈＲＮＡからなる群から選択される少なくとも１つを有効成分として含有する医薬組成物。
　［２］　ＦＧＦ４遺伝子増幅腫瘍は、食道癌である［１］に記載の医薬組成物。
　［３］　抗ＦＧＦ４抗体が、ＦＧＦ４に特異性を有する中和抗体である［１］又は［２］に記載の医薬組成物。
　［４］　対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、
　対象のＦＧＦ４遺伝子が増幅している場合に［１］から［３］のいずれかに記載の医薬組成物の治療的投与の有効性が予測されると前記対象を分類すること、又は、対象のＦＧＦ４遺伝子が増幅していない場合に［１］から［３］のいずれかに記載の医薬組成物の治療的投与の有効性が予測されないと前記対象を分類することを含む、方法。
　［５］　ＦＧＦ４遺伝子増幅レベルの検出が、蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によって行われる［４］記載の方法。
　［６］　前記腫瘍組織又は腫瘍細胞が、対象の食道から採取されたものである［４］又は［５］に記載の方法。
　［７］　蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によりＦＧＦ４遺伝子増幅レベルの検出を行うための試薬及びＦＧＦ４遺伝子にハイブリダイズするヌクレオチドを備える［４］から［６］のいずれかに記載の方法に使用するためのキット。
　［８］　［１］から［３］のいずれかに記載の医薬組成物を製造するための、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４　ｓｉＲＮＡ、及びＦＧＦ４　ｓｈＲＮＡからなる群から選択される少なくとも１つの使用。
　［９］　［１］から［３］のいずれかに記載の医薬組成物を必要とされる対象に投与することを含む、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法。
　［１０］　［９］のＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法における、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４　ｓｉＲＮＡ、及びＦＧＦ４　ｓｈＲＮＡからなる群から選択される少なくとも１つの使用。
　［１１］　対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、対象のＦＧＦ４遺伝子が増幅している場合にＦＧＦ４タンパク質の発現を抑制又は機能阻害する物質を有効成分とする医薬組成物を前記対象に投与することを含む、ＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法。
　［１２］　［１１］　記載のＦＧＦ４遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療方法における［１］から［３］のいずれかに記載の医薬組成物の使用。 The present disclosure further relates to one or more of the following embodiments.
[1] A pharmaceutical composition for improvement, suppression of progression and / or treatment of FGF4 gene-amplified tumor, which is selected from the group consisting of anti-FGF4 antibody, FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA. A pharmaceutical composition containing at least one of these as an active ingredient.
[2] The pharmaceutical composition according to [1], wherein the FGF4 gene-amplified tumor is esophageal cancer.
[3] The pharmaceutical composition according to [1] or [2], wherein the anti-FGF4 antibody is a neutralizing antibody having specificity for FGF4.
[4] detecting the gene amplification level of the FGF4 gene in the tumor tissue sample or tumor cell sample of the subject; and
Classifying the subject when the efficacy of therapeutic administration of the pharmaceutical composition according to any one of [1] to [3] is predicted when the subject's FGF4 gene is amplified, or Categorizing the subject as not predicting the effectiveness of therapeutic administration of the pharmaceutical composition of any of [1] to [3] when the FGF4 gene is not amplified.
[5] The method according to [4], wherein the FGF4 gene amplification level is detected by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR.
[6] The method according to [4] or [5], wherein the tumor tissue or tumor cell is collected from the subject's esophagus.
[7] A reagent for detecting the FGF4 gene amplification level and the FGF4 gene are hybridized by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR. A kit for use in the method according to any one of [4] to [6], comprising nucleotides.
[8] At least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA for producing the pharmaceutical composition according to any one of [1] to [3] One use.
[9] A method for improving, suppressing progression and / or treating an FGF4 gene-amplified tumor, comprising administering the pharmaceutical composition according to any one of [1] to [3] to a subject in need.
[10] At least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA in the improvement, suppression of progression, and / or treatment of the FGF4 gene-amplified tumor of [9] One use.
[11] Detecting the gene amplification level of the FGF4 gene in the target tumor tissue sample or tumor cell sample, and effectively inhibiting the function or inhibiting the function of the FGF4 protein when the target FGF4 gene is amplified A method for improving, suppressing progression and / or treating FGF4 gene-amplified tumor, comprising administering a pharmaceutical composition as a component to the subject.
[12] Use of the pharmaceutical composition according to any one of [1] to [3] in the improvement, suppression of progression, and / or treatment of the FGF4 gene-amplified tumor according to [11].
　以下、実施例及び比較例に基づいて説明するが、本開示はこれに限定されるものではない。 Hereinafter, although described based on Examples and Comparative Examples, the present disclosure is not limited thereto.
　［食道癌手術標本のＦＧＦ４遺伝子コピー数解析］
　１９４例の食道癌手術標本においてＦＧＦ４遺伝子のコピー数解析を行った。
　（標本）
　被験試料として、手術時切除凍結標本又はＦＦＰＥ（ホルマリン固定パラフィン包埋）標本である食道癌（ＥＳＣＣ：食道扁平上皮癌）の標本を使用した。
　（コピー数アッセイ）
　食道癌標本からＤＮＡを抽出し、TaqMan Copy Number Assays (Applied Biosystems, Foster City, CA)を用いてコピー数アッセイを行った。用いたプライマーのＩＤは、HS01235235#cn（Forward: 5'- GAG CAG CAA GGG CAA GCT CTA -3'：SEQ ID No.1、Reverse：5'- ACC TTC ATG GTG GGC GAC A -3'：SEQ ID No.2）である。また、ＴＥＲＴローカスを内部コントロールとした（Forward: 5'- GCA CCG TCA AGGCTG AGA AC -3'：SEQ ID No.3、Reverse：5'- ATG GTG GTG AAG ACG CCA GT -3'：SEQ ID No.4）。また、Human Genomic DNA (Clontech) を正常コントロールとして使用した。その結果を図１に示す。 [Analysis of FGF4 gene copy number in surgical specimen of esophageal cancer]
Copy number analysis of the FGF4 gene was performed in 194 esophageal cancer surgical specimens.
(Sample)
A specimen of esophageal cancer (ESCC: esophageal squamous cell carcinoma) which was a frozen specimen excised at surgery or a FFPE (formalin fixed paraffin embedded) specimen was used as a test sample.
(Copy number assay)
DNA was extracted from esophageal cancer specimens and copy number assays were performed using TaqMan Copy Number Assays (Applied Biosystems, Foster City, Calif.). The ID of the primer used was HS01235235 # cn (Forward: 5'-GAG CAG CAA GGG CAA GCT CTA-3 ': SEQ ID No.1, Reverse: 5'-ACC TTC ATG GTG GGC GAC A-3': SEQ ID No.2). In addition, TERT locus was used as an internal control (Forward: 5′-GCA CCG TCA AGGCTG AGA AC-3 ′: SEQ ID No. 3, Reverse: 5′-ATG GTG GTG AAG ACG CCA GT-3 ′: SEQ ID No .Four). In addition, Human Genomic DNA (Clontech) was used as a normal control. The result is shown in FIG.
　図１は、１９４例の食道癌標本のＦＧＦ４遺伝子のコピー数を測定した結果の一例を示すグラフである。横軸が標本サンプルでコピー数の順に配置されている。縦軸が測定されたコピー数である。同図に示すとおり、食道癌標本の約４０％においてＦＧＦ４遺伝子のコピー数が５以上を示した。なお、同じ食道癌標本をＣＧＨ（comparative genomic hybridization）解析法及びＦＩＳＨで解析した場合も、ＦＧＦ４遺伝子の増幅が同様の傾向で認められた（データ示さず）。なお、ＣＧＨ法は、商品名：Oncoscanを用いて行い、ＦＩＳＨは、ヒトゲノムクローンライブラリ（GSP Laboratory, Kawasaki, Japan）から得たプローブを使用した。 FIG. 1 is a graph showing an example of the results of measuring the FGF4 gene copy number of 194 esophageal cancer specimens. The horizontal axis is the sample sample and is arranged in order of copy number. The vertical axis represents the measured copy number. As shown in the figure, the copy number of the FGF4 gene was 5 or more in about 40% of the esophageal cancer specimens. In addition, when the same esophageal cancer specimen was analyzed by the CGH (comparative genomic hybridization) method and FISH, amplification of the FGF4 gene was observed in the same tendency (data not shown). The CGH method was performed using the trade name: Oncoscan, and FISH used a probe obtained from a human genomic clone library (GSP Laboratory, Kawasaki, Japan).
　［ＦＧＦ４過剰発現株の作製］
　大腸癌の細胞株ＷｉＤｒ、肺癌の細胞株Ａ５４９、及び、食道癌の細胞株ＫＹＳＥ－２７０に、それぞれ、ＦＧＦ４遺伝子を導入してＦＧＦ４タンパク質を過剰発現する細胞株を作製した。作製されたＦＧＦ４過剰発現株を、それぞれ、ＷｉＤｒ／ＦＧＦ４、Ａ５４９／ＦＧＦ４、及び、ＫＹＳＥ－２７０／ＦＧＦ４とする。
　一方、これらのコントロールとして、ＥＧＦＰ遺伝子が導入されＥＧＦＰタンパク質が過剰発現される細胞株ＷｉＤｒ／ＥＧＦＰ、Ａ５４９／ＥＧＦＰ、及び、ＫＹＳＥ－２７０／ＥＧＦＰを作製した。
　（腫瘍形成能の評価）
　作製したＦＧＦ４過剰発現株の腫瘍形成能を調べた。具体的には、約５×１０6個の細胞をマウスの皮下に移植し、培養後６，１０，１１，１３，１５，１８，２１日目、又は、４，７，１１，１４，１８，２１日目で腫瘍の大きさを測定した。腫瘍の大きさはノギスで測定した。その結果を図２～４に示す。 [Preparation of FGF4 overexpression strain]
The FGF4 gene was introduced into the colon cancer cell line WiDr, the lung cancer cell line A549, and the esophageal cancer cell line KYSE-270 to prepare cell lines that overexpress the FGF4 protein. The prepared FGF4 overexpressing strains are referred to as WiDr / FGF4, A549 / FGF4, and KYSE-270 / FGF4, respectively.
On the other hand, cell lines WiDr / EGFP, A549 / EGFP, and KYSE-270 / EGFP in which the EGFP gene was introduced and the EGFP protein was overexpressed were prepared as these controls.
(Evaluation of tumorigenicity)
The prepared FGF4 overexpression strain was examined for tumorigenicity. Specifically, about 5 × 10 6 cells are transplanted subcutaneously into mice, and 6, 10, 11, 13, 15, 18, 21 days after culture, or 4, 7, 11, 14, 18 On the 21st day, tumor size was measured. Tumor size was measured with calipers. The results are shown in FIGS.
　図２は、大腸癌の細胞株ＷｉＤｒにＦＧＦ４遺伝子を導入して過剰発現させた培養した場合細胞株（ＷｉＤｒ／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。図３は、肺癌の細胞株Ａ５４９にＦＧＦ４遺伝子を導入して過剰発現させた培養した場合細胞株（Ａ５４９／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。図４は、食道癌の細胞株ＫＹＳＥ－２７０にＦＧＦ４遺伝子を導入して過剰発現させた培養した場合細胞株（ＫＹＳＥ－２７０／ＦＧＦ４）の腫瘍の大きさを経時的に測定した結果の一例を示すグラフである。いずれの場合も、ＥＧＦＰ遺伝子を導入したコントロールに対して有意な腫瘍形成能の亢進が認められた。また、これらの結果は、ＦＧＦ４遺伝子が発がん遺伝子として機能（oncogenic activity）を発揮しうることを示す。 FIG. 2 is a graph showing an example of the results of measuring the tumor size of a cell line (WiDr / FGF4) over time when FGF4 gene is introduced into a colon cancer cell line WiDr and overexpressed in culture. . FIG. 3 is a graph showing an example of the results of measuring the tumor size of the cell line (A549 / FGF4) over time when the FGF4 gene was introduced into the lung cancer cell line A549 and overexpressed. FIG. 4 shows an example of the results of measuring the tumor size of a cell line (KYSE-270 / FGF4) over time when the FGF4 gene was introduced into the cell line KYSE-270 of esophageal cancer and overexpressed. It is a graph to show. In any case, significant enhancement of tumor-forming ability was observed with respect to the control into which the EGFP gene was introduced. Moreover, these results show that the FGF4 gene can exert a function (oncogenic activity) as an oncogene.
　［抗ＦＧＦ４抗体のin vivoにおけるＦＧＦ４過剰発現腫瘍に対する効果］
　ＦＧＦ４過剰発現腫瘍に対する抗ＦＧＦ４抗体の効果をin vivoで確認した。
　抗ＦＧＦ４抗体として、市販品（商品名：AB-235-NA）を使用した。なお、この抗体のＦＧＦ４中和能は、ＩＣ50＝１０～２０μｇ／ｍｌであった（データ示さず）。
　ＦＧＦ４過剰発現食道癌細胞株ＫＹＳＥ－２７０／ＦＧＦ４を移植した担がんマウスモデルを作製した。具体的には、ＫＹＳＥ－２７０／ＦＧＦ４細胞をヌードマウスの皮下に移植してマウスモデルを作製した。マウスを４匹ずつ２群に分け、コントロール群にはヤギのポリクローナルＩｇＧを、もう一方の群には抗ＦＧＦ４抗体をマウスの腹腔内に投与した（５mg/kg、２回／週、２週間）。数日おきに腫瘍径と体重を測定した。 [Effect of anti-FGF4 antibody on FGF4-overexpressing tumor in vivo]
The effect of anti-FGF4 antibody on FGF4 overexpressing tumors was confirmed in vivo.
As the anti-FGF4 antibody, a commercially available product (trade name: AB-235-NA) was used. The FGF4 neutralizing ability of this antibody was IC 50 = 10 to 20 μg / ml (data not shown).
A tumor-bearing mouse model in which the FGF4-overexpressing esophageal cancer cell line KYSE-270 / FGF4 was transplanted was prepared. Specifically, a mouse model was prepared by transplanting KYSE-270 / FGF4 cells subcutaneously into nude mice. Mice were divided into 2 groups of 4 mice, goat polyclonal IgG was administered to the control group, and anti-FGF4 antibody was administered intraperitoneally to the other group (5 mg / kg, 2 times / week, 2 weeks). . Tumor diameter and body weight were measured every few days.
　図５は、マウスに移植したＫＹＳＥ－２７０／ＦＧＦ４の腫瘍に対する抗ＦＧＦ４抗体の効果を確認した結果の一例を示すグラフである。図５左図は、縦軸が腫瘍の大きさ（ｍｍ3）であり、横軸が培養時間（日）である。図５右図は、縦軸がマウスの体重（ｇ）であり、横軸が培養時間（日）である。図５左図に示すとおり、抗ＦＧＦ４抗体に有意な抗腫瘍活性（Ｔ／Ｃ＝０．７）が認められた。また、図５右図に示す通り、抗ＦＧＦ４抗体を投与しても、マウスの体重の減少は認められなかった。 FIG. 5 is a graph showing an example of the result of confirming the effect of the anti-FGF4 antibody on the tumor of KYSE-270 / FGF4 transplanted into a mouse. In the left diagram of FIG. 5, the vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days). In the right diagram of FIG. 5, the vertical axis represents the mouse body weight (g), and the horizontal axis represents the culture time (days). As shown in the left diagram of FIG. 5, significant antitumor activity (T / C = 0.7) was observed for the anti-FGF4 antibody. In addition, as shown in the right diagram of FIG. 5, even when the anti-FGF4 antibody was administered, the weight of the mouse was not reduced.
　［ＦＧＦＲ阻害剤のin vivoにおけるＦＧＦ４過剰発現腫瘍に対する効果］
　ＦＧＦ４過剰発現腫瘍に対するＦＧＦＲ阻害剤の効果をin vivoで確認した。
　ＦＧＦＲ阻害剤として、市販品（商品名：PD173074）を使用した。なお、このPD173074は、ＦＧＦＲのシグナル経路を阻害する（データ示さず）。
　ＦＧＦ４過剰発現肺癌細胞株Ａ５４９／ＦＧＦ４を移植した担がんマウスモデルを作製した。この担がんマウスモデルに、前記ＦＧＦＲ阻害剤を５０mg/kg、７回／週の腹腔内投与を１週間行った。コントロールとして、ＦＧＦＲ阻害剤に換えてＤＭＳＯを使用した。その結果を図６に示す。 [Effect of FGFR inhibitor on FGF4-overexpressing tumor in vivo]
The effect of FGFR inhibitors on FGF4 overexpressing tumors was confirmed in vivo.
A commercially available product (trade name: PD173074) was used as the FGFR inhibitor. The PD173074 inhibits the FGFR signal pathway (data not shown).
A tumor-bearing mouse model in which the FGF4 overexpressing lung cancer cell line A549 / FGF4 was transplanted was prepared. This tumor-bearing mouse model was intraperitoneally administered with the FGFR inhibitor at 50 mg / kg, 7 times / week for 1 week. As a control, DMSO was used instead of the FGFR inhibitor. The result is shown in FIG.
　図６は、マウスに移植したＦＧＦ４過剰発現肺癌細胞株Ａ５４９／ＦＧＦ４に対するＦＧＦＲ阻害剤の効果を確認した結果の一例を示すグラフである。縦軸が腫瘍の大きさ（ｍｍ3）であり、横軸が培養時間（日）である。同図に示すとおり、ＦＧＦＲ阻害剤は有意な抗腫瘍活性を示した。 FIG. 6 is a graph showing an example of the results of confirming the effect of an FGFR inhibitor on the FGF4-overexpressing lung cancer cell line A549 / FGF4 transplanted into mice. The vertical axis represents the tumor size (mm 3 ), and the horizontal axis represents the culture time (days). As shown in the figure, the FGFR inhibitor showed significant antitumor activity.

Claims (8)

  1. 　ＦＧＦ４（Fibroblast growth factor 4）遺伝子増幅腫瘍の改善、進行抑制、及び／又は、治療のための医薬組成物であって、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４ｓｉＲＮＡ、及びＦＧＦ４ｓｈＲＮＡからなる群から選択される少なくとも１つを有効成分として含有する医薬組成物。 A pharmaceutical composition for FGF4 (Fibroblast growth factor 増 幅 4) gene amplification tumor improvement, progression inhibition, and / or treatment, selected from the group consisting of anti-FGF4 antibody, FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA A pharmaceutical composition containing at least one of the above as an active ingredient.
  2. 　ＦＧＦ４遺伝子増幅腫瘍は、食道癌である、請求項１記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the FGF4 gene-amplified tumor is esophageal cancer.
  3. 　抗ＦＧＦ４抗体が、ＦＧＦ４に特異性を有する中和抗体である、請求項１又は２に記載の医薬組成物。 The pharmaceutical composition according to claim 1 or 2, wherein the anti-FGF4 antibody is a neutralizing antibody having specificity for FGF4.
  4. 　対象の腫瘍組織試料又は腫瘍細胞試料におけるＦＧＦ４遺伝子の遺伝子増幅レベルを検出すること、及び、
    　対象のＦＧＦ４遺伝子が増幅している場合に請求項１から３のいずれかに記載の医薬組成物の治療的投与の有効性が予測されると前記対象を分類すること、又は、対象のＦＧＦ４遺伝子が増幅していない場合に請求項１から３のいずれかに記載の医薬組成物の治療的投与の有効性が予測されないと前記対象を分類することを含む、方法。     Detecting the level of gene amplification of the FGF4 gene in a tumor tissue sample or tumor cell sample of interest; and
    When the subject FGF4 gene is amplified, the subject is classified as being effective for the therapeutic administration of the pharmaceutical composition according to any one of claims 1 to 3, or the subject FGF4 gene A method comprising classifying said subject as not predicting the effectiveness of therapeutic administration of a pharmaceutical composition according to any of claims 1 to 3 when is not amplified.
  5. 　ＦＧＦ４遺伝子増幅レベルの検出が、蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によって行われる、請求項４記載の方法。 The method according to claim 4, wherein the detection of the FGF4 gene amplification level is performed by a method selected from the group consisting of fluorescence in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR.
  6. 　前記腫瘍組織又は腫瘍細胞が、対象の食道から採取されたものである、請求項４又は５に記載の方法。 The method according to claim 4 or 5, wherein the tumor tissue or tumor cell is collected from the subject's esophagus.
  7. 　蛍光 in situ ハイブリダイゼーション（ＦＩＳＨ）、次世代シークエンサー、ＤＮＡチップ、及びリアルタイム定量ＰＣＲからなる群から選択される方法によりＦＧＦ４遺伝子増幅レベルの検出を行うための試薬及びＦＧＦ４遺伝子にハイブリダイズするヌクレオチドを備える、請求項４から６のいずれかに記載の方法に使用するためのキット。 A reagent for detecting the FGF4 gene amplification level by a method selected from the group consisting of fluorescent in situ hybridization (FISH), next-generation sequencer, DNA chip, and real-time quantitative PCR, and a nucleotide that hybridizes to the FGF4 gene A kit for use in the method according to any one of claims 4 to 6.
  8. 　請求項１から３のいずれかに記載の医薬組成物を製造するための、抗ＦＧＦ４抗体、ＦＧＦ受容体阻害剤、ＦＧＦ４ｓｉＲＮＡ、及びＦＧＦ４ｓｈＲＮＡからなる群から選択される少なくとも１つの使用。 Use of at least one selected from the group consisting of an anti-FGF4 antibody, an FGF receptor inhibitor, FGF4 siRNA, and FGF4 shRNA for producing the pharmaceutical composition according to any one of claims 1 to 3.
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