WO2003083117A2 - Gene regulant la vascularisation - Google Patents

Gene regulant la vascularisation Download PDF

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Publication number
WO2003083117A2
WO2003083117A2 PCT/JP2003/004163 JP0304163W WO03083117A2 WO 2003083117 A2 WO2003083117 A2 WO 2003083117A2 JP 0304163 W JP0304163 W JP 0304163W WO 03083117 A2 WO03083117 A2 WO 03083117A2
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Prior art keywords
vegf
protein
expression
vegf receptor
activity
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PCT/JP2003/004163
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English (en)
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WO2003083117A3 (fr
Inventor
Hiromitsu Nagumo
Akio Matsuda
Toshinori Sugahara
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Asahi Kasei Pharma Corporation
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Priority claimed from JP2002098679A external-priority patent/JP2006166702A/ja
Application filed by Asahi Kasei Pharma Corporation filed Critical Asahi Kasei Pharma Corporation
Priority to AU2003226444A priority Critical patent/AU2003226444A1/en
Publication of WO2003083117A2 publication Critical patent/WO2003083117A2/fr
Publication of WO2003083117A3 publication Critical patent/WO2003083117A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a protein having an activity of promoting expression of VEGF and/or VEGF receptor, a DNA encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein.
  • the present invention also relates to use of the protein, DNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive expression of VEGF and/or VEGF receptor or the inhibition of this expression.
  • the present invention also relates to a method for screening a substance capable of inhibiting or promoting expression of VEGF and/or VEGF receptor by using the protein, DNA, recombinant vector and transformant.
  • vascularization means the formation of a fresh vascular plexus after processes; such as digestion or disruption of basal membranes by protease in existing blood vessels, migration, proliferation or adhesion to extracellular matrices of vascular endothelial cells, and lumen formation caused by vascular endothelial cell differentiation.
  • processes such as digestion or disruption of basal membranes by protease in existing blood vessels, migration, proliferation or adhesion to extracellular matrices of vascular endothelial cells, and lumen formation caused by vascular endothelial cell differentiation.
  • vascularization a phenomenon of constructing a fresh vascular system, is involved in the onset or development of many diseases. For example, in order for a solid tumor to grow, it is essential to supply oxygen or nutrients and remove wastes through vascularization. Also, vascularization plays an important role in the metastasis of cancer cells. Furthermore, in the case of diabetic retinopathy, neovascularity disrupts retinal tissue, and becomes a major cause of acquired blindness. Therefore, the inhibition of pathologic vascularization has been considered to lead to the treatment or prevention of the above diseases, and at present several vascularization inhibitors are being studied in terms of clinical efficacy.
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • VEGF is a representative growth factor to promote in vivo vascularization.
  • VEGF is a specific vascular growth factor which regards almost only vascular endothelial cells as targets (Ferrara et al., Endocr. Rev., 13:18-32 (1992), Dovorak et al., Am. J. Pathol., 146:1029-1039 (1995), Thomas et al., J. Biol. Chem., 271:603-606 (1996)).
  • VEGF is involved in normal vascularization in growing tissues such as in fetus growth (Peter et al., Proc Natl. Acad. Sci.
  • VEGF and KDR are essential for vascularization, and the elucidation of an expression control mechanism for these protein molecules provides a very important means for the development of novel pharmaceuticals or treatment methods of pathologic vascularization or ischemic diseases. This has an extremely important significance.
  • VEGF has a sequence at the upstream of its gene, which is called the "Hypoxia responsible element (HRE)" and expresses promoter activity in response to hypoxia, and it is known that this promoter has an important role in the VEGF expression (Forsythe et al, Mol. Cell. Biol. 16:4604-4613 (1996)).
  • HRE Hapoxia responsible element
  • the object of the present invention is to identify a new gene and protein useful as mentioned above, which has an activity of promoting vascularization, and to provide a method of use of them in medicaments, diagnostics and therapy. That is, an object of the present invention is to provide a new protein having an activity of promoting expression of VEGF and/or VEGF receptor, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
  • Another object of the present invention is to provide a method for screening a substance capable of inhibiting or promoting expression of VEGF and/or VEGF receptor using the protein, the DNA, the recombinant vector or the transformant, a kit for the screening, a substance capable of inhibiting or promoting expression of VEGF and/or VEGF receptor which is obtained by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or promoting expression of VEGF and/or VEGF receptor.
  • the present inventors have intensively studied to solve the above problems. As a result, the present inventors have succeeded in constructing a full-length cDNA library by using the oligo-capping method; establishing a gene function assay system by expression cloning using ECV304 cells; and isolating a new DNA (cDNA) encoding a protein having an activity of promoting expression of VEGF and/or VEGF receptor by using the assay system.
  • This new DNA molecule induced an activity of promoting expression of VEGF and/or VEGF receptor by its expression in ECV304 cells.
  • This result shows that this new DNA is a signal transduction molecule involved in the promotion of expression of VEGF and/or VEGF receptor.
  • the present invention has been completed based on these findings.
  • the present invention provides the followings:
  • a purified protein selected from the group consisting of:
  • a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76;
  • VEGF Vascular endotherial growth factor
  • a purified protein that has an activity of promoting expression of VEGF and/or VEGF receptor and comprises an amino acid sequence having at least 95% identity to the protein according to (1) over the entire length thereof.
  • a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76;
  • VEGF Vascular endotherial growth factor
  • a polynucleotide sequence which encodes a protein that has an activity of promoting expression of VEGF and/or VEGF receptor and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75.
  • a polynucleotide sequence which encodes a protein that has an activity of promoting expression of VEGF and/or VEGF receptor and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75.
  • An isolated polynucleotide comprising a polynucleotide sequence which encodes a protein that has an activity of promoting expression of VEGF and/or VEGF receptor and has at least 95% identity to the polynucleotide sequence according to (3) over the entire length thereof.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that has an activity of promoting expression of VEGF and/or VEGF receptor and has at least 95% identity to the polynucleotide sequence according to (4) or (5) over the entire length thereof.
  • a recombinant vector which comprises a polynucleotide according to any one of (3) to (7).
  • a agent for gene therapy which comprises the recombinant vector according to (9) as an active ingredient.
  • a membrane of the cell according to (11) which has the protein according to (1) or (2), which is a membrane protein.
  • a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of (1), (2) or (8) in a subject comprising the steps of:
  • a method for screening compounds having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor which comprises the steps of:
  • a method for screening compounds having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor which comprises the steps of:
  • a compound having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor which is selected by the method for screening according to (15) or (16).
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a kit for screening a compound having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor which comprises:
  • a transformed cell comprising a gene encoding a protein that promotes expression of VEGF and/or VEGF receptor according to (1), (2) or (8) and a gene encoding a signal which can detect promotion of expression of VEGF and/or VEGF receptor;
  • (21) A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to (1), (2) or (8).
  • An antisense oligonucleotide having a sequence complementary to a part of the polynucleotide according to any one of (3) to (7), which prevents the expression of a protein which promotes expression of VEGF and/or VEGF receptor.
  • a ribozyme or deoxyribozyme capable of inhibiting an activity of promoting expression of VEGF and/or VEGF receptor which has an action of cleavage of RNA that encodes the protein according to (1), (2) or (8) or an action of cleavage of RNA that encodes a protein which promotes expression of VEGF and/or VEGF receptor
  • a method for treating a disease associated with abnormal expression of VEGF and VEGF receptor which comprises administering to a subject a compound screened by the process according to (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22), and/or an antisense oligonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25) in an effective amount to treat a disease selected from the group consisting of solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, and Buerger's disease.
  • a disease selected from the group consisting of solid tumors, tumor metastasis, inflammations, ps
  • a method of treating solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with an activity of inhibiting or pomoting expression of VEGF and/or VEGF receptor.
  • (31) A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22) as an active ingredient.
  • a pharmaceutical composition which comprises an antisense oligonucleotide according to (24) as an active ingredient.
  • a pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
  • a pharmaceutical composition or a gene therapy agent which comprises a double strand RNA according to (26) or a vector capable of expressing said double strand RNA, an active ingredient.
  • composition according to any one of (29) to (31) for the treatment and/or prevention of a disease which is selected from the group consisting of solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76
  • a method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to (36) with data of said other nucleotide sequences and/or amino acid sequences.
  • polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76, are fixed.
  • a full-length cDNA was produced from mRNA prepared from human normal umbilical vein endothelial cell (purchased from Sanko Junyaku Co., Ltd.) by using the oligo-capping method, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864).
  • the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone.
  • the pGL3HRE-Luc reporter plasmid containing HRE (Hypoxia responsible element), a major promoter of VEGF, at the uptream of a gene encoding luciferase
  • the pGL3KDR-Luc reporter plasmid containing 5' -region of KDR gene which is VEGF receptor at the uptream of a gene encoding luciferase, and the above full-length cDNA plasmid were cotransfected into ECV302 cells (Invitrogen).
  • luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA) was selected (the selected plasmid showed a 2-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was determined. It can be expected that the protein encoded by the cDNA thus obtained may be highly possibly a signal transduction molecule involved in promotion of vascularization.
  • a protein which promotes expression of VEGF and/or VEGF receptor and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in the above amino acid sequences;
  • VEGF vascular endothelial growth factor
  • VEGF receptor means receptors which transmit a signal of VEGF to the inside of the cell upon binding with VEGF so as to promote vascularization.
  • VEGF receptors are usually present on cell membranes, and examples thereof include tyrosine kinase type receptor, KDR, present on the membrane of vascular endotjelial cells.
  • promotion of expression of VEGF and/or VEGF receptor used herein broadly means that the expression level of VEGF and/or VEGF receptor in cells is promoted at gene level and/or protein level.
  • identity used herein is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)).
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990)
  • Altschul SF Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990)
  • Altschul SF Madden TL
  • Schaffer AA Zhang Z, Miller W, Lipman DJ
  • the expected value is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
  • the Examples described below demonstrate that the protein consisting of an amino acid sequence of any one of the above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76 has an activity of promoting expression VEGF and/or VEGF receptor.
  • the present invention further provides the following isolated polynucleotides:
  • a polynucleotide comrising a nucleotide sequence which encodes a protein having an activity of promoting expression VEGF and/or VEGF receptor, and comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences;
  • a polynucleotide comprisng a nucleotide sequence which encodes a protein having an activity of promoting expression VEGF and/or VEGF receptor, and having an amino acid sequence which has at least 95% identity, preferably, at least 97-99% identity, to the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76.
  • Polynucleotides which are identical or almost identical to nucleotide sequences contained in the above nucleotide sequences may be used as hybridization probes to isolate full-length cDNA and genomic clones encoding the protein of the present invention, or cDNA or genomic clones of other genes that have a high sequence similarity to the above sequences, or genomic clones, or may be used as primers for nucleic acid amplification reactions.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences.
  • the probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
  • the polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA.
  • the polynucleotide may be single- stranded or double-stranded.
  • the double-stranded polynucleotides may be double- stranded DNA, double-stranded RNA or DNA:RNA hybrid.
  • the single- stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
  • a protein having the same activity that promotes expression of VEGF and/or VEGF receptor as the protein having an amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76, by means of appropriate substitution of an amino acid in the protein using known methods.
  • One such method involves using conventional mutagenesis procedures for the DNA encoding the protein.
  • Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.).
  • the present invention also includes a mutated protein having an activity of promoting expression of VEGF and/or VEGF receptor which has at least one amino acid deletion, substitution or addition relative to the protein of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76, and the DNA encoding the protein.
  • the number of mutations is preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3.
  • substitutions of amino acids are preferably conservative substitutions, specific examples of which are substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
  • DNA e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75
  • a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76 or a fragment thereof
  • those skilled in the art can routinely isolate a DNA with a high sequence similarity to these nucleotide sequences by using hybridization techniques and the like, and can obtain proteins having the same activity of promoting expression of VEGF and/or VEGF receptor as the protein having of an amino acid sequence of any one of SEQ ID NO
  • the present invention also includes a protein that has an activity of promoting expression of VEGF and/or VEGF receptor and comprises an amino acid sequence having a high identity to the amino acid sequence of any one of the above SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76.
  • High identity refers to an amino acid sequence having an identity of at least 95%, preferably at least 97 to 99% over the entire length of an amino acid sequence expressed by any one of the above SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76.
  • the proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques.
  • the protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
  • proteins which are encoded by the genes of the present invention include secretion proteins (growth factors, cytokines, hormones, and the like), protein modification enzymes (protein kinase, protein phosphatase, protease, and the like), signal transduction molecules (protein-protein interaction moleculaes and the like), nuclear proteins (nuclear receptor, transcription factors and the like), and membrane proteins .
  • the membrane proteins include receptors, cell adhesion molecules, ion channels, and transporters.
  • the present invention also includes a polynucleotide encoding the above protein of the present invention.
  • nucleotide sequences encoding a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76 include nucleotide sequences of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75.
  • the DNA includes cDNA, genomic DNA, and chemically synthesized DNA.
  • at least one nucleotide in the nucleotide sequence encoding a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76 can be substituted with other nucleotides without altering the amino acid sequence of the protein produced from the gene. Therefore, the DNA sequences of the present invention also include nucleotide sequences altered by substitution based on the degeneracy of the genetic code. Such DNA sequences can be synthesized using known methods.
  • the DNA of the present invention includes a DNA which encodes a protein having an activity of promoting expression of VEGF and/or VEGF receptor and hybridizes under stringent conditions with the DNA sequence of the above nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75, or any complementary sequence thereof.
  • Stringent conditions are apparent to those skilled in the art, and can be easily attained in accordance with various laboratory manuals such as T. Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory 1982, 1989.
  • stringent conditions refer to overnight incubation at 37 °C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate), 5 x Denhardt's solution, 0.5% SDS, 100 ⁇ g/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 1 x SSC, 0.1% SDS for 10 minutes at 37 °C (low stringency).
  • Preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 42 °C (moderate stringency). Most preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency).
  • the DNA sequence thus obtained must encode a protein having an activity of promoting expression of VEGF and/or VEGF receptor.
  • the present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein having an activity of promoting expression of VEGF and/or VEGF receptor and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5).
  • these nucleotide sequence are 95% identical, preferably 97% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
  • the above DNA of the present invention can be used to produce the above protein using recombinant DNA techniques.
  • the DNA and peptide of the present invention can be obtained by:
  • Techniques for cloning the DNA encoding the protein of the present invention in the above step (A) include, in addition to the methods described in the specification of the present application, PCR amplification using a synthetic DNA having a part of the nucleotide sequence of the present invention (e.g., any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75) as a primer, and selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or full coding region of the protein of the present invention or a labeled synthetic DNA.
  • a synthetic DNA having a part of the nucleotide sequence of the present invention e.g., any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
  • RNAs or mRNA fractions prepared from cells or tissues can be direct amplification from total RNAs or mRNA fractions prepared from cells or tissues, using the reverse transcriptase polymerase chain reaction (RT-PCR method).
  • RT-PCR method reverse transcriptase polymerase chain reaction
  • a DNA inserted into a suitable vector for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used.
  • the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker.
  • the DNA thus obtained may have a nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75, or a polynucleotide of above items (3) to (7).
  • the DNA sequence to be inserted into an expression vector in the above step (B) may be a full-length cDNA or a DNA fragment encoding the above full-length protein, or a DNA fragment constructed so that it expresses a part thereof.
  • the present invention also provides a recombinant vector, which comprises the above DNA sequence.
  • the expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
  • Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses so long as such vectors are replicable.
  • the vectors should be selected to be compatible with microorganisms or cells used as hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
  • Plasmid vectors compatible with these microorganisms are generally used as replicable expression vectors for recombinant DNA molecules.
  • the plasmids pBR322 and pBR327 can be used to transform E. coli.
  • Plasmid vectors normally contain an origin of rephcation, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA.
  • promoters include a ⁇ -lactamase promoter, lactose promoter and tryptophan promoter.
  • marker genes include an ampicillin resistance gene, and a tetracycline resistance gene.
  • suitable expression vectors include the plasmids pUC18 and pUC19 in addition to pBR322, ⁇ BR327.
  • YEp24 can be used as a replicable vector.
  • the plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene.
  • promoters in expression vectors for yeast cells include promoters derived from genes for 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
  • promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin.
  • suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
  • promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter.
  • expression vectors which are suitable for insect cells include baculo virus vector.
  • Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as an origin of replication, a promoter to be placed upstream of the DNA of the present invention, a ribosome-binding site, a polyadenylation site and a transcription termination sequence.
  • Such functional sequences which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses and viral substances.
  • Examples of such functional sequences include an SR promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR ⁇ promoter can be preferably used.
  • any promoters can be used so long as they are suitable for use in the above host-vector systems.
  • origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed.
  • Suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.
  • the present invention also provides a transformed cell, which comprises the above recombinant vector.
  • Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector as memtioned above. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
  • any of prokaryotes e.g., E. coli
  • microorganisms e.g., yeast and fungi
  • insect and animal cells can be used so long as such hosts are compatible with the expression vectors used.
  • microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coli JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E.
  • yeast such as Salmonella typhimurium or Serratia marcescens
  • various strains belonging to the genus Pseudomonas include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • yeast examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • fungi examples include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
  • insect cells for example, Spodoptera frugiperda (Sf cells), High FiveTM cells derived from eggs of Trichoplusiani, etc.
  • Sf cells Spodoptera frugiperda
  • High FiveTM cells derived from eggs of Trichoplusiani, etc. can be used when the virus is AcNPV
  • animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells.
  • CHO cells and HEK 293 cells are preferred.
  • combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
  • Transformation of hosts in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form.
  • Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, lipofection, etc.
  • selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated.
  • transformants can be selected using the above selectable marker as an indication of successful transformation.
  • the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced.
  • the present invention also provides a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein, and recovering the protein from the culture (that is, cells or culture medium).
  • animal cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.).
  • animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended.
  • attachment culture such as Petri dish culture, multitray type culture and module culture
  • suspension culture in which productive cells themselves are suspended.
  • media for use in the culture include media commonly used for animal cell culture, such as D-MEM and RPMI 1640.
  • suitable combinations of per se known separation and purification methods can be used.
  • methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing.
  • a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
  • the protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention.
  • any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein.
  • proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His).
  • GST glutathione S-transferase
  • 6 x His hexa-histidine sequence
  • membrane protein When the protein of the present invention is a membrane protein, a transformed cell into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane.
  • the membrane which is prepared from such transformed cells and contains the protein of the present invention is also included within the present invention.
  • membrane of a cell includes cell membrane, and membrane of cell organelle.
  • the membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformed cells are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads.
  • the obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare a membrabe fraction.
  • the condition for ultracentrifugation can be suitably selected depending on the type of membrane and the like.
  • the present invention also includes a protein capable of inhibiting the activity of the protein of the present invention.
  • proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
  • the present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody.
  • “specifically” means that closs-reactivity is low, more preferably closs-reactivity is not present.
  • the antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention.
  • examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies.
  • Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab') 2 fragments, Fab' fragments, Fab fragments and Fv fragments.
  • a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) or epitope-bearing fragments as an antigen to a non-human animal.
  • the antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera.
  • Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • polyclonal antibodies for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected.
  • the polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • mice In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting hybrid cells (i.e. hybridoma cells), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma cells can be implanted intraperitoneally into mice, the ascitic fluid recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • a reagent such as polyethylene glycol.
  • the resulting antibody When the resulting antibody is used to administer it to humans, it is preferably used as a humanized antibody or human antibody in order to reduce its immunogenicity.
  • the humanized antibody can be produced using transgenic mice or other mammals.
  • Humanized chimeric antibodies can be produced by linking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other than a complementarity-determining region (CDR) from a mouse monoclonal antibody. In addition, human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
  • these antibodies can be used to purify the protein of the present invention from a cell extract or transformed cells producing the protein of the present invention.
  • These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems. These assay systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human.
  • a disease characterized by undesirable vascularization resulting from (expression) abnormality of the protein of the present invention, such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, and Buerger's disease.
  • a standard value that is, a normal value for the expression of the protein of the present invention
  • a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention).
  • the presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention.
  • These antibodies can also be used as reagents for studying functions of the protein of the present invention.
  • the antibody of the present invention can be used as a medicament as mentioned below.
  • an antibody capable of inhibiting the activity of promoting expression of VEGF and/or VEGF receptor possessed by the present invention that is, neutralizing antibody.
  • the antibodies of the present invention can be purified and then administered to patients of a disease characterized by undesirable vascularization resulting from (expression) abnormality of the protein of the present invention, such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, and Buerger's disease.
  • the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and a method for therapy and/or prevention using the antibody of the present invention.
  • the active ingredient may be combined with other therapeutically or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and excipients.
  • active ingredients or inactive ingredients e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants
  • physiologically non-toxic stabilizers and excipients e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants
  • physiologically non-toxic stabilizers and excipients e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants
  • Administration to a patient can be intra-arterial administration, intravenous administration and subcutaneous adniinistration, which are well known to those skilled in the art.
  • the dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art.
  • These antibodies exhibit therapeutic activity by inhibiting undesirable vascularization mediated by the protein of the present invention.
  • the antibody of the present invention is useful as a medicament for treating or preventing a disease associated with expression abnormality of VEGF and VEGF receptor such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, and Buerger's disease.
  • a disease associated with expression abnormality of VEGF and VEGF receptor such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, and Buerger's disease.
  • the DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes.
  • the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention from cDNA or genome DNA library.
  • prey a protein which can associate with the protein of the present invention from cDNA or genome DNA library.
  • proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention.
  • the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein to isolate a protein which can associate with the protein of the present invention.
  • the present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
  • the diagnostic assays offer a process for diagnosing diseases or deterrnining a susceptibility to the diseases through detection of mutation in a gene for the protein of the present invention which has an activity of promoting expression of VEGF and/or VEGF receptor.
  • diseases may be diagnosised by analyzing expression level of the gene in a sample derived from a subject at protein or mRNA level, and detecting an abonormally decreased or increased level of the expression.
  • Determination of the presence or absence of a mutation in the gene encoding the protein of the present invention which has an activity of promoting expression of VEGF and/or VEGF receptor may involve RT-PCR using a part of the nucleotide sequences of genes as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation.
  • PCR-SSCP Genetics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
  • Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR and methods such as RNase protection assay, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays.
  • the antibody of the present invention mentioned above can be used.
  • the degree of abnormality of expression level of gene in a sample is not particularly limited.
  • the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the mutation may cause a disease associated with the control of expression of VEGF and/or VEGF receptor.
  • the abnormal expression of the novel protein of the present invention which has an activity of promoting expression of VEGF and/or VEGF receptor may be responsible for diseases associated with the expression of VEGF and/or VEGF receptor.
  • the present invention also relates to a method for screening compounds which inhibit or promote expression of VEGF and/or VEGF receptor by the protein of the present invention.
  • the method for screening comprises the steps of:
  • an activator compound a compound that increases said detectable signal 2-fold or higher than normal
  • an inhibitor compound a compound that decreases said detectable signal half or less than normal
  • genes encoding a signal which can detect promotion of expression of VEGF and/or VEGF receptor include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest to be tested. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • Any peptide or protein can be used so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA) of the reporter genes. For example, enzymatic activity of chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase, etc., can be measured.
  • the reporter plasmids which is used to evaluate promotion of expression of VEGF and/or VEGF receptor include those obtained by inserting a major promoter of VEGF, HRE, into the upstream of a reporter gene and those obtained by inserting a KDR upstream sequence into the upstream of a reporter gene, and these reporter plasmids can be introduced into a host.
  • Any host cells may be used so long as promotion of expression of VEGF and/or VEGF receptor can be detected in the host cells.
  • Preferred host cells are mammalian cells such as ECV304 cells. Transformation and culture of the cells can be carried out as described above.
  • the method for screening a compound which inhibits or promotes expression of VEGF and/or VEGF receptor comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • test compounds include, but not limited to, low molecular weight compounds and peptides.
  • Test compounds may be artificially synthesized compounds or naturally occurring compounds.
  • Test compounds may be a single compound or mixtures.
  • Usable examples includes a library of low molecular weight compounds, a compound library which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product.
  • the test substance which shows an activity of inhibiting or promoting expression of VEGF and or VEGF receptor can be further screened to isolate a single substance having the activity. Isolation and purification of a desired compound from a mixture can be carried out by using any knonw method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
  • the method for screening according to the present invention can be carried out by the following steps:
  • the expression of VEGF and/or VEGF receptor can be measured by ELISA assay or western blotting assay using an antibody which detects VEGF or VEGF receptor, instead of using a reporter gene assay as a method for detecting a detectable signal.
  • the present invention further provided a method of producing a pharmaceutical composition, which comprises the following steps (a) to (e):
  • step (d) of the method of producing a pha ⁇ naceutical composition it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal half or less than normal.
  • the protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
  • the present invention also provides a compound which is selected by the above screening method.
  • This compound has an activity of inhibiting or promoting expression of VEGF and or VEGF receptor. More specifically, this compound has an activity of inhibiting or promoting expression of VEGF and or VEGF receptor which is promoted by the protein of the present invention. Since the compounds obtained by the above screening methods have an activity of inhibiting or promoting expression of VEGF and or VEGF receptor, they are useful as therapeutic or preventive pharmaceuticals for the treament of diseases resulting from unfavorable expression of VEGF and/or VEGF receptor.
  • a compound which is obtained in the form of a salt can be purified as it is.
  • a compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dispersing or dissolving the compound into a suitable solvent and then adding a desired acid or base.
  • Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition include methods of formulating according to ordinary processes such as the following.
  • the above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed.
  • a form of formulation suitable for the mode of administration is selected.
  • a composition suitable for oral adrninistration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion.
  • a form useful for parenteral administration includes sterile solution, dispersion, emulsion and suspension.
  • the above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as com, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil,alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol.
  • examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
  • the present invention also provides a kit for screening compounds having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor.
  • the kit comprises: (a) a transformed cell comprising a gene encoding a protein that promotes expression of VEGF and/or VEGF receptor according to the present invention and a gene encoding a signal which can detect promotion of expression of VEGF and/or VEGF receptor; and (b) reagents for measuring the signal.
  • the kit comprises reagents necessary for screening compounds having an activity of inhibiting or promoting expression of VEGF and/or VEGF receptor.
  • the present invention relates to a diagnostic kit which comprises:
  • a polynucleotide of the present invention having a nucleotide sequence expressed by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75;
  • a protein of the present invention having an amino acid seqeunce expressed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76, or a fragment thereof; or
  • a kit comprising at least one of (a) to (d) is useful for diagnosing a disease or susceptibility to a disease such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • a disease or susceptibility to a disease such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • Bexause vascularization is involved in various pathologic conditions such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease, it is an attractive target for drug design and therapeutic intervention.
  • the present invention also relates to a method of using a compound which inhibits the activity of the protein having an activity of promoting expression of VEGF and/or VEGF receptor as mentioned above, for inhibiting an abnormal vascularization.
  • the compound obtained by the above screening method which inhibits expression of VEGF and/or VEGF receptor, is useful as a medicament to treat or prevent diseases characterized by undesirable vascularization such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • diseases characterized by undesirable vascularization such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • Vascularization is known to sometime improve certain clinical conditions of ischemia, and a compound which promotes expression of VEGF and/or VEGF receptor is expected to hava an effect of treating myocardial infarction, cerebral infarction, vascular dementia, arteriosclerosis obliterans and the like.
  • the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • Gene therapy refers to administering into the human body a gene or a cell into which a gene has been introduced for the purpose of therapy of diseases.
  • the protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes.
  • the present invention provides a agent for gene therapy which comprises a gene encoding the protein of the present invention.
  • the present invention provides a vector for gene therapy which expresses double strand RNA having a gene sequence encoding the protein of the present invention.
  • the form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer.
  • the pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent.
  • the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome.
  • the agent for gene therapy may be applied using a catheter.
  • the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
  • the dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 ⁇ g/kg to about 1000 mg/kg, more preferably about 10 ⁇ g/kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult.
  • the number of administration is not particularly limited.
  • the compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures.
  • the formulations thus obtained are safe and of low toxicity, and can be administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys). Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection.
  • the dosage and administration mode may vary with the weight and age of the patient, but those skilled in the art can appropriately select suitable administration mode and can appropriately select suitable dosage depending on the administration mode.
  • the compound can be encoded by DNA
  • the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out.
  • the present invention relates to a medicament which comprises the above-mentioned compound.
  • the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • the present invention also relates to a medicament comprising the above mentioned compound for tumors, inflammations, ischemia disease and the like.
  • the compound is useful as a therapeutic and/or prophylactic drug against, for example, solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • solid tumors tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • the present invention also relates to the use of the above compound for manufacturing a medicament for the therapy and/or prevention of the diseases characterized by undesirable vascularization, such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • undesirable vascularization such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • the present invention also provides an antisense oligonucleotide against the polynucleotide of any one of above items (3) to (7).
  • An antisense oligonucleotide refers to an oligonucleotide complementary to the target gene sequence.
  • the antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions.
  • the antisense oligonucleotide may be RNA or DNA.
  • the DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention.
  • an antisense ohgonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No.4 (1994)).
  • the oligonucleotide containing an antisense coding sequence against a gene encoding the protein of the present invention can be introduced into a cell by standard methods.
  • the oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
  • the antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.10, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicien) Vol. 14, No. 4 p85-95(1996)].
  • the oligonucleotide may have modified sugar moieties or inter- sugar moieties.
  • modified forms include phosphothioates and other sulfur-containing species used in the art.
  • at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
  • Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure.
  • the antisense oligonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturally occurring purine and pyrimidine.
  • the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-alkyl and 2'-halogen substituted nucleotides.
  • sugar moieties at their 2-position examples include OH, SH, SCH 3 , OCH 3 , OCN or O(CH 2 ) n CH 3 , wherein n is 1 to about 10, and other substituents having similar properties. All the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
  • the antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 20 nucleotides.
  • the oligonucleotide of the present invention can be produced by the well-known solid phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other oligonucleotides such as phosphothioates can also be produced by methods known in the art.
  • the antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention.
  • Those skilled in the art can easily design an antisense oligonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.10 pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)).
  • antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initaiation site, are most effective for the inhibition of the expression of a gene.
  • the length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search.
  • the evaluation of whether the designed antisense oligonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
  • Another method includes the triple helix technique. This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage.
  • the oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention. Such RNA, DNA and oligonucleotide can be produced using known synthesizers.
  • the antisense oligonucleotide may be introduced into the cells containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, electroporation,lipofection, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses.
  • An antisense oligonucleotide expression vector can be prepared using a suitable retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
  • the DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique to inhibit vascularization mediated by the protein of the present invention.
  • the antisense oligonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable vascularization, such as solid tumors, tumor metastasis, inflammations, psoriasis, chronic rheumatoid arthritis, hemangioma, diabetic retinopathy, angiofibroma, macular degeneration, myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, arteriosclerosis obliterans, or Buerger's disease.
  • the present invention also provides a medicament which comprises the above antisense oligonucleotide as an active ingredient.
  • the antisense oligonucleotide can also be used to detect such diseases using northern hybridization or PCR.
  • the present invention also provides a ribozyme or deoxyribozyme which inhibits expression of VEGF and/or VEGF receptor.
  • a ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA).
  • the ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention).
  • the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed.
  • Such ribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention.
  • the level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
  • ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme.
  • hammerhead ribozymes or deoxyribozymes have been well studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Iida et al., Saibou Kougaku Vol.16, No.3, ⁇ .438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)].
  • the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II forming a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites.
  • the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res.
  • the suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994).
  • the ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
  • the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymatically synthesize an RNA molecule in vitro.
  • a suitable RNA polymerase promoter e.g., T7 or SP6
  • Such ribozymes can be introduced into cells by gene transfer methods such as microinjection.
  • Another method involves inserting a ribozyme DNA into a suitable expression vector and introducing the vector into cell strains, cells or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell.
  • vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors).
  • animal virus vectors e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors.
  • ribozymes or deoxy ribozymes has an activity of inhibiting expression of VEGF and/or VEGF receptor mediated by the protein of the present invention.
  • a double- stranded RNA which inhibits function having an activity of promoting or inhibitng expression of VEGF or VEGF receptors.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • RNAi RNAi RNAi
  • siRNA Expression Vector available from Ambion can be used (Morita Takashi et al., Protein, Nucleic Acid and Enzyme, Vol.4 No.14 p. 1939-p. 1945 (2001); Sugimoto Asako, Kagaku to Seibutu (Chemistry and Biology), Vol.40 No.ll pp.713-718).
  • the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream the cDNA can be identified.
  • the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene.
  • the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid.
  • the reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene. For example, such screening can be carried out by transforming a suitable cell with the reporter plasmid, culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added. These methods are also included in the scope of the present invention.
  • the present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75 or a coding region thereof, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76.
  • the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences.
  • the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology.
  • These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG program package (Devereux, J, et al., Nucleic Aids Research 12(1):387 (1984)). In this manner, a sequence in a database having a certain homology can be easily found.
  • the computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commercially available floppy disks, tapes, chips, hard drives, compact disks and video disks.
  • the data on the medium allows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one- second polynucleotide or polypeptide sequence to identify the homology.
  • the present invention also relates to an insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75 are fixed.
  • a plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrate such as slide glass to form a DNA microarray and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes.
  • the data obtained is analyzed and the gene expression is determined.
  • the present invention further relates to an insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76, are fixed.
  • polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76.
  • Example 1 Construction of a full-length cDNA library using the oligo-capping method (1) Preparation of RNA from human umbilical vein endothelial cell (HUVEC) Human umbilical vein endothelial cell (HUVEC; purchased from Sanko Junyaku
  • RNA extraction reagent ISOGEN purchased from NIPPON GENE
  • poly A + RNA was obtained from the total RNA by using an oligo-dT cellulose column according to Maniatis et al., supra.
  • a full-length cDNA library was constructed from the above-mentioned poly A + RNA by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki, Y. & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • the full-length cDNA library constructed in the above (2) was transformed into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 ⁇ g/ml of ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the colonies grown on ampicillin-containing LB agar medium.
  • Example 2 Cloning of DNA capable having an activity of promoting expression of VEGF and/or VEGF receptor
  • ECV304 cells purchased from Invitrogen
  • DMEM medium containing 10% FBS in a 96 well cell culture plate to a final cell density of 1 x 10 4 cells/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • 75ng of pGL3HRE-Luc, 5ng of pGL3KDR-Luc, and 2 ⁇ 1 of the full-length cDNA prepared in above Example 1 (2) were cotransfected into the cells in a well using FuGENE 6 (purchased from Roche) according to the manufacturer's protocol.
  • luciferase activity was measured using long-term luciferase assay system,PIKKA GENE LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using Wallac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • sequencing primer 5'-CGACCTGCAGCTCGAGCACA-3' (SEQ ID NO: 78)
  • Thermo Sequenase II Dye Te ⁇ ninator Cycle Sequencing Kit Amersham Pharmacia Biotech
  • BigDye Terminator Cycle Sequencing FS Ready Reaction Kit Applied Biosystems
  • nucleotide sequences was subjected to BLAST (Basic local augment search tool) (S.F.Altschul et al., J. Mol.Biol., 215: 403-410) search in GenBank.
  • BLAST Basic local augment search tool
  • the result shows the the domes are genes encoding new proteins having an activity of promoting expression of VEGF and or VEGF receptor.
  • the full-length DNA sequences for the 37 new clones were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 and 75).
  • the amino acid sequences of the protein coding regions were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 76).
  • Example 3 Assay of reporter activity corrected with an internal standard
  • ECV304 cells were inoculated in DMEM medium containing 10% FBS in a 96-well cell culture plate so as to have a cell number of lxlO 4 cells/100 ⁇ l/well, and cultured at 37°C for 24 hours in the presence of 5% CO 2 .
  • reporter activity firefly luciferase activity
  • reporter activity that reflects the VEGF expression and reporter activity (sea pansy luciferase) as an internal standard
  • luciferase activity was assayed using Wallac ARVOTMST 1420 MULTILABEL COUNTER manufactured by Perkin Elmer.
  • Table 1 shows S/B values which were obtained by dividing the reporter activity reflecting the VEGF expression which was corrected with the internal standard reporter activity when the expression plasmids containing the above gene were introduced, by the same reporter activity when empty vectors were introduced.
  • Example 4 Screening compounds which inhibit expression of VEGF
  • ECV304 cells were seeded on M199medium containing 10% FBS in a 96-well cell culture plate to a final cell density of 1 x 10 4 cells/100 ⁇ 1 well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 . Then, lOOng of the plasmid comprising the gene encoding Serum/glucocorticoid regurated kinase, which was obtained in the screening of the above Example 2 and was revealed to have an activity of promoting expression of VEGF, and 75ng of pGL3HRE-Luc Promega were cotransfected into the cells in a well using FuGENE 6. After 1 hour, 1000 compound for screening were added to each well to final concentrations of 10 ⁇ M.
  • the reporter activity was measured using PUKKA GENE LT2.0. As a result, one compound inhibited 50% or more of the reporter activity. Upon analysys of the structure of this compound, it was found that the compound was staurosporine which strongly inhibits various protein kinases.
  • the present invention provides industrially highly useful proteins having an activity of promoting expression VEGF and/or VEGF receptor and genes encoding the proteins.
  • the proteins of the present invention and the genes encoding the proteins are likely to have an activity of promoting industrially useful vascularization.
  • the proteins of the present invention and the genes thereof allow screening for compounds useful for treating and preventing diseases associated with undesirable vascularization, and also production of diagnostics for such diseases.
  • the genes of the present invention are also useful as a gene source used for gene therapy.

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Abstract

L'invention concerne des protéines ayant une activité de vascularisation qui sont utilisées pour le diagnostic, le traitement ou la prévention des maladies associées à la vascularisation. Au moyen des plasmides pGL3HRE-Luc et pGL3KDR-Luc, on a cloné à partir d'une bibliothèque d'ADNc construite de cellules endothéliales de veine ombilicale chez l'humain un ADNc codant pour une protéine ayant une activité de promotion de l'expression de VEGF et/ou d'un récepteur de VEGF à des fins de clonage, et la séquence d'ADN ainsi que la séquence d'acide aminé qui en a été déduite. La protéine, l'ADN codant pour la protéine, le vecteur recombinant contenant l'ADN et un transformant contenant le vecteur recombinant sont utiles pour cribler une substance inhibant ou favorisant la vascularisation.
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WO2007089445A3 (fr) * 2006-01-27 2008-04-10 Amgen Inc Combinaisons d'inhibiteurs d'ang2 et de vegf

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WO2007089445A3 (fr) * 2006-01-27 2008-04-10 Amgen Inc Combinaisons d'inhibiteurs d'ang2 et de vegf

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