WO2007037299A1 - Substance inhibant l’expression de hb24 ou son activité de régulation transcriptionnelle, son utilisation, et procédé de criblage de la substance - Google Patents

Substance inhibant l’expression de hb24 ou son activité de régulation transcriptionnelle, son utilisation, et procédé de criblage de la substance Download PDF

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WO2007037299A1
WO2007037299A1 PCT/JP2006/319242 JP2006319242W WO2007037299A1 WO 2007037299 A1 WO2007037299 A1 WO 2007037299A1 JP 2006319242 W JP2006319242 W JP 2006319242W WO 2007037299 A1 WO2007037299 A1 WO 2007037299A1
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seq
amino acid
acid sequence
base sequence
polynucleotide
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PCT/JP2006/319242
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Japanese (ja)
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Masayuki Okada
Sachi Suzuki
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Eisai R & D Management Co., Ltd.
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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns

Definitions

  • the present invention relates to novel angiogenesis inhibitors and Z or antitumor agents.
  • the present invention also relates to a novel angiogenesis inhibitor and a screening method for Z or an antitumor agent.
  • Angiogenesis inhibitors have been shown to be useful as antitumor agents.
  • bevacizumab a neutralizing antibody preparation against VEGF, which plays an important role in angiogenic factors, has been reported to have an antitumor effect on colorectal cancer in clinical trials.
  • Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metast atic colorectal cancer N Engl J Med. 2004, 350, 2335-42.
  • HB24 is a homebox domain derived from a cDNA library of activated B cells in 1991.
  • a diverged homeobox gene is involved in t he proliferation and lineage commitment of human hematopoietic progenitors ana hig hly expressed in acute myelogenous leukemia.
  • High level expression of the homeobox gene HB24 in a human T—eel 1 line confers the ability to form tumors in nude mouse.Deguchi Y et al .; and (iv) J. Biol.Chem. 1993; 268 (5): 3646— 3653.
  • a human homeobox gene, HB24 inhibits devel opment of CD4 + T cells and impairs thymic involution in transgenic mice.Degichi Y et al.), Endothelial cells, angiogenesis
  • HB24 is associated with proliferation, migration, and lumen formation of vascular endothelial cells. That is, it was found that by reducing the expression of HB24, proliferation, migration, and lumen formation of vascular endothelial cells can be suppressed. For this reason, it was found that substances having an HB24 expression-lowering action and substances having an action to suppress the transcriptional regulatory activity of Z or HB24 are useful as angiogenesis inhibitors and Z or antitumor agents. Furthermore, since HB24 affects the proliferation, migration, and lumen formation of vascular endothelial cells, it has proved to be useful for screening substances having angiogenesis inhibitory action and Z or antitumor action. The present invention is based on these findings.
  • an object of the present invention is to provide an angiogenesis inhibitor and an antitumor agent, and a screening method for a substance having an angiogenesis inhibitory action and Z or an antitumor action.
  • the angiogenesis inhibitor and Z or antitumor agent according to the present invention (that is, the angiogenesis inhibitor composition and Z or antitumor agent composition) comprise a substance having an HB24 expression-reducing action as an active ingredient. .
  • the angiogenesis inhibitor and / or antitumor agent according to the present invention comprises a substance having a substance having an action of suppressing the transcriptional regulatory activity of HB24 as an active ingredient.
  • angiogenesis inhibition and Z or Z comprising a continuous base sequence of at least 10 bases in length.
  • a polynucleotide for use in the treatment or prevention of a tumor is provided:
  • nucleotide sequence that also has the ability to polynucleotide to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions;
  • the first force is also a base sequence complementary to the base sequence described in the 819th or 997th to 1464th positions,
  • the polynucleotide has the ability to hybridize under stringent conditions to the polynucleotide having the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions.
  • nucleotide sequence set forth in SEQ ID NO: 1 the nucleotide sequence complementary to the nucleotide sequence having 90% or more identity to the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions .
  • the vector according to the present invention produces the polynucleotide according to the present invention described above.
  • the antibody according to the present invention is an antibody against a protein having the amino acid sequence ability described in SEQ ID NO: 2, which is used for inhibiting angiogenesis and treating or preventing Z or tumor.
  • a substance having an HB24 expression lowering action or a substance having an action of suppressing the transcriptional regulatory activity of HB24 in the present invention for producing an angiogenesis inhibitor and Z or an antitumor agent There is provided the use of a polynucleotide according to the invention, a vector according to the invention, or an antibody according to the invention.
  • a substance having an HB24 expression lowering action or a substance having an action of suppressing the transcriptional regulatory activity of HB24 in the present invention for an angiogenesis inhibitor and Z or an antitumor agent according to the present invention
  • a polynucleotide, a vector according to the invention, or an antibody according to the invention is provided.
  • a substance having an HB24 expression-reducing action or a substance having an action of suppressing the transcriptional regulatory activity of HB24, a polynucleotide according to the present invention, a vector according to the present invention, or the present invention A method of inhibiting angiogenesis and a method of treating Z or cancer comprising administering to a patient an effective amount of an antibody is provided. That is, according to the present invention, a method for inhibiting angiogenesis or a method for treating or preventing cancer is provided.
  • FIG. 1 shows proliferation (A) and migration (B) of HB24 highly expressing PAE cell line.
  • FIG. 2 shows the effect of anti-HB24 antisense oligonucleotide on HUVEC migration.
  • FIG. 3 shows the effect on migration of PAE by anti-HB24 antisense oligonucleotide.
  • FIG. 4 shows the effect of HB24 siRNA on the migration of vascular endothelial cells.
  • FIG. 5 shows the effect of HB24 siRNA on vascular endothelial cell lumen formation.
  • FIG. 6 shows the effect of HB24 siRNA expressing recombinant lentivirus on HB24 expression reduction in vascular endothelial cells (A) and the effect on survival and proliferation of vascular endothelial cells (B).
  • FIG. 7 shows the effect of Luminacin D to decrease HB24 expression in vascular endothelial cells.
  • FIG. 8 shows the effect of Luminacin D on migration of vascular endothelial cells.
  • FIG. 9 shows the effect of Luminacin D on vascular endothelial cell lumen formation.
  • HB24 includes a polypeptide having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 (GenBank accession number: NM-0221958). [0018]
  • the polypeptide having the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 is specifically selected from, for example, the following groups (i) to (iv) Things are listed:
  • polypeptide consisting of an amino acid sequence having 90% or more, preferably about 95% or more, more preferably about 98% or more identity (sometimes referred to as homology) to the amino acid sequence shown in SEQ ID NO: 2
  • a polypeptide preferably a polypeptide having an amino acid sequence having the same identity, and further having substantially the same activity as HB24.
  • “having substantially the same activity as HB24” indicates that the activity is comparable to that of the protein having an amino acid sequence ability described in SEQ ID NO: 2. “Similar” means that the activity is homogeneous in nature. For example, “same degree” is substantially when the transcriptional regulatory activity of the protein has 10% or more, preferably 30% or more of the transcriptional regulatory activity of the protein having the amino acid sequence ability represented by SEQ ID NO: 2. It can be said that it has the same transcriptional regulation activity.
  • the method for measuring transcriptional regulatory activity can be measured according to a conventional method.
  • the method for measuring the transcriptional regulatory activity in the test protein includes the following.
  • a protein having an amino acid sequence ability described in SEQ ID NO: 2 introduces a vector containing a promoter and a reporter gene (for example, luciferase, alkaliphosphatase, j8-galactosidase, etc.) including a transcriptional regulatory region that regulates transcriptional activity into a cell.
  • the host cell is not particularly limited, but preferably a cell line, for example, B3 00, CHO, BHK, COS7, NIH3T3, HEK293, etc.
  • a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a test protein is expressed.
  • the protein can be expressed by genetic engineering techniques according to conventional methods.
  • the transcriptional regulatory activity can be measured by quantifying the amount of protein produced by the reporter gene (eg, luciferase, alkaline phosphatase, j8-galactosidase, etc.).
  • polypeptide having substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2 of GO include:
  • (I) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2 A deleted amino acid sequence,
  • amino acids 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids are added to the amino acid sequence represented by SEQ ID NO: 2. Or the inserted amino acid sequence,
  • (III) 1-9 (for example, 1-5, preferably 1-3, more preferably 1-2, more preferably 1) amino acids in the amino acid sequence represented by SEQ ID NO: 2
  • An amino acid sequence substituted with another amino acid
  • deletion of amino acid means a mutation in which one or more amino acid residues in the sequence are deleted, and deletion includes deletion of amino acid residues in the amino acid sequence. And those in which amino acid residues in the middle of the amino acid sequence are deleted.
  • addition of an amino acid means a variation in which one or more amino acid residues are added to the sequence, and an amino acid residue is added to the end of the amino acid sequence. And those with amino acid residues added in the middle of the amino acid sequence. What is added in the middle is sometimes called “insertion”.
  • substitution of an amino acid means a mutation in which one or more amino acid residues in the sequence are changed to different types of amino acid residues.
  • substitution By such substitution, the amino acid sequence of ⁇ 24
  • a conservative substitution is a change in sequence to encode an amino acid with similar properties to the amino acid before substitution.
  • Amino acid properties include, for example, nonpolar amino acids (Ala, lie, Leu, Met, Phe, Pro, Trp, Val), uncharged amino acids (Asn, Cys, Gin, Gly, Ser, Thr, Tyr), acidic amino acids (Asp, Glu), basic amino acids (Arg, His, Lys), neutral amino acids (Ala, Asn, Cys, Gin, Gly, lie, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val ), Aliphatic amino acids (Ala, Gly), branched amino acids (lie, Leu, Val), hydroxyamino acids (Ser, Thr), amide-type amino acids (Gin, Asn), sulfur-containing amino acids (Cys, Met), It can be classified into aromatic amino acids (His, Phe, Trp, Tyr), heterocyclic amino acids (His, Trp), imino acids (Pro, 4Hy p) and the like.
  • substitution with nonpolar amino acids or uncharged amino acids is preferred.
  • substitutions between Ala, Val, Leu and lie, between Ser and Thr, between Asp and Glu, between Asn and Gin, between Lys and Arg, and between Phe and Tyr depend on the nature of the protein. Is preferred as a substitution for retaining There is no particular limitation on the number and site of amino acids to be mutated.
  • HB24 can also be said to include a protein encoded by a polynucleotide having the same or substantially the same base sequence as the base sequence shown in SEQ ID NO: 1.
  • the nucleotide sequence substantially identical to the nucleotide sequence represented by SEQ ID NO: 1 is 90% or more, preferably 95% or more, more preferably 98% or more identity with the nucleotide sequence represented by SEQ ID NO: 1.
  • the base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 is, for example, one or more (for example, one or several) in the base sequence represented by SEQ ID NO: 1.
  • Examples include nucleotide sequences that have mutations such as deletions, substitutions or additions in nucleic acids, and that encode proteins that have substantially the same transcriptional regulatory activity.
  • the base sequence substantially the same as the base sequence represented by SEQ ID NO: 1 includes, for example,
  • nucleic acid in the nucleotide sequence represented by SEQ ID NO: 1 Deleted base sequence
  • nucleic acid in the nucleotide sequence represented by SEQ ID NO: 1 Deleted base sequence
  • nucleic acid 1 to 9 eg 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1 nucleic acid is added to the nucleotide sequence represented by SEQ ID NO: 1.
  • base sequence for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1 preferably 1 to 2, more preferably 1 nucleic acid is added to the nucleotide sequence represented by SEQ ID NO: 1.
  • (III) 1 to 9 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) nucleic acid in the base sequence represented by SEQ ID NO: 1 Nucleotide sequences substituted with other nucleic acids,
  • a polynucleotide that is hybridized under stringent conditions specifically includes FASTA, BLAST, Smith-Waterman [Meth. Enzym., 164, 76 5 (1988)].
  • the base sequence represented by SEQ ID NO: 1 is at least 90% or more, preferably 95% or more, more preferably 97 when calculated using the default (initial setting) parameters. % Or more, more preferably 98% or more, and even more preferably 99% or more of a polynucleotide having identity.
  • stringent conditions for example, “2 X SSC, 0.1% SDS, 50 ° C.”, “2 X SSC, 0.1% SDS, 42.
  • the hybridization can be performed according to a known method.
  • it can be performed according to the method described in the attached instruction manual.
  • identity (sometimes referred to as homology) with respect to amino acid sequences is used to mean the degree of coincidence of amino acid residues constituting each sequence between sequences to be compared. It is done. At this time, the existence of gaps and the nature of amino acids are considered (Wil bur, Natl. Acad. Sci. U.S.A. 80: 726-730 (1983)).
  • commercially available software such as BLAST (Altschul: J. Mol. Biol. 215: 403-410 (1990)), FASTA (Peasron: Methods in Enzymology 183: 63-69 (1990), etc. Can be used
  • BLAST Basic local alignment search tool
  • NCBI National Center for Biotechnology Information
  • SEQ ID NO: 4 GenBank accession number: NM-008250
  • SEQ ID NO: 6 GenBank
  • Accession number: XM—344184 amino acid sequences substantially identical to these amino acid sequences are also included. The meaning of “substantially the same” is the same as in the case of SEQ ID NO: 2 above.
  • HB24 is preferably a protein having an amino acid sequence ability represented by SEQ ID NO: 2.
  • a substance having an action of reducing HB24 expression and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24 are useful as active ingredients of angiogenesis inhibitors and antitumor agents.
  • the substance having an HB24 expression-reducing action include antisense oligonucleotides, ribozymes, double-stranded RNA, non-peptide compounds, synthetic compounds, fermentation products, and the like.
  • the substance having the action of reducing HB24 expression is preferably a polynucleotide, more preferably an antisense oligonucleotide, a ribozyme, or a double-stranded RNA.
  • the substance having an action of suppressing the transcriptional regulatory activity of HB24 include anti-HB24 antibody, non-peptide compound, synthetic compound, fermentation product and the like.
  • the “HB24 expression-reducing action” means, for example, that a test substance is treated! /, V, compared to the case, the expression level of HB24 mRNA and Z or protein is at least It can be said to have an effect of lowering 10% or more, preferably 30% or more, more preferably 50% or more.
  • the action of suppressing the transcriptional regulatory activity of HB24 means, for example, that the transcriptional regulatory activity of HB24 is at least 10% or more, preferably 30% or more, more preferably 5 It can refer to the action of inhibiting 0% or more.
  • the substance having an action of reducing HB24 expression and the substance having an action of suppressing the transcriptional regulatory activity of Z or HB24 include an antisense oligonucleotide for HB24, a ribozyme for HB24, and a double strand for HB24, which will be described later. Forces that include RNA, anti-HB24 antibody, etc. Other examples include low molecular weight compounds such as Luminacin D.
  • an antisense oligonucleotide against HB24 can be mentioned as a substance having an action of reducing HB24 expression and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • Antisense oligonucleotides against HB24 suppress the expression of HB24 in cells by binding to mRNA or DNA.
  • the antisense oligonucleotide included in the present invention may be a polynucleotide that suppresses gene expression by any of the mechanisms (1) to (11) described above, that is, only the translation region encoding HB24. Including an antisense sequence to the sequence of the untranslated region It may be.
  • a polynucleotide encoding an antisense against HB24 can be used by ligating under an appropriate control sequence that allows its expression.
  • the antisense oligonucleotide for HB24 only needs to effectively inhibit the expression of HB24, rather than being completely complementary to the translated or untranslated region of the HB24 gene.
  • “Effectively inhibits the expression of HB24” means, for example, a cell that has an antisense oligonucleotide against HB24, and the amount of HB24 expression in the cell cannot contain an antisense oligonucleotide against HB24. Compared to the above, it can be said that it is suppressed by at least 10% or more, preferably 20% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more.
  • the antisense oligonucleotide for HB24 is preferably selected from the following (a) to (j): In any one of the base sequences, it is at least 15 bases long (preferably 18 bases long, more preferably 21 bases long). (Long) polynucleotides, and more preferably, the following (f) to (j): Polynucleotides containing at least 15 consecutive base sequences among the selected base sequences Is mentioned.
  • amino acid sequence shown in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted, substituted, inserted or added, or the amino acid sequence is altered by a combination thereof.
  • nucleotide sequence that also has the ability to polynucleotide to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions;
  • nucleotide sequence complementary to a nucleotide sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 1.
  • the first force is also a base sequence complementary to the base sequence described in the 819th or 997th to 1464th positions,
  • amino acid sequence set forth in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted from the amino acid sequence described in the 1st to 273rd or 333th to 488th positions, A base sequence complementary to a base sequence consisting of a polynucleotide encoding a polypeptide which also has an amino acid sequence ability, which is mutated by substitution, insertion or addition, or a combination thereof,
  • the polynucleotide has the ability to hybridize under stringent conditions to the polynucleotide having the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions.
  • nucleotide sequence set forth in SEQ ID NO: 1 90% or more, preferably 95% or more, more preferably 98% or more with respect to the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions A nucleotide sequence complementary to a nucleotide sequence having the same identity.
  • the antisense oligonucleotide for HB24 is selected from (f) to (j) above. More preferred is a polynucleotide comprising a continuous base sequence having a length of at least 15 bases.
  • the antisense oligonucleotide for HB24 includes, for example, positive nucleotide S of at least 15 bp to 3000 bp, preferably 2000 bp of positive nucleotides such as lOObp, and more preferably 500 bp to lOOObp. A polynucleotide may be mentioned.
  • An antisense oligonucleotide against HB24 can be prepared by the phosphorothioate method (Stein (1988) Nucleic Acids Res. 16: 3209-21) based on the sequence shown in SEQ ID NO: 1.
  • a ribozyme for HB24 can be mentioned as a substance having an action of reducing HB24 expression and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • the Ribozymes for HB24 suppress the expression of HB24 in cells by binding to mRNA or DNA.
  • a ribozyme is a general term for catalysts having RNA as a constituent component, and is roughly classified into a large ribozyme and a small liboyme.
  • Large ribozyme is an enzyme that cleaves the phosphate ester bond of nucleic acid and leaves a 5'-phosphate and 3'-hydroxyl group at the reaction site after the reaction.
  • ribozymes are further divided into (1) group I intron RNA that undergoes transesterification at the 5 'splice site with guanosine, (2) group ⁇ intron RNA that undergoes self-splicing in a two-step reaction via a Lariat structure, and (3 ) It is classified as the RNA component of ribonuclease P, which cleaves the tRNA precursor by hydrolysis on the 5th side.
  • a small ribozyme is a relatively small structural unit (about 40 bp) that cleaves RNA to form a 5′-hydroxyl group and a 2′-3 ′ cyclic phosphate.
  • Small ribozymes include hammerhead (Koizumi et al.
  • Ribozyme is, for example, a hammerhead type that recognizes and cleaves the base sequence UC, UU or UA in the target RNA by designing the substrate binding part of the ribozyme to be complementary to the RNA sequence near the target site. Ribozymes can be made (Koizumi et al. (1988) FEBS Lett.
  • Hairpin ribozymes can also be designed and manufactured according to known methods (Kikuchi and Sasaki (1992) Nucleic Acids Res. 19: 6751; Hiroshi Kikuchi (1992) Chemistry and Biology 30: 112) .
  • the ribozyme for HB24 may be any as long as it effectively inhibits the expression of HB24. “Effectively inhibits the expression of HB24” means, for example, that in cells that have ribozyme against HB24, the expression level of HB24 in the cell is at least 10%, preferably at least 10% compared to cells that do not add ribozyme to HB24. What can be suppressed is 20% or more, more preferably 30% or more, still more preferably 50% or more, and particularly preferably 70% or more.
  • the ribozyme for HB24 is preferably selected from the following (a) to (; j) force
  • a polynucleotide containing a base sequence of at least 15 bases in length preferably 18 bases in length, more preferably 21 bases in length
  • a polynucleotide containing a base sequence of at least 15 bases in length preferably 18 bases in length, more preferably 21 bases in length
  • Strength Among the selected nucleotide sequences, there may be mentioned polynucleotides containing a base sequence having a length of at least 15 bases.
  • amino acid sequence shown in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted, substituted, inserted or added, or the amino acid sequence is altered by a combination thereof.
  • nucleotide sequence that also has the ability to polynucleotide to a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions;
  • nucleotide sequence complementary to a nucleotide sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 1.
  • the first force is also a base sequence complementary to the base sequence described in the 819th or 997th to 1464th positions,
  • amino acid sequence set forth in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted from the amino acid sequence described in the 1st to 273rd or 333th to 488th positions, A base sequence complementary to a base sequence consisting of a polynucleotide encoding a polypeptide which also has an amino acid sequence ability, which is mutated by substitution, insertion or addition, or a combination thereof,
  • the polynucleotide has the ability to hybridize under stringent conditions to the polynucleotide having the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions.
  • Base sequence, and (j) Of the nucleotide sequence set forth in SEQ ID NO: 1, 90% or more, preferably 95% or more, more preferably 98% or more with respect to the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions A nucleotide sequence complementary to a nucleotide sequence having the same identity.
  • double-stranded RNA against HB24 can be mentioned as a substance having an action of reducing HB24 expression and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • Double-stranded RNA against HB24 is caused by RNA interference (Fire et al. (1998) Nature 391: 806 -811; Morita Takashi et al. (2002) Protein Nucleic Acid Enzyme 47: 1939-1945). Suppresses the expression of HB24 in cells.
  • the double-stranded RNA for HB24 may be any one that effectively inhibits the expression of HB24 without necessarily being completely complementary to the HB24 gene. “Effectively inhibits the expression of HB24” means, for example, that in a cell with a double-stranded RNA against HB24, the expression level of HB24 in the cell must not be double-stranded with respect to HB24. ! /, Which can be suppressed by at least 10% or more, preferably 20% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more, compared to cells.
  • the double-stranded RNA for HB24 is preferably at least 10 bases long (preferably 15 bases long, more preferably) in any of the following base sequences that are also selected from (a) to (; j): Is a polynucleotide containing a base sequence of 18 bases long, and more preferably, a base sequence of at least 10 bases continuous among base sequences selected from the following (f) to (j): Examples thereof include polynucleotides.
  • amino acid sequence shown in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted, substituted, inserted or added, or the amino acid sequence is altered by a combination thereof.
  • nucleotide sequence complementary to a nucleotide sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 1.
  • the first force is also a base sequence complementary to the base sequence described in the 819th or 997th to 1464th positions,
  • amino acid sequence set forth in SEQ ID NO: 2 one or more (for example, one or several) amino acids are deleted from the amino acid sequence described in the 1st to 273rd or 333th to 488th positions, A base sequence complementary to a base sequence consisting of a polynucleotide encoding a polypeptide which also has an amino acid sequence ability, which is mutated by substitution, insertion or addition, or a combination thereof,
  • the polynucleotide has the ability to hybridize under stringent conditions to the polynucleotide having the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions.
  • nucleotide sequence set forth in SEQ ID NO: 1 90% or more, preferably 95% or more, more preferably 98% or more with respect to the nucleotide sequence described in the 1st to 819th or 997th to 1464th positions A nucleotide sequence complementary to a nucleotide sequence having the same identity.
  • the double-stranded RNA for HB24 is a combination of a sense strand and an antisense strand for the sequence of HB24, and includes, for example, at least 10 to 100 polynucleotides, preferably 15 to 75. Examples include polynucleotides, more preferably 18 to 50 polynucleotides, and even more preferably 20 or 25 polynucleotides.
  • Double-stranded RNA against HB24 can be introduced into cells by a known method.
  • a polynucleotide that encodes a sense strand and an antisense strand constituting a double-stranded RNA for HB24 on a single strand is designed, the polynucleotide is incorporated into an expression vector, and the expression vector is introduced into a cell.
  • To double-stranded RNA for HB24 Can be introduced into cells.
  • Plasmid expression vectors that continuously produce double-stranded RNA for HB24 by transfection can also be designed (eg, RNAi-Ready pSIREN Vector, RNAi-Ready pSI REN-RetroQ Vector (BD Biosciences Clontech)).
  • RNA for HB24 should be designed using the computer program of Ambion website (http: ///www.ambion.com/techlib/misc/siRNA—finder.html), for example. Can do.
  • Antisense oligonucleotides against HB24, ribozymes against HB24 and double-stranded RNAs against HB24 are vectors derived from viruses such as retroviruses, adenoviruses, adeno-associated viruses, etc. in order to control gene expression in cells.
  • viruses such as retroviruses, adenoviruses, adeno-associated viruses, etc.
  • non-viral vectors using liposomes, etc., or naked DNA can be used by the ex vivo method or the in vivo method.
  • Vectors can be prepared by referring to known methods (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 11.4— ⁇ ⁇ .11; Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Press (1989) Section 5.61-5.63).
  • Confirmation of the base sequence of the antisense oligonucleotide for HB24, the ribozyme for HB24, and the double-stranded RNA for HB24 can be performed by sequencing by conventional methods. For example, it can be carried out by the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). It is also possible to analyze the sequence using an appropriate DNA sequencer.
  • the HB24 expression-reducing action can be measured by a known technique, such as an immunological method (eg, flow cytometry, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoassay method). (RIA), ELISA, SDS-PAGE, Western blot, etc.), molecular biological analysis methods (eg, Northern blot, RT-PCR, quantitative RT-PCR, in situ hybridization, etc.) .
  • an immunological method eg, flow cytometry, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoassay method). (RIA), ELISA, SDS-PAGE, Western blot, etc.
  • molecular biological analysis methods eg, Northern blot, RT-PCR, quantitative RT-PCR, in situ hybridization, etc.
  • an angiogenesis comprising a base sequence having a length of at least 10 consecutive bases in any one of the base sequences selected from the following (a) to (j): Provides polynucleotides used for inhibition and treatment or prevention of Z or tumors Is done.
  • the polynucleotide has a base length of 15, more preferably 18.
  • the vector for producing a polynucleotide is not particularly limited in its type and production method as long as it can produce a polynucleotide such as an antisense oligonucleotide, a ribozyme, or a double-stranded RNA. Any of them may be used.
  • the vector is preferably a retroviral vector or an adenovirus vector.
  • an anti-HB24 antibody can be mentioned as a substance having an action of suppressing the transcriptional regulatory activity of HB24.
  • the anti-HB24 antibody is an antibody against the HB24 or a partial fragment thereof, preferably an antibody against a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (or an antibody having an affinity for the protein), more preferably Of the amino acid sequence U described in SEQ ID NO: 2, the antibody against the polypeptide consisting of the 1st force and the 273rd or the 333th force and the 488th amino acid sequence U force.
  • Anti-antibody 24 antibodies are, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies (scFV) (Huston et la. (1988) Proc. Natl. Acad. Sci.
  • the anti-HB24 antibody may be modified with polyethylene glycol (PEG) or the like, if necessary.
  • PEG polyethylene glycol
  • the anti-HB24 antibody can be produced as a fusion protein with 13-galatatosidase, MBP, GST, GFP, etc., and is detected without using a secondary antibody in the ELISA method. You may be able to make it out.
  • the antibody may be recovered using avidin, streptavidin, etc. by labeling the antibody with piotin, etc.
  • the anti-HB24 antibody can be produced according to a conventional method using HB24 or a partial fragment thereof (hereinafter sometimes referred to as "HB24 polypeptide fragment") or cells expressing them as a sensitizing antigen. ("Current Protocols in Molecular Biology” (J. hn Wiley & Sons (1 987) Section 11.4-11.13)).
  • the polypeptide fragment of HB24 may be a fusion protein with Fc region, GST, MBP, GFP, AP or the like.
  • the polypeptide fragment of HB24 may be a polypeptide comprising an amino acid sequence that is identical to a part of the amino acid sequence of HB24.
  • Examples of the polypeptide fragment of HB24 include the same polypeptide as a part of the protein having the amino acid sequence of SEQ ID NO: 2, and preferably at least 6 amino acid residues (for example, 8, 10, 12). Or a polypeptide fragment containing 15 amino acid residues or more).
  • the polypeptide fragment of the present invention may be a mutant such as a substitution of the above polypeptide! /.
  • polypeptide fragment of HB24 can be produced by a conventional method.
  • the polypeptide fragment of the present invention can also be prepared as described in Example 2, for example.
  • the measurement of the action of suppressing the transcriptional regulatory activity by the anti-HB24 antibody can be performed by a known technique.
  • a vector containing a promoter and a reporter gene eg, luciferase, alkaline phosphatase, j8-galactosidase, etc.
  • a transcriptional regulatory region known to regulate transcriptional activity by HB24 is introduced into the cell.
  • the host cell for example, B300, CHO, BHK, COS7, NIH3T3, HEK293, etc., which cell lines are preferred can be used.
  • a protein derived from a single reporter gene for example, luciferase, alkaline phosphatase, ⁇ -galatatosidase, etc.
  • the expression level of HB24 is regulated by the transcriptional regulator T- bet (Mullen et a 1. (2002) Nature Immunology 3 (7): 652, GenBank accession number: NM—01335 Do, Substances that have T- bet expression-reducing action and substances that suppress Z or T- bet transcriptional regulation activity are used as substances that have HB24 expression-lowering action and substances that have the action of inhibiting Z or HB24 transcriptional regulation activity. Examples of substances having T- bet expression-reducing action include antisense oligonucleotides, ribozymes, double-stranded RNAs, non-peptide compounds, synthetic compounds, fermentation products, etc. T bet transcriptional regulation Examples of the substance that suppresses the activity include an anti-T bet antibody, a non-peptide compound, a synthetic compound, and a fermentation product.
  • the substance having an HB24 expression-reducing action or the substance having an action of suppressing the transcriptional regulatory activity of HB24 is a transcriptional regulatory factor that controls the expression of HB24. It is a substance having a bet expression-reducing action or a substance that suppresses T bet transcriptional regulatory activity.
  • an antisense oligonucleotide against T bet can be mentioned as a substance having an HB24 expression lowering action and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • Antisense oligonucleotides against T-bet are those that reduce HB24 expression through T-bet expression lowering action.
  • An antisense oligonucleotide against T-bet suppresses the expression of T-bet in cells by binding to mRNA or DNA.
  • the antisense oligonucleotide for T bet may include an antisense sequence for the sequence of the untranslated region as well as the translation region encoding T bet.
  • the antisense oligonucleotide only needs to effectively inhibit the expression of T bet without needing to be completely complementary to the translated region or untranslated region of the T bet gene. “Effectively inhibits expression of T-bet” means, for example, expression of T bet in a cell containing an antisense oligonucleotide against T-bet.
  • the antisense oligonucleotide for T bet is preferably selected from the following (a,) to (e,): a base sequence having a length of at least 15 consecutive bases in any base sequence And a polynucleotide containing (preferably 18 bases long, more preferably 21 bases long).
  • (d ′) a base sequence that also has a polynucleotide ability to hybridize under stringent conditions to a polynucleotide comprising the base sequence set forth in SEQ ID NO: 7.
  • (e ′) a base sequence complementary to a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the base sequence shown in SEQ ID NO: 7.
  • antisense oligonucleotides against T-bet include polynucleotides of at least 15 bp to 3000 bp, preferably lOObp to 2000 bp, and more preferably 500 bp to lOOObp. Nucleotides are mentioned.
  • Antisense oligonucleotides against T bet can be prepared by the phosphorothioate method (Stein (1988) Nucleic Acids Res. 16: 3209-21) based on the sequence shown in SEQ ID NO: 1.
  • a ribozyme for T bet can be mentioned as a substance having a HB24 expression lowering action and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • Ribozyme against T-bet reduces HB24 expression through T- bet expression-reducing action. It is something to be lowered.
  • Ribozymes for T-bet suppress T bet expression in cells by binding to mRNA or DNA.
  • the ribozyme for T-bet only needs to effectively inhibit the expression of T-bet.
  • Effectively inhibits the expression of T- bet means, for example, that in a cell bearing a ribozyme against T- bet, the expression level of T bet in the cell is higher than that without adding a ribozyme against T bet. In other words, it can be suppressed at least 10% or more, preferably 20% or more, more preferably 30% or more, further preferably 50% or more, particularly preferably 70% or more.
  • the ribozyme for T-bet preferably has a base sequence of at least 15 bases in length (preferably 18 bases long) in any of the following base sequences selected from (a ') to (e'): And more preferably a polynucleotide containing 21 bases in length).
  • (d ′) a base sequence that also has a polynucleotide ability to hybridize under stringent conditions to a polynucleotide comprising the base sequence set forth in SEQ ID NO: 7.
  • (e ′) a base sequence complementary to a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the base sequence shown in SEQ ID NO: 7.
  • double-stranded RNA against T bet can be mentioned as a substance having an action of reducing HB24 expression and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24.
  • Double-stranded RNA against T bet induces a decrease in HB24 expression through a T- bet expression lowering action, thereby suppressing the transcriptional regulatory activity of HB24.
  • Double-stranded RNA against T-bet causes intracellular interference by degrading mRNA by causing RNA interference. Suppresses the expression of HB24.
  • the double-stranded RNA for T bet need only be one that effectively inhibits T bet expression without necessarily being completely complementary to the T bet gene. “Effectively inhibits T bet expression” means, for example, that in cells added with double-stranded RNA against T bet, the expression level of T bet within the cell is double-stranded RNA against T bet. It can be said that it is suppressed at least 10% or more, preferably 20% or more, more preferably 30% or more, even more preferably 50% or more, particularly preferably 70% or more, compared to cells that cannot.
  • the double-stranded RNA against T bet is preferably at least 10 bases long (preferably 15 bases long, more preferably) in any base sequence in which (a ') to (e') forces are also selected. And a polynucleotide containing a base sequence of 18 bases long).
  • (d ′) a base sequence that also has a polynucleotide ability to hybridize under stringent conditions to a polynucleotide comprising the base sequence set forth in SEQ ID NO: 7.
  • (e ′) a base sequence complementary to a base sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity to the base sequence shown in SEQ ID NO: 7.
  • the double-stranded RNA for T bet is a combination of a sense strand and an anti-sense strand for the sequence of T bet, and includes, for example, at least 10 to 100 polynucleotides, preferably 15 to 75. More preferably, 18 to 50 polynucleotides, and even more preferably 20 to 25 polynucleotides.
  • Double-stranded RNA against T-bet can be introduced into cells by a known method.
  • a polynucleotide that encodes a sense strand and an antisense strand constituting a double-stranded RNA against T-bet on a single strand is designed, the polynucleotide is incorporated into an expression vector, and the expression vector is incorporated into a cell.
  • double-stranded RNA for T bet can be introduced into the cell.
  • Plasmid expression vectors that continuously produce double-stranded RNA against T-bet by transfection can also be designed (eg, RNAi-Ready pSIREN Vector, RNAi-Ready pSI REN-RetroQ Vector (BD Biosciences Clontech)).
  • RNA for T bet is designed using, for example, the computer program of Ambion website (http: ///www.ambion.com/techlib/misc/siRNA—finder.html) be able to.
  • Antisense oligonucleotides to T- bet, ribozymes to T- bet and double-stranded RNA to T bet are used to control the expression of genes in cells, retroviruses, adenoviruses, adeno-associated viruses. It can also be used as a vector derived from viruses such as non-viral vectors utilizing ribosomes, etc., or naked DNA by the ex vivo method or the in vivo method.
  • Confirmation of the base sequence of the antisense oligonucleotide for T-bet, the ribozyme for T-bet, and the double-stranded RNA for T bet can be carried out by sequencing by conventional methods. For example, it can be performed by the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463). It is also possible to analyze the sequence using an appropriate DNA sequencer.
  • Measurement of T-bet expression-reducing action can be performed by a known technique.
  • immunological methods eg, flow cytometry, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoimmunity
  • RIA enzyme immunoassay
  • ELISA electrophoressay
  • SDS-PAGE Western blot
  • molecular biological analysis methods eg, Northern blot, RT-PCR, quantitative RT-PCR, in situ hybridization, etc.
  • a substance having an HB24 expression-reducing action and transcription of Z or HB24 Anti-T-bet antibody is mentioned as a substance which has the effect
  • the anti-T bet antibody is an antibody against the T bet or a partial fragment thereof, preferably an antibody against the T bet or a partial fragment thereof, which has an effect of suppressing the transcriptional regulatory activity of T bet.
  • Anti-T-bet antibodies are, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies (scFV) (Huston et la. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-83; Tne Pharmacology of Monoclonal Antibody, vol.113, Rosenburg and Moore ed., Springer Verlag (1994) pp.269-315), hyalin antibody, multispecific antibody (LeDoussal et al.
  • the bet antibody may be modified with polyethylene glycol (PEG) or the like as necessary.
  • the anti-T-beta antibody can be produced as a fusion protein with 13-galatatosidase, MBP, GST, GFP, etc., and may be detected without using a secondary antibody in an ELISA method or the like.
  • the antibody may be modified so that it is recovered by using avidin, streptavidin or the like by labeling the antibody with piotin or the like.
  • the anti-T-bet antibody is prepared by a conventional method using T-bet or a partial fragment thereof (hereinafter sometimes referred to as "T-bet polypeptide fragment") or cells expressing them as a sensitizing antigen.
  • T-bet polypeptide fragment a partial fragment thereof
  • the polypeptide fragment of T-beta may be a fusion protein with Fc region, GST, MBP, GFP, AP or the like.
  • the polypeptide fragment of T bet may be a polypeptide containing an amino acid sequence that is identical to a part of the amino acid sequence of T bet.
  • examples of the polypeptide fragment of T bet include the same polypeptide as a part of the protein having the amino acid sequence set forth in SEQ ID NO: 4, preferably at least 6 amino acid residues (for example, 8, 10, 12, Or a polypeptide fragment containing 15 amino acid residues or more).
  • T bet polybepetit The fragment may be a mutant such as a substitution of the above polypeptide.
  • the polypeptide fragment of T bet can be produced by a conventional method.
  • the measurement of the effect of suppressing the transcriptional regulatory activity by the anti-T bet antibody can be performed by a known technique.
  • a vector containing a promoter and a reporter gene for example, luciferase, alkaline phosphatase, ⁇ -galatatosidase, etc.
  • a transcriptional regulatory region known to regulate transcriptional activity by T-beta is introduced into the cell.
  • the host cell for example, B300, CHO, BHK, COS7, NIH3 T3, HEK293, etc., which cell lines are preferred can be used.
  • the protein mass derived from the produced reporter gene for example, luciferase, alkaline phosphatase, etc.
  • the protein mass derived from the produced reporter gene for example, luciferase, alkaline phosphatase, etc.
  • ⁇ -galatatosidase etc.
  • a substance having an HB24 expression lowering action or a substance having an action of suppressing the transcriptional regulatory activity of HB24 has an angiogenesis inhibitory action and Z or antitumor action.
  • an angiogenesis inhibitory action and a Z or antitumor action are exhibited by a decrease in HB24 expression or suppression of transcriptional regulatory activity of HB24.
  • an angiogenesis inhibitor and / or an antitumor agent comprising a substance having an HB24 expression lowering action as an active ingredient.
  • an angiogenesis inhibitor and a Z or antitumor agent comprising a substance having a substance having an action of suppressing the transcriptional regulatory activity of HB24 as an active ingredient.
  • the angiogenesis inhibitor and / or the antitumor agent can also be referred to as an angiogenesis inhibiting composition and Z or an antitumor composition.
  • the angiogenesis inhibitor and the Z or antitumor agent comprise a vector for producing the polynucleotide according to the present invention as an active ingredient.
  • Inhibiting angiogenesis can be expected to have an antitumor effect such as a tumor cell growth inhibitory effect, a cancer prognosis improving effect, or a cancer recurrence preventing effect.
  • antitumor Tumors include tumor cell growth inhibitors, cancer prognostics, cancer recurrence preventives, etc.
  • Antiangiogenic and antitumor effects are based on findings such as radiographs, CT, MRI, PET, and biopsy pathologies It can be confirmed by histological diagnosis or by the value of a tumor marker.
  • angiogenesis inhibitor and a Z or antitumor agent comprising a substance having an HB24 expression-reducing action and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24 as an active ingredient (hereinafter referred to as "the blood vessel according to the present invention").
  • Anti-neoplastic and Z or anti-tumor agents may be suitable for mammals (eg, humans, rats, rabbits, hidges, pigs, ushi, cats, nu, monkeys, etc.) Can be administered.
  • the angiogenesis inhibitor and Z or antitumor agent according to the present invention can be used for the treatment or prevention of any of solid tumors and humoral tumors.
  • solid tumors include brain tumors, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, spleen cancer, gastric cancer, colon cancer (colon cancer, rectal cancer), small intestine or duodenal cancer, bladder cancer, Renal cancer, liver cancer, prostate cancer, uterine cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma (eg, osteosarcoma, chondrosarcoma, force positive sarcoma, myoma, angiosarcoma, fibrosarcoma, etc.), malignant lymphoma , And melanoma.
  • sarcoma eg, osteosarcoma, chondrosarcoma, force positive sarcoma, myoma, angiosarcoma,
  • the target disease of the angiogenesis inhibitor and Z or antitumor agent according to the present invention is preferably a solid tumor. Therefore, the angiogenesis inhibitor and Z or antitumor agent according to the present invention are preferably, for example, brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, spleen cancer, stomach cancer, colon cancer (colon cancer, direct cancer). Intestinal cancer), small intestine or duodenal cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer, ovarian cancer, thyroid cancer, gallbladder cancer, pharyngeal cancer, sarcoma (e.g.
  • osteosarcoma chondrosarcoma, force positive sarcoma, Myoma, angiosarcoma, fibrosarcoma, etc.
  • malignant lymphoma melanoma force
  • melanoma force can be used to treat or prevent at least one selected disease.
  • the administration method can be either oral or parenteral administration! /, Which can be shifted, preferably parenteral administration.
  • injection, nasal administration, pulmonary administration, trans For example, skin administration.
  • injections can be systemically or locally administered by, for example, intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and the like.
  • angiogenesis inhibitor and Z or antitumor agent according to the present invention a substance having an HB24 expression-reducing action and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24 can be used as they are, but suitable It is possible to use a product formulated with additives.
  • additives examples include excipients, binders, lubricants, disintegrants, colorants, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, etc., which are generally used in medicine.
  • examples include tonicity agents, buffering agents, preservatives, antioxidants, stabilizers, absorption promoters, and the like, and these can be used in appropriate combinations as desired.
  • Examples of the additive include the following:
  • Excipients For example, lactose, sucrose, glucose, corn starch, mannitol, sorbitol, starch, pregelatinized starch, dextrin, crystalline cellulose, light anhydrous carboxylic acid, aluminum silicate, calcium silicate, magnesium aluminate metasilicate Calcium hydrogen phosphate;
  • Binders For example, polybulal alcohol, methylcellulose, ethylcellulose, arabic gum, tragacanth, gelatin, shellac, hydroxypropylmethylcellulose, hydroxypropylcellulose, sodium carboxymethylcellulose, polybulurpyrrolidone, macrogol;
  • Lubricants For example, magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethylene glycol, colloidal silica;
  • Disintegrants for example, crystalline cellulose, agar, gelatin, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, low-substituted hydroxypropyl cellulose, canoleoxy methinorescenellose, canoleboxoxymethinole Senorelose canolecium, croscarmellose sodium, carboxymethyl starch, carboxymethyl starch sodium;
  • Coloring agents for example, ferric sesquioxide, yellow ferric oxide, carmine, caramel, ⁇ -strength rotin, titanium oxide, talc, sodium riboflavin phosphate, yellow aluminum lake, etc., which are permitted to be added to pharmaceutical products ;
  • Flavoring agent for example, cocoa powder, heart force brain, aroma powder, heart force oil, dragon brain, cinnamon powder; emulsifier or surfactant: for example, stearyl triethanolamine, sodium lauryl sulfate Thorium, laurylaminopropionic acid, lecithin, glyceryl monostearate, sucrose fatty acid ester, glycerin fatty acid ester;
  • emulsifier or surfactant for example, stearyl triethanolamine, sodium lauryl sulfate Thorium, laurylaminopropionic acid, lecithin, glyceryl monostearate, sucrose fatty acid ester, glycerin fatty acid ester;
  • Solubilizing agents for example, polyethylene glycol, propylene glycol, benzoic acid benzyl, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate, polysorbate 80, nicotinamide;
  • Suspending agent for example, in addition to the surfactant, hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose;
  • hydrophilic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose;
  • Isotonic agents for example, glucose, sodium chloride salt, mantol, sorbitol
  • Buffers for example, buffers such as phosphates, acetates, carbonates, citrates
  • Preservatives for example methylparaben, Propylparaben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid;
  • Antioxidants for example, sulfate, ascorbic acid, a-tocopherol;
  • Stabilizers for example, those commonly used in medicine
  • Absorption enhancers For example, those commonly used in medicine.
  • blend components such as vitamins and an amino acid, as needed.
  • the above preparations include tablets, powders, granules, fine granules, capsules, syrups, troches, inhalants and other oral preparations, suppositories, ointments, ophthalmic ointments, tapes. And ophthalmic preparations, nasal drops, ear drops, poultices, lotions and the like.
  • Oral preparations can be formulated by appropriately combining the above additives. You can also coat these surfaces as needed! /.
  • external preparations include excipients, binders, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, preservatives, antioxidants, among others.
  • excipients include excipients, binders, flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, preservatives, antioxidants, among others.
  • binders include flavoring agents, emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, preservatives, antioxidants, among others.
  • an appropriate combination of an agent, a stabilizer or an absorption promoter can be prepared.
  • injections are emulsifiers, surfactants, solubilizers, suspending agents, isotonic agents, buffers, preservatives, antioxidants, stabilizers, or absorption enhancers. It can be formulated by appropriately combining agents.
  • An angiogenesis inhibitor and / or an antitumor agent according to the present invention is an antisense oligonucleotide.
  • the administration method can be either oral or parenteral administration, but preferably is parenteral administration.
  • Nasal administration can be systemically or locally administered by, for example, intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and the like.
  • the dose can be appropriately selected depending on the age and symptoms of the patient.
  • the dosage is, for example, in the range of 1 to 5 times per day, and is 0.1.OOlmg / kg of body weight per kilogram of body weight, preferably 0.01 mg to 100 mg, more preferably 0. lmg ⁇ : L0 mg is mentioned.
  • the dose may be, for example, 0.001 mgZbody to 10000 OmgZbody per patient, preferably 0.1 mg to L0000 mg, more preferably 1 mg to LOOOmg.
  • the angiogenesis inhibitor and the Z or antitumor agent according to the present invention are not necessarily limited to the number and dose of these administrations.
  • the use of a substance having an action of decreasing the expression of HB24 and a substance having an action of suppressing the transcriptional regulatory activity of Z or HB24 for producing an angiogenesis inhibitor and Z or an antitumor agent Is also included.
  • the angiogenesis inhibitor and Z or antitumor agent of the present invention comprise an angiogenesis inhibitor and Z or antitumor containing a substance having an action of suppressing the transcriptional regulatory activity of HB24 as an active ingredient. It is a tumor agent.
  • the substance having an HB24 expression lowering action and the substance having an action to suppress the transcriptional regulatory activity of Z or HB24 are preferably substances having an HB24 expression lowering action and anti-HB24 antibody, more preferably an antisense oligosaccharide against HB24. Nucleotides, ribozymes for HB24, and double-stranded RNA for HB24.
  • an angiogenesis inhibition method and cancer treatment comprising administering to a patient an effective amount of an angiogenesis inhibitor and Z or an antitumor agent according to the present invention.
  • a preventive method is provided.
  • a substance having an HB24 expression lowering action or a substance having an action of suppressing the transcriptional regulatory activity of HB24 in the present invention, a polynucleotide according to the present invention, a vector according to the present invention, or An angiogenesis comprising administering to a patient an effective amount of an antibody according to the invention. Live inhibition methods and methods of treating Z or cancer are provided.
  • the substance having an HB24 expression lowering action and the substance having an action to suppress the transcriptional regulatory activity of Z or HB24 are preferably a substance having an HB24 expression lowering action and an anti-HB24 antibody, more preferably. Includes antisense oligonucleotides against HB24, ribozymes against HB24, and double-stranded RNA against HB24.
  • the administration route and administration method of the angiogenesis inhibitor and Z or antitumor agent according to the present invention are not particularly limited, but refer to the description of the angiogenesis inhibitor and Z or antitumor agent according to the present invention. be able to.
  • treatment generally means obtaining a desired pharmacological effect and Z or physiological effect.
  • the effect is prophylactic in terms of completely or partially preventing the disease and Z or symptoms and is therapeutic in terms of partial or complete cure of the adverse effects caused by the disease and Z or disease.
  • Treatment includes any treatment of disease in mammals, particularly humans, for example
  • IA to prevent a disease or symptom from occurring in a patient who may be predisposed to the disease or symptom but has not yet been diagnosed
  • IC includes alleviation of disease symptoms, ie, regression of disease or symptoms, or reversal of progression of symptoms.
  • a method for screening a substance having an angiogenesis inhibitory action and Z or antitumor action comprising: HB24 mRN
  • a method comprising at least a step of contacting a test substance with a cell expressing A and Z or HB24 protein.
  • this method further comprises the step of selecting a substance that decreases the expression of HB24 mRNA using the amount of HB24 mRNA as an index.
  • the method further comprises the step of selecting a substance that decreases the protein of HB24 as an indicator of the amount of HB24 protein.
  • cells expressing HB24 mRNA and Z or HB24 protein are reacted in the presence and absence of a test substance, and HB24 mRNA and Z or HB24 protein amount is used as an index.
  • an angiogenesis inhibitor and Z or an anti-tumor agent can be screened by selecting a test substance that reduces the expression of HB24 protein.
  • the screening standard is, for example, that the expression level of HB24 mRNA and Z or protein in the cell in the presence of the test substance is at least 10% or more, preferably 20 in the presence of the test substance compared to the absence of the test substance. % Or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more can be selected.
  • the expression levels of HB24 mRNA and protein can be measured by known techniques, such as immunological methods (eg, flow cytometry, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoassay, etc. Measurement methods (RIA), ELISA, SDS-PAGE, Western blot, etc., molecular biological analysis methods (eg, Northern plot, RT-PCR, quantitative RT-PCR, in situ hybridization, etc.) Can be performed.
  • immunological methods eg, flow cytometry, fluorescent antibody method, enzyme immunoassay (EIA), radioimmunoassay, etc.
  • Measurement methods RIA
  • ELISA enzyme immunoassay
  • SDS-PAGE Western blot
  • Western blot etc.
  • molecular biological analysis methods eg, Northern plot, RT-PCR, quantitative RT-PCR, in situ hybridization, etc.
  • the screening method further comprises a step of selecting a substance that decreases the transcriptional regulatory activity by HB24 using the transcriptional regulatory activity by HB24 as an index.
  • cells that can measure transcriptional regulatory activity by HB24 are reacted in the presence and absence of the test substance, and transcriptional regulatory activity by HB24 is reduced using transcriptional regulatory activity by HB24 as an index.
  • an angiogenesis inhibitor and Z or antitumor agent can be screened.
  • Screening criteria are, for example, the presence of the test substance compared to the absence of the test substance. Below, it is possible to select those that suppress the transcriptional regulatory activity by HB24 at least 10% or more, preferably 20% or more, more preferably 30% or more, still more preferably 50% or more, particularly preferably 70% or more. it can.
  • Measurement of transcriptional regulatory activity by HB24 can be performed by a known technique.
  • a vector containing a promoter and a reporter gene eg, luciferase, alkaline phosphatase, 13 galactosidase, etc.
  • a transcriptional regulatory region known to regulate transcriptional activity by HB24 is introduced into the cell.
  • the host cell for example, B300, CHO, BHK, COS7, NIH3T3, HEK293, etc., which cell lines are preferred can be used.
  • the amount of protein derived from the produced reporter gene eg, luciferase, alkaline phosphatase, 13 galactosidase, etc.
  • the amount of protein derived from the produced reporter gene eg, luciferase, alkaline phosphatase, 13 galactosidase, etc.
  • transcriptional regulatory activity by HB24 can be measured.
  • a screening method for a substance having an angiogenesis inhibitory action and a Z or antitumor action wherein a change in transcriptional activity by a transcriptional regulatory region of HB24 is observed.
  • a method comprising at least a step of bringing a measurable cell into contact with a test substance, and a step of selecting a substance that decreases the transcriptional activity of the transcriptional regulatory region of HB24 using the transcriptional activity of the transcriptional regulatory region of HB24 as an index.
  • cells that can measure changes in transcriptional activity in the transcriptional regulatory region of HB24 are reacted in the presence and absence of the test substance, and transcriptional activity in the transcriptional regulatory region of HB24 is used as an index to transcribe HB24.
  • an angiogenesis inhibitor and Z or an antitumor agent can be screened.
  • the screening criterion is, for example, that the transcriptional regulatory activity of the transcriptional regulatory region of HB24 in the presence of the test substance is at least 10% or more, preferably 20% or more, in the presence of the test substance. More preferably 30% or more, further preferably 50% or more, particularly preferably 70% or more can be selected.
  • the transcriptional activity in the transcriptional regulatory region of HB24 can be measured by a known technique. wear.
  • a vector containing a promoter containing a transcriptional regulatory region of HB24 and a reporter gene (for example, luciferase, alkaline phosphatase, j8-galactosidase, etc.) is introduced into cells.
  • a reporter gene for example, luciferase, alkaline phosphatase, j8-galactosidase, etc.
  • the host cell for example, B300, CHO, BHK, COS7, NIH3T3, HEK293, etc., which cell lines are preferred can be used.
  • the mass of the protein derived from the reporter gene (for example, luciferase, alkaline phosphatase, ⁇ -galatatosidase, etc.) is quantified. By doing so, transcriptional activity by ⁇ 24 can be measured.
  • the reporter gene for example, luciferase, alkaline phosphatase, ⁇ -galatatosidase, etc.
  • test substances include peptides, proteins, non-peptide compounds, synthetic compounds, polynucleotides, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, and the like. It is.
  • Polynucleotides include, for example, antisense oligonucleotides, ribozymes, double stranded RNA, and the like.
  • ⁇ 24 is preferably a polypeptide selected from the group consisting of the following (i) to (xii) forces.
  • the substance obtained by the screening method of the present invention further measures at least one activity selected from a tube formation inhibitory activity, an angiogenesis inhibitory activity and an antitumor activity.
  • the measurement of the tube formation inhibitory activity can be performed by a known technique, for example, sandwich tube formation Atsusei (Cancer Research. 2002; 62 (21): 6116-23., Clinical Cancer Research. 2004; 10 (4): 1430—8), rat aorta Tube Formation Atssey (Biol Pharm Bull. 2 002; 25 (4): 446-51, Vascular Research. 2005; 42 (2): 111-119) be able to.
  • the angiogenesis inhibitory activity can be measured by a known technique, for example, DAS atsy (Oncol Res. 1999; 11 (7): 319-29.), Matrigel Atsy (Clinical Cancer Research. 2 004; 10 (4): 1430-8.), would healing Atsey (J Biol Chem. 2004; 279 (18): 18600-7.), Dorsal skinfold chamber model (Cancer Res, 52: 6553-60, 1992) It can be carried out.
  • the antitumor activity can be measured by a known technique, for example, a cancer cell subcutaneous transplantation model (Clin Exp Metastasis. 1987; 5 (2): 135-46.), A cancer cell orthotopic transplantation model (CI Clini cal Cancer Research. 2003; 9 (15): 5532- 9.).
  • the substance obtained by the screening method of the present invention has a substance having an angiogenesis inhibitory action and Z or antitumor action. Therefore, the obtained substance is also an angiogenesis inhibitor and Z or anti-tumor containing these as an active ingredient in the same manner as the substance having an HB24 expression-reducing action or the substance inhibiting the transcriptional regulatory activity of HB24 in the present invention. It can be a tumor agent.
  • Human umbilical vein endothelial cells (hereinafter referred to as “HUVEC”) (clinically obtained), human aortic endothelial cells (obtained from Sanko Junyaku Co., Ltd., product name: Cryo HAEC), human lung microvascular endothelial cells (Sanko Junyaku) Obtained from KK, product name: Cryo HMVEC-L) VEGF (10ngZml), F GF (10ng / ml), EGF (10ng / ml) (above Invitrogen) bloodless medium for endothelial cells (Invitrogen) (Hereinafter, referred to as “growth factor-containing serum-free medium”).
  • HMVEC Human umbilical vein endothelial cells
  • Cryo HAEC human aortic endothelial cells
  • human lung microvascular endothelial cells (Sanko Junyaku) Obtained from KK, product name: Cryo HMVEC-L) VEGF (10ng
  • hemocyte cell line KG-1 (ATCC) expressing HB24 was cultured in IMDM medium containing 10% FCS.
  • RNA was extracted by the guanidine isothiocyanate method according to a conventional method.
  • Quantitative RT-PCR was performed using a Taqman Gold RT-PCR kit (Applied Biosystems) according to the accompanying Mayorole. The random hexmer included in the kit was used as a primer for cDNA synthesis, and Hs00172035 and Human ACTB (both from Applied Biosy stems) were used as the HB24 and j8-actin probes and primers, respectively.
  • the expression level of HB24 is calculated by calculating the amount of HB24 mRNA per ⁇ -actin mRNA amount for each cell, and taking the value of HB24 mRNA amount per 13-actin mRNA amount in KG-1 cells as one unit. Calculated.
  • H B24 human aortic endothelial cells, and human lung microvascular endothelial cells
  • H B24 the expression levels of H B24 were 8.5 units, 4.3 units, and 8.5 units, respectively.
  • the skin cells were found to express HB24.
  • the vector was purified from an E. coli clone (MGC-1567 O (ATCC)) containing the HLX1 (HB24) insertion vector (pOTB7) according to a conventional method.
  • the purified HLX1 insertion vector (pOTB7) is used as a cage, and primers with oligonucleotides having the sequences described in SEQ ID NO: 9 (BamHl added to the 5 'end) and SEQ ID NO: 10 (EcoRl added to the 5' end) are used as primers.
  • PCR was performed according to a conventional method. Then, cDNA corresponding to the 482th to 901st positions in the sequence described in SEQ ID NO: 1 was obtained.
  • chimeric protein was expressed in E. coli using the expression vector.
  • a central fragment of HB24 protein was obtained from the Escherichia coli using dartathione sepharose 4B (Amasham) according to the attached manual.
  • a central fragment of HB24 protein was immunized to New Zealand white rabbits to obtain a rabbit rabbit anti-HB24 antiserum.
  • the obtained Usagi anti-HB24 antiserum was subjected to ammonium sulfate precipitation and then purified on a affinity column coupled with the central fragment (antigen) of HB24 protein to obtain an anti-HB24 antibody. .
  • SEQ ID NO: 10 CG-GAATTC (EcoRl) -TTA-GTACTTCTGAATCTCAAA
  • Example 3 Expression of HB24 in vascular endothelial cells (Western plot)
  • HUVEC nuclear protein was extracted using the Nuclear extract kit (Active motiff). The amount of protein was measured using Protein assay kit II (manufactured by BioRad). Dissolve the nucleoprotein in the sample buffer and perform 10 ⁇ g / lane of SDS polyacrylamide gel electrophoresis with Nupage 7% Tris-acetate gel (Invitrogen) and transfer to PVDF membrane (Millipore) did. Next, the PVDF membrane was blocked with 5% skim milk.
  • Example 2 the anti-HB24 antibody prepared in Example 2 (10 ⁇ g / ml , 5% skim milk) at room temperature for 2 hours, and then incubated with HRP-labeled anti-rabbit IgG antibody (Amersham) for 2 hours at room temperature. Then, ECL Plus Western Blotting Dection Reagents (manufactured by Amersham) was irradiated with light and developed with Hyperfirm ECL (manufactured by Amersham dry soil).
  • vascular endothelial cells express HB24 protein.
  • the vector was purified from an E. coli clone (MGC-15670 (ATCC)) containing the HLX1 (HB24) insertion vector (pOTB7) according to a conventional method.
  • E. coli clone MCC-15670 (ATCC)
  • pOTB7 purified HLX1 insertion vector
  • the HB24 full-length cDNA is obtained. Obtained.
  • the cDNA was incorporated into pCDNA3.1 (—) (manufactured by Invitrogen) to prepare an HB24 expression vector.
  • the porcine aortic endothelial cell line (hereinafter referred to as “PAE”) is an endothelial cell line capable of infinite growth.
  • PAE porcine aortic endothelial cell line
  • the above HB24 expression vector or pcDNA3.1 (—) was transfected into PAE using Lipofectamine 2000 (Invitrogen). Two days later, drug selection with G418 (lmgZml) was carried out to obtain a HB24-expressing PAE cell line and a pcD NA3.1 (—)-introduced PAE cell line.
  • RNA was extracted by the guanidine isocyanate method according to a conventional method.
  • the amount of HB24 mRNA and the amount of Ribosomal RNA (rRNA) were measured by quantitative RT-PCR.
  • the FAM-modified oligonucleotide (Applied Biosystems) described in SEQ ID NO: 11 was used as a probe, and the oligonucleotides described in SEQ ID NO: 12 and SEQ ID NO: 13 were used as primers. Use It was.
  • Ribosomal RNA control reagents (Applied Biosystems) were used for the measurement of the amount of rRNA.
  • the expression level of HB24 was calculated for each cell by calculating the amount of HB24 mRNA per rRNA, and the value in KG-1 cells as 1 unit.
  • HB24 highly expressing PAE cell line expressing about 25 times higher HB24 mRNA than endogenous HB24 mRNA was obtained.
  • SEQ ID NO: 11 FAM-TTCCAGGTCCCTATGCTGTGCTCACG
  • SEQ ID NO: 13 TGT ACG TCT GCG GCAT GGT
  • HB24 highly expressing PAE cell line or pcDNA3.1 (—) introduced in Example 4 was plated on 96 well plates per well, and cultured in DMEM medium (100 1) containing 10% FCS. did. After confirming cell adhesion, the medium was replaced with a new medium, and immediately after (dayO) or after culturing for 1 to 3 days, Alamer Blue (manufactured by IWAKI) was added at 5 ⁇ l / well. After further incubation for 3 hours, the absorbance at a detection wavelength of 590 nm was measured under an excitation wavelength of 560 nm. The number of cells after 1 to 3 days of culturing of each cell was calculated by taking the measured value at dayO of each cell as 1, and calculating the ratio to this.
  • the HB24 highly expressing PAE cell line had a renewed growth ability compared to the pcDNA3.1 (—) introduced cell line (FIG. 1A). Therefore, it was revealed that HB24 is involved in the proliferation of vascular endothelial cells.
  • PAE cell lines or pcDNA3 HB24 high expression prepared in Example 4 PAE cell lines or pcDNA3 1.
  • (-) introduced PAE cell lines 12 ⁇ El plates coated with collagen (manufactured by IWAKI Co., Ltd.) ⁇ This 1 seeded 2Xl0 5 per Ueru, with 10% FCS The cells were cultured in DMEM medium (500 / zl) containing them until they became subconfluent. Thereafter, the medium was replaced with 0.1% BSAZDMEM, and further cultured for 24 hours until confluent. Then, pipette tip (200 1, Quality This cell was removed by pulling with the tip of Science Plastics) to form a band-like cell-free region having a width of about 0.1 mm.
  • the cells were further cultured with 0.1% BSAZDMEM. Observed with a culture microscope (manufactured by Olympus Corporation) over time, the cells on both sides migrated toward the center in the area without the band-like cells, and the condition was recorded.
  • the HB24 highly expressing PAE cell line had a faster migration rate than the pcDNA3.1 (—) introduced PAE cell line (FIG. 1B). Therefore, it was revealed that HB24 is involved in endothelial cell migration.
  • Example 6 Inhibition of vascular endothelial cells by anti-HB24 antisense oligonucleotide
  • Antisense oligonucleotide for human HB24 (hereinafter referred to as “anti-human HB24 antisense oligo”), antisense oligonucleotide for porcine HB24 (hereinafter referred to as “anti-porcine HB24 antisense oligo”), and control nonsense oligonucleotide (Hereinafter referred to as “nonsense oligo”) were oligonucleotides having the sequences described in SEQ ID NOs: 14, 15, and 16, respectively. These oligonucleotides were phosphothioate-modified, and the oligonucleotides were commissioned and synthesized by Nippon Bioservice Co., Ltd.
  • SEQ ID NO: 15 GTG TGT TGC CTT CCT TGA
  • the medium was again replaced with fresh growth factor-containing serum-free medium and HB24 antisense oligo, anti-pig HB24 antisense oligo or nonsense oligo was added at the same concentration.
  • remove the cells in this area by pulling about the center of the well with the tip of a pipette tip (2001, Quality Science Plastics).
  • a 1 mm band-free area was formed. Wash with endothelium serum-free medium (manufactured by Invitrogen) to remove debris, and again prepare growth factor-containing serum-free medium, and add anti-human HB24 antisense oligo, anti-porcine HB24 antisense oligo, or nonsense oligo.
  • the culture was continued with the same concentration added. Observation was performed with a culture microscope (manufactured by Olympus Co., Ltd.) over time, and the cells on both sides migrated toward the center and recorded the state without any band-like cells.
  • siRNA small interference double-stranded RNA
  • SEQ ID NOs: 11 to 25 are sequences containing a part of the sequence shown in SEQ ID NO: 1.
  • the oligonucleotides described and oligonucleotides having complementary sequences to each were synthesized (manufactured by Nippon Bioservice Co., Ltd.) and then annealed.
  • a control siRNA was prepared by synthesizing an oligonucleotide shown in SEQ ID NO: 26 and an oligonucleotide having a complementary sequence to each of them (Nippon Bioservice) and then annealing.
  • HUVEC was cultured in a collagen-coated 24-well plate, and the siRNA was added to each cell using Effectene (manufactured by QIAGEN) (added so as to be 20 ⁇ each in 500 ⁇ l of medium). Two days later, the mRNA level of HB24 was measured by quantitative RT-PCR in the same manner as in Example 1. When a control siRNA was introduced, the mRNA level of HB24 did not change. When this value is taken as 100%, when siRNA consisting of the sequences shown in SEQ ID NOs: 1 to 25 is prepared, the amount of HB24 mRNA is 56.1, 71.8, 43.7, 64.1, 64.8, 22.1, 29.9, 56.9, 68.7, 54.5, 46.
  • siRNA consisting of a part of IJ IJ No. 1 described in IJ No. 1 has a HB24 expression lowering effect.
  • SEQ ID NO: 17 GCC CUC CUU CUG CAU CGC A
  • SEQ ID NO: 20 AGA AGG CAA CAC GCU GAG A
  • SEQ ID NO: 21 GGC AAC ACG CUG AGA GAU C
  • SEQ ID NO: 22 CGA GGC UUC UGC AAU CCU G
  • SEQ ID NO: 23 AGG AAG CGU UCA UGG UCG C
  • SEQ ID NO: 24 GGC AAC ACG CUG AGA GAU C
  • SEQ ID NO: 25 GGA AAG GCC UGG AGA AAA G
  • SEQ ID NO: 26 UCU CUA GAU CCC AUU AAC GAG GCU U
  • SEQ ID NO: 27 GGC UUC UGC AAU CCU GAG UCC CUU A
  • SEQ ID NO: 28 CCC UUA AAC UCG AAC CCA AGA AAU U
  • SEQ ID NO: 29 CCA AGA CAC GUU UCC AGG UCC CUA U
  • SEQ ID NO: 30 CCA GCA GUC UUA GUA GCA GCA
  • Sequence number 31 AGU UCA GCA GUU CCA AGA
  • SEQ ID NO: 32 GAA CCC GGA CUU CUG GAA C
  • Example 8 Effect of HB24 siRNA on vascular endothelial cell migration
  • HUVEC-coated collagen-coated 12-well plates (IWAKI) were spread on lx10 5 per well and cultured in EGM-2 medium (500 1). Thereafter, the medium was changed to a growth factor-containing serum-free medium (see Example 1), and at the same time, the oligonucleotide described in SEQ ID NO: 22 and its complementary sequence ability siRNA also had an oligonucleotide ability or SEQ ID NO: 32 as a control.
  • SiRNA consisting of the oligonucleotide described in 1) and an oligonucleotide consisting of its complementary sequence were introduced into cells using Effectene (QIAG EN).
  • the medium was replaced with a growth factor-containing serum-free medium (see Example 1), and at the same time, the oligonucleotide described in SEQ ID NO: 22 and its oligonucleotide SiRNA with complementary sequence strength siRNA or siRNA with an oligonucleotide strength consisting of the oligonucleotide shown in SEQ ID NO: 32 and its complementary sequence as a control (added to each 1 ml of medium to 20 nM) Effectene (QIAGEN) was used to introduce the cells. After 24 hours, cells were collected with trypsin EDTA (Invitrogen).
  • type I collagen solution 5 times concentration RPMI 1640, and reconstitution buffer (made by Nitta Gelatin Co., Ltd.) are mixed at a mixing ratio of 7: 2: 1. ))
  • Restitution buffer made by Nitta Gelatin Co., Ltd.
  • Example 1 Start of three-dimensional culture. Thereafter, a growth factor-containing serum-free medium (see Example 1) was further added onto the gel, followed by culturing, and the HU over time with a culture microscope (manufactured by Olympus Corporation). The state where the VEC moved and formed a lumen was recorded.
  • a recombinant lentivirus that continuously expresses siRNA against HB24 was prepared.
  • Each of the various siRNAs against HB24 prepared in Example 7 has a relatively strong knockdown effect, and based on the sequence of the siRNA that has a relatively strong knockdown effect, also has the oligonucleotide strength described in SEQ ID NO: 33 and SEQ ID NO: 34.
  • the DNA and the double-stranded DNA consisting of the oligonucleotides described in SEQ ID NO: 35 and SEQ ID NO: 36 were consigned and synthesized (manufactured by Invitrogen).
  • a double-stranded DNA consisting of the oligonucleotides described in SEQ ID NO: 37 and SEQ ID NO: 38 based on siRNA for the luciferase gene was consigned and synthesized (manufactured by Invitorgen) (designated U numbers 33, 35, 37). 5 and the end are BamHI, and the 5 and the end of IJ numbers 34, 36 and 38 are Hind III).
  • shRNA short hairpin RNA
  • siRNA homologous to the siRNA consisting of the nucleotide and its complementary sequence is generated. These three types of double-stranded DNAs were inserted into the BamHlZHind III site, a vector for lentivirus production, which was partially modified from pLenti6ZBL OCK-it-GW / U6 (manufactured by Invitrogen).
  • Example 11 HBEC cattle with HB24 siRNA expression group Lentivirus. Existence and 3 ⁇ 4t 3 ⁇ 4
  • HB24 siRNA-expressing recombinant lentivirus A, HB24 siRNA-expressing recombinant lentivirus B or control lentivirus prepared in Example 9 was added, followed by culturing. From the 3rd day of infection, fluorescence microscopy was performed, and the majority of each HUVEC expressed GFP, confirming that these viruses were sufficiently infected. Each cell was subcultured to a plurality of dishes, and after 8 days of infection, nucleoproteins were extracted from some dishes with the same method as in Example 3, and HB2 4 protein was analyzed by Western blotting.
  • HB24 protein was expressed in HB24 siRNA expressing recombinant lentivirus A. And HB24 siRNA-expressing recombinant lentivirus B was inhibited compared to control lentivirus infection ( Figure 6A).
  • HB24 siRNA-expressing recombinant lentivirus A HBECs infected with HB24 siRNA-expressing recombinant lentivirus B or control lentivirus were spread on 2000 day on 2000 day per uel plate on 2000 days.
  • the cells were cultured in EGM-2 medium (100 1).
  • the medium was changed to a growth factor-containing serum-free medium (see Example 1), and immediately after (0 day) or after 6 days of culture, Alamer Blue (IWAKI) was added in 5 ⁇ l Zwell. After further incubation for 3 hours, the absorbance at a detection wavelength of 590 nm was measured under an excitation wavelength of 560 nm.
  • the number of cells after 6 days of culture was expressed as a percentage of the value of the group infected with the control lentivirus, with the measured value at dayO of each group being 1.
  • Luminacin D (Luminacin D) is a compound represented by the following formula (I).
  • Lumincain D is the deposited actinomycetes (Streptomycesbsp. Mer-VD1207) (Independent Administrative Agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) 305-8566))) on September 27, 1996, It was purified from the fermentation product of the strain deposited internationally under the Budapest Treaty under the accession number: FERM BP-6633 on April 5, 1999.
  • HUVEC, human aortic endothelial cells, human lung microvascular endothelial cells, and PAE were cultured in the same manner as in Example 1.
  • the medium was changed to a growth factor-containing serum-free medium (see Example 1), and 24 hours later, Luminacin D (4 / z g / ml) was added to the medium, followed by further culturing for 8 hours or 24 hours.
  • RNA was extracted in the same manner as in Example 1, and the amount of HB24 mRNA was quantified.
  • Luminacin D decreased the mRNA amount of HB24 to 5 to 10% in the case of no additive after 8 hours and 24 hours.
  • nucleoprotein was extracted in the same manner as in Example 3, and HB24 protein was analyzed by Western blotting.
  • Luminacin D decreased the amount of HB24 protein after 24 hours compared to the case of no additive force (Fig. 7).
  • Luminacin D has an action of significantly reducing the expression of HB24 in endothelial cells.
  • Example 12 since it became clear that Luminacin D suppressed the expression of HB24 in endothelial cells, the effect on migration of Luminacin D in HUVEC was investigated.
  • HUVEC were seeded 2 xlO 5 per 1 Ueru to 12 ⁇ El plate coated with collagen (IWAKI Co., Ltd.), were cultured in EGM- 2 medium (500 ⁇ 1). Thereafter, the medium was replaced with a growth factor-containing serum-free medium (see Example 1) and cultured for 24 hours. After confirming that the cells are confluent, remove the cells in this area by pinching the center of the well with the tip of a pipette tip (2001, Quality Science Plastics). A zone was formed, not a band-like cell.
  • Luminacin D (0 to 4 ⁇ g / ml) was added to the medium. Observation with a culture microscope (manufactured by Olympus Co., Ltd.) was performed over time, and the state in which cells on both sides migrated toward the center was recorded in an area without band-like cells. [0161] As a result, HUVEC migration was inhibited in a dose-dependent manner by Luminacin D supplementation (Fig. 8).
  • Luminacin D which has an HB24 expression-reducing action, also suppresses the migration of vascular endothelial cells.
  • Type I collagen mixture (see Example 9) 250 1 was added to a 12 well plate and allowed to gel by incubation at 37 ° C for 4 hours. On top of that, 5 ⁇ 20 ⁇ 20 per well were cultured with HUVEC together with a growth factor-containing serum-free medium (see Example 1) and further cultured for 12 to 24 hours. After confirming that the cells have become confluent on the gel, remove the supernatant, place the type I collagen mixture, 250 1 gently on it, and incubate at 37 ° C for 4 hours to allow gelation. (Start of three-dimensional culture). Thereafter, growth factor-containing serum-free medium (see Example 1) was added onto the gel, and at the same time, Luminacin D (0-4 ⁇ g / ml) was added. The culture was continued, and the state of HUVEC moving and forming a lumen over time was recorded with a culture microscope (manufactured by Olympus Corporation).
  • HUVEC had a mesh structure by 24 hours after the start of three-dimensional culture in unattached cages, and a lumen was formed by 2 days later.
  • HUVEC suppressed the tube formation and the cells remained paved (Fig. 9). Therefore, it has been clarified that Luminacin D, which has an effect of reducing HB24 expression, also suppresses the formation of luminal endothelial cells.

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Abstract

L’invention concerne un inhibiteur d’angiogenèse et/ou un agent anti-tumoral contenant, en tant que principe actif, une substance qui présente un effet de réduction de l’expression de HB24. L’invention concerne également un inhibiteur d’angiogenèse et/ou un agent anti-tumoral contenant, en tant que principe actif, une substance qui présente un effet inhibant l'activité de régulation transcriptionnelle de HB24. Elle concerne, en outre, un procédé de criblage d’un nouvel inhibiteur d’angiogenèse utile et/ou d’un agent anti-tumoral qui comprend la sélection d’une substance présentant un effet de réduction de l’expression de HB24 et/ou d’une substance présentant un effet d’inhibition de l’activité de régulation transcriptionnelle de HB24. Ainsi, il devient possible de fournir un nouvel inhibiteur d’angiogenèse et/ou un agent anti-tumoral.
PCT/JP2006/319242 2005-09-28 2006-09-28 Substance inhibant l’expression de hb24 ou son activité de régulation transcriptionnelle, son utilisation, et procédé de criblage de la substance WO2007037299A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013080999A1 (fr) 2011-11-29 2013-06-06 興和株式会社 Agent destiné à inhiber l'expression de l'arnm de npc1l1 et/ou de lipg et agent destiné à prévenir et/ou traiter l'obésité

Citations (1)

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Publication number Priority date Publication date Assignee Title
JP2005519889A (ja) * 2001-12-28 2005-07-07 エーザイ株式会社 ルミナシン類似物およびそれら用途

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JP2005519889A (ja) * 2001-12-28 2005-07-07 エーザイ株式会社 ルミナシン類似物およびそれら用途

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DEGUCHI Y. ET AL.: "A diverged homeobox gene is involved in the proliferation and lineage commitment of human hematopoietic progenitors and highly expressed in acute myelogenous leukemia", BLOOD, vol. 79, no. 11, 2002, pages 2841 - 2848, XP002053236 *
DEGUCHI Y. ET AL.: "Stable expression of HB24, a diverged human homeobox gene, in T lymphocytes induces genes involved in T cell activation and growth", J. BIOL. CHEM., vol. 267, no. 12, 1992, pages 8222 - 8229, XP003011085 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013080999A1 (fr) 2011-11-29 2013-06-06 興和株式会社 Agent destiné à inhiber l'expression de l'arnm de npc1l1 et/ou de lipg et agent destiné à prévenir et/ou traiter l'obésité

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