WO2008066032A1 - Facteur inhibiteur de la lymphangiogenèse - Google Patents

Facteur inhibiteur de la lymphangiogenèse Download PDF

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Publication number
WO2008066032A1
WO2008066032A1 PCT/JP2007/072838 JP2007072838W WO2008066032A1 WO 2008066032 A1 WO2008066032 A1 WO 2008066032A1 JP 2007072838 W JP2007072838 W JP 2007072838W WO 2008066032 A1 WO2008066032 A1 WO 2008066032A1
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Prior art keywords
lymphangiogenesis
vasohibin
inhibitor
cells
protein
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PCT/JP2007/072838
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English (en)
Japanese (ja)
Inventor
Yasufumi Sato
Hikaru Sonoda
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Tohoku University
Shionogi & Co., Ltd.
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Priority to JP2008546995A priority Critical patent/JP5154438B2/ja
Publication of WO2008066032A1 publication Critical patent/WO2008066032A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors

Definitions

  • the present invention relates to a lymphangiogenesis inhibitor. More specifically, production of a lymphangiogenesis inhibitor comprising vasohibin, a pharmaceutical composition for treating a disease requiring an inhibitory action on lymphangiogenesis, and a pharmaceutical composition for treating a disease requiring an inhibitory action on lymphangiogenesis, comprising the inhibitor.
  • the present invention relates to a method for treating a disease requiring a lymphangiogenesis-inhibiting action, comprising the use of vasohibin for the purpose and a step of administering the inhibitor.
  • Cancer cells are known to increase by obtaining oxygen and nutrients by secreting VEGF (vascular endothelial growth factor) and promoting tumor angiogenesis in the cancer tissue.
  • VEGF vascular endothelial growth factor
  • many cancer cells secrete VEGF-C.
  • VEGF-C or VEGF-D acts on VEGFR3 expressed in existing lymphatic endothelial cells to form lymphatic vessels that have expanded and proliferated around or within the tumor (hereinafter, referred to as tumor lymphatic vessels).
  • This tumor lymphangiogenesis makes it easier for cancer cells to enter lymphatic vessels, resulting in lymph node metastasis of cancer cells.
  • cancer cells that have metastasized to lymphatic vessels have also been found to induce lymphangiogenesis and angiogenesis, resulting in further distant metastasis.
  • Patent Document 2 JP-A-2004-532281
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-528378
  • the present invention provides:
  • a lymphangiogenesis inhibitor comprising vasohibin
  • a lymphangiogenesis inhibitor comprising a vector comprising a polynucleotide encoding nosohibin
  • a pharmaceutical composition for treating a disease requiring an inhibitory action on lymphangiogenesis comprising the inhibitor according to [1] or [2],
  • Vasohibin for the manufacture of a pharmaceutical composition for the treatment of a disease requiring a lymphangiogenesis-inhibiting action
  • [6] A method for treating a disease requiring a lymphangiogenesis-inhibiting action, comprising a step of administering the inhibitor according to [1] or [2].
  • FIG. 1 shows suppression of lymphangiogenesis induced by bFGF by Vasohibin.
  • FIG. 2 shows inhibition of lymphangiogenesis induced by VEGF-A by Vasohibin.
  • FIG. 3 shows inhibition of lymphangiogenesis induced by VEGF-C by Vasohibin.
  • FIG. 4 shows inhibition of lymphangiogenesis induced by PDGF—BB by vasohibin.
  • the present invention relates to a lymphangiogenesis inhibitor comprising a substance other than a VEGFR3 inhibitor, a pharmaceutical composition for treatment of a disease requiring a lymphangiogenesis inhibitory action, comprising the inhibitor, lymphatic vessel
  • the present invention relates to the use of Vasohibin for the production of a pharmaceutical composition for the treatment of a disease requiring an anti-neoplastic action, and a method for treating a disease requiring an anti-angiogenic action comprising the step of administering the inhibitor.
  • lymphangiogenesis can be suppressed.
  • vasohibin acts as a negative feedback regulator in angiogenesis.
  • the action of vasohibin in the lymphatic vessels is unknown, and as a result of intensive studies by the present inventors, it was found that vasohibin can be used to suppress lymphangiogenesis and the present invention was completed.
  • the present invention has a great feature in using vasohibin in the suppression of lymphangiogenesis, and the lymphangiogenesis inhibitor of the present invention encodes an inhibitor composed of vasohibin (Aspect 1) and vasohibin.
  • Examples include inhibitors comprising a vector containing a polynucleotide (Aspect 2), and are used from the viewpoint of inhibiting lymphangiogenesis.
  • Vasohibin is expressed in vascular endothelial cells by angiogenesis-promoting factors (VEGF, FGF-2, etc.) secreted from tumor cells, stromal cells, macrophages, etc., and acts on the endothelial cells themselves in an autocrine manner. It is a substance that suppresses neoplasia.
  • vasohibin By utilizing the excellent effect of vasohibin relating to angiogenesis inhibition, and administering vasohibin protein or vasohibin gene from outside the body, angiogenesis can be more strongly inhibited.
  • Vasohibin is also considered to act on lymphatic endothelial cells and suppress lymphangiogenesis in lymphatic vessels. Therefore, administration of Vasohibin protein or Vasohibin gene from outside the body suppresses lymphangiogenesis. It is estimated that it is possible. You can As disclosed in WO02 / 090546, WO2006 / 073052, etc., nosohibin 1 and vasohibin 2 are different genes that exist on different chromosomes. The amino acid sequence of the protein encoded by these genes is 58.
  • Vasohibin 1 is a protein encoded by the KIAA1036 polynucleotide consisting of the nucleotide sequence represented by the 386th A force of SEQ ID NO: 1, and the 1480th C, and the KIAA1036 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2.
  • Vasohibin 2 is the base represented by the 1st A to 1068th G of SEQ ID NO: 3.
  • the vasohibin in the present invention preferably contains the amino acid sequence or a polynucleotide encoding the amino acid sequence.
  • the polynucleotide in the present invention includes a KIAA1036 polynucleotide comprising the base sequence represented by SEQ ID NO: 1, an AY834202 polynucleotide comprising the base sequence represented by SEQ ID NO: 3, and the polynucleotide or a complementary strand thereof. And polynucleotides that can hybridize under stringent conditions.
  • Polynucleotide that can hybridize under stringent conditions refers to a method known in the art, for example, colony hybridization, using a polynucleotide fragment as a probe. , Plaque hybridization method, Southern blotno, hybridization method, etc., specifically, a membrane on which a colony or plaque-derived polynucleotide is immobilized. Use: 0.7 ⁇ ; 1. After hybridization at 65 ° C in the presence of OM NaCl, 0.;!
  • a polynucleotide that can be hybridized means the above-mentioned hybridizate.
  • such a polynucleotide is at least 60% or more, preferably 80% or more, more preferably 95%, with the KIAA1036 polynucleotide represented by SEQ ID NO: 1 or the AY834202 polynucleotide represented by SEQ ID NO: 3. It is possible to list polynucleotides having a homology of at least%.
  • the homology is determined by, for example, using the search program BLAST, which uses the algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)), as a similarity score. Is shown.
  • the polynucleotide can be prepared according to a known method, for example, according to the method disclosed in WO02 / 090546. It can also be prepared by chemically synthesizing DNA encoding KIAA1036 polypeptide or AY834202 polypeptide based on the amino acid sequence. Chemical synthesis of DNA can be performed by using a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, a DNA synthesizer model 392 manufactured by Parkin Elma Co. using the phosphoramidite method, or the like.
  • the polypeptide in the present invention includes a KIAA1036 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, an AY834202 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, one or several amino acids in the amino acid sequence Examples include polypeptides having amino acid residue deletions, additions, insertions or substitutions, and derivatives thereof, and salts thereof.
  • polypeptide derivative refers to, for example, polypeptide acetylenolation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succination.
  • N-terminal acetylation, C-terminal amidation, and C-terminal methylation impart resistance to exopeptidases that degrade polypeptides from the terminus, and can also be achieved by glycosylation or polyethylene glycolation. Is also preferable since stability in the living body is expected to increase.
  • salt refers to any pharmacologically acceptable salt of a polypeptide or a derivative thereof (including inorganic salt and organic salt).
  • sodium salts, potassium salts, and phosphates are preferable.
  • the polypeptide can be prepared according to a known method, for example, according to the method disclosed in WO02 / 090546, WO2006 / 073052, and the like.
  • a salt of the above polypeptide can also be easily prepared by a person skilled in the art by any method known in the art.
  • the inhibitor of aspect 1 is substantially composed of the vasohibin.
  • the inhibitor of aspect 2 is composed of a vector containing the polynucleotide encoding the vasohibin.
  • the vector is capable of autonomous replication in a host cell, and at the same time is composed of a promoter, a ribosome binding sequence, a gene encoding vasohibin, and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained. Suitable vectors used in the present invention include the vectors described below.
  • the vector can be prepared according to a known method, for example, according to the method disclosed in WO02 / 090 546, WO2006 / 073052, and the like.
  • the present invention also provides a pharmaceutical composition for treating a disease requiring a lymphangiogenesis-inhibiting action, ie, a therapeutic agent, comprising the inhibitor comprising the above-mentioned bathohibin.
  • the disease requiring a lymphangiogenesis-inhibiting action in the present invention is not particularly limited as long as it has a therapeutic effect by inhibiting lymphangiogenesis.
  • myeloid leukemia, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma examples include myeloid leukemia, acute myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma.
  • lymphangitis, lymphadenitis, lymphangioma, nevus, and pulmonary lymphangioleiomyomatosis are not expected to have a therapeutic effect even if angiogenesis is suppressed, so the therapeutic agent of the present invention is expected to be applied. Is done.
  • Examples of the therapeutic agent of the present invention include those prepared by combining the inhibitor of the present invention with a known pharmaceutical carrier.
  • Vasohibin can also be blended with other components that can be used for the same purpose as Vasohibin, for example, a component having an action of suppressing known lymphangiogenesis, such as a VEGFR3 inhibitor.
  • the therapeutic agent of the present invention is usually produced by blending the inhibitor of the present invention with a pharmaceutically acceptable liquid or solid carrier, and if necessary, a solvent, a dispersant, an emulsifier, a mild agent. Add impactant, stabilizer, excipient, binder, disintegrant, lubricant, etc., solids such as tablets, granules, powders, powders, capsules, etc. Ordinary solutions, suspensions, emulsions Or the like. Further, it can be made into a dry product that can be made liquid by adding an appropriate carrier before use, or other external preparations.
  • the pharmaceutical carrier can be selected according to the administration form and formulation of the therapeutic agent, and is not particularly limited.
  • the therapeutic agents in various preparation forms as described above can be appropriately produced by conventional methods using known pharmaceutical carriers and the like.
  • the content of the inhibitor of the present invention in the therapeutic agent is not particularly limited as long as the desired effect of the present invention can be obtained in consideration of its administration form, administration method and the like. It is not limited.
  • the content of the inhibitor of the present invention in the therapeutic agent of the present invention is usually about 1 to 100% by weight.
  • the therapeutic agent of the present invention is administered by an appropriate administration method according to the preparation form.
  • the administration method is not particularly limited, for example, it can be administered by internal use, external use or injection.
  • the therapeutic agent of the present invention when administered by injection, it can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, etc., and when administered by external use, it is suitable as an externally applied agent such as a suppository. Do it according to the method of administration!
  • the dosage of the therapeutic agent of the present invention is appropriately set according to the preparation form, administration method, purpose of use and age, weight, and symptom of the patient to whom the therapeutic agent is administered, and is not constant.
  • administration can be performed once or several times within a day within the desired dosage range. It may be done separately.
  • the administration period is also arbitrary.
  • the present invention also provides a method for treating a disease requiring an inhibitory action on lymphangiogenesis, comprising administering Vasohibin to a subject.
  • the subject is preferably a human that requires a lymphangiogenesis inhibitory action, but may be a pet animal or the like.
  • an effective amount means that when Vasohibin is administered to the subject.
  • the amount of nosohibin that exerts an inhibitory effect on lymphangiogenesis compared to a subject who has not been administered nosohibin is appropriately set depending on the administration form, administration method, purpose of use, age, weight, symptom, etc. of the subject and is not constant.
  • an effective amount of nosohibin may be administered to the subject as it is, or as a medical agent such as the above therapeutic agent. It may be administered. Also, there is no limitation on the administration method. For example, it may be administered by oral administration, injection or the like, as in the case of the above-mentioned medicine.
  • the disease targeted by the therapeutic agent of the present invention can be treated.
  • the effect of treating a disease caused by lymphangiogenesis is effective. Can be demonstrated.
  • the lymphangiogenesis inhibitor comprising a vector comprising the polynucleotide encoding vasohibin of the present invention is used for gene therapy in a patient having a disease caused by lymphangiogenesis. be able to.
  • an in vivo method in which the inhibitor is directly introduced into the body and a certain type of cell from a human being taken out, the DNA is transferred to the cell outside the body.
  • an ex vivo method that introduces and returns the cells to the body [Nikkei Science, April issue, 20-45 (1994), Monthly Pharmaceutical Affairs, 36, 23-48 (1994), experimental medicine, lj, 12, 15 (1994)].
  • the in vivo method is preferred.
  • the in vivo method When administered by the in vivo method, it is administered by an appropriate administration route according to the disease to be treated, the target organ and the like. For example, it can be administered directly to a tissue in which a lesion is observed, or can be administered by vein, artery, subcutaneous, intramuscular, intraperitoneal, endoscopic, aerosol or the like. As an administration method, intravenous or intraperitoneal administration is preferable. Also lesions Also preferred is direct injection of tissue that can be seen! Taking an image of a lesioned tissue using any of those available in the art, such as nuclear magnetic resonance imaging or computed tomography, and administering the inhibitor comprising the vector of the present invention by, for example, stereotaxic injection Can
  • the inhibitor when used as a gene therapy vector, the inhibitor can be in various dosage forms suitable for each of the above administration forms.
  • the injection can be prepared by a conventional method.
  • the base used in the gene therapy agent is not particularly limited as long as it is a base that is usually used for injections, and is a salt solution of distilled water, sodium chloride, or a mixture of sodium chloride and an inorganic salt, mannitol, ratatose.
  • a solution such as dextran and glucose, an amino acid solution such as glycine and arginine, an organic acid solution, or a mixed solution of a salt solution and a glucose solution.
  • these bases are used with an auxiliary agent such as an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil and soybean oil, or a surfactant such as lecithin or a nonionic surfactant.
  • injectables may be prepared as solutions, suspensions or dispersions. These injections can be made into preparations for use and dissolution by operations such as powdering and freeze-drying.
  • the content of DNA encoding Vasohibin in the preparation varies depending on the disease to be treated, administration site, number of administrations, desired treatment period, patient age, body weight, etc., and can be adjusted as appropriate.
  • the weight of DNA encoding Vasohibin is generally about 0.0;! To 2000 mg, preferably ⁇ is 0 .;! To lOO mg.
  • the gene for Vasohibin is expressed in the host cell and produced.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • a DNA in which the base is replaced so that the base sequence of the protein-encoding portion becomes the optimal codon for host expression is prepared. The DNA is useful for improving the production rate of the protein.
  • Recombinant DNA (expression plasmid) is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector. By introducing the expression plasmid into a host cell suitable for the expression vector, a transformant producing Vasohibin can be obtained.
  • any prokaryotic cell, animal cell, insect cell, etc. can be used as long as it can express the target gene.
  • the expression vector a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position suitable for transcription of a gene encoding vasohibin is used.
  • the expression vector of nosohibin protein is capable of autonomous replication in prokaryotes, and at the same time is composed of a promoter, a ribosome-binding gene IJ, a gene encoding vasohibin, and a transcription termination sequence. ! / The gene that controls the promoter is included!
  • Examples of expression vectors include pBTrp2, pBTacl, pBTac2 (Roche Diagnostatus), Bluescript II SK (+), pBluescript II SK (—) (Stratagene), pSTV28, pUC118, pUC19 (Takara Shuzo), pKK233_2 (Amersham Neuscience), pSE280, pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), pGEMEX- ⁇ (Promega), pQE-8 (Qiagen) ), PGEX (manufactured by Falmasia), pET system (manufactured by Novagen), pMAL-c2 (manufactured by New England Biolabs), pKYP10 (Japanese Patent Laid-Open No.
  • the promoter may be any as long as it can be expressed in a host cell such as Escherichia coli.
  • a promoter derived from E. coli or phage such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SPOl promoter, SP02 promoter, penP promoter, etc. I can do it.
  • An artificially designed and modified promoter such as a promoter in which two Ptrps are connected in series (Ptrpx2), a tac promoter, a lacT7 promoter, and a letl promoter can also be used.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance, for example, 6 to 18 bases.
  • a transcription termination sequence is not necessarily required for the expression of the vasohibin gene! // It is preferable to place a transcription termination sequence directly under the structural gene.
  • Examples of host cells include prokaryotes such as Escherichia, Serratia, Bacillus, Brevibacterium lus, Corynebacterium ⁇ , Microbacterium J P, Pseudomonas J ⁇ , and Escherichia genus, such as XL1-Blue strain, XL2 — Blue, DH1, MC1000, KY3276, W1485, JM109, HB101, No.49, W3110, NY49, BL21 (DE3), BL21 (DE3) pLysS, HMS 174 (DE3) strain and HMS 174 (DE3) pLysS strain isobaric Serratia genus, S. ficaria strain, S.
  • prokaryotes such as Escherichia, Serratia, Bacillus, Brevibacterium lus, Corynebacterium ⁇ , Microbacterium J P, Pseudomonas J ⁇ , and Escherichia genus
  • any method for introducing an expression plasmid any method can be used as long as it is a method for introducing DNA into the host cell.
  • expression vectors include, for example, pcDNAl / Amp, pcDNAl, pCDM8, pREP4 (Invitrogen), pHM6 (Roche Diagnostics), pKK223-3, pGEX (Amersham Bioscience) PAGElO 7 (Cytotechnology, technology, 13d (1990)), pAGE103 (Tne Journal of Biochem istry, 101, 1307 (1987)), pAMo, pAMoA (pAMoPRSA) (The Journal of Biological Chemistry, 268, 22782— 22787 (1993)), pAS3-3 (JP-A-2-22705) and the like can be used.
  • Any promoter can be used as long as it can be expressed in the host.
  • human cytomegalovirus (hCMV) IE immediate-early gene promoter
  • SV40 early promoter SV40 early promoter
  • Murine Leulemia Virus long “Terminal repeat” promoter (Long Terminal Repeat Promoter), retrowinores promoter, HSP promoter, SRa promoter, and metamouth thionein promoter.
  • an enhancer of IE gene of hCMV may be used together with a promoter.
  • Animal cells used as hosts include human-derived cell lines HEK293 (human embryonic kidney cells, AT CC: CRL-1573), Namalwa (Burkitt lymphoma, ATCC: CRL-1432), HeLa (cervical cervix) Cancer cells, ATCC: CCL-2), HBT5637 (leukemia cells, JP-A 63-299), BALL-1 (leukemia cells) and HCT-15 (colon cancer cells); mouse-derived cell line S p2 / 0 -A g 14 (mouse myeloma cell, ATCC: CRL- 1581) and NSO (mouse myeloma cell); monkey-derived cell line COS- 1 (African green monkey kidney cells (SV40 transformed cells), ATCC: CRL- 1650) and COS-7 (African green monkey kidney cells (SV40 transformed) ATCC: CRL-1651); Hamster-derived cell line CHO—K1 (Chinese hamster ovary cell, ATCC: CCL—
  • any method can be used as long as it is a method for introducing DNA into a host.
  • the electopore position method (Cytotechnology, 3, 133, (1990))
  • the calcium phosphate method JP-A-2-22705
  • the ribofusion method (Proceedings of the National Academy of Sciences, USA, 84, 7413 (1987), Virology, 52, 456 (1973)).
  • examples of expression vectors include pVL1392, pV L1393, pBlueBacIII, pFASTBacl (Invitrogen), etc.
  • Infectious viruses include, for example, baculovirus (Vaculovirus) Autog rapha California nuclear polyhedrosis virus ⁇ AcMNPV) Bac-N-Blue DNA.
  • Insect cell transformation methods include, for example, Baculovirus Expression Vector: A Laboratory Manual (1992) (WH Freeman and Company), Molecular Cloning: A Laboratory Manual, Second Edition (1 989) (Cold Spring Harbor Laboratory Press) , Current Protocols in Molecular Biology 994) (Wiley—Interscience), Biotechnology, 6, 47 (1 988) and the like.
  • An insect vector is added to an insect cell culture medium containing an expression vector containing the target gene and baculovirus DNA for infection of the insect cell, and the recombinantly produced virus expressing the target gene is infected with the insect cell.
  • Vasohibin can be expressed.
  • insect cells used as the host include Spodoptera frugiperda-derived cell lines, Trichoplusia ni (Irakusakkinno-derived cell lines), and specifically, S • frugiperda-derived cells include Sf9 ( ATCC: CRL— 1711, ovarian cells), Sf 21 (ovarian cell), etc., as T. ni-derived cell lines, High Five: ⁇ — ⁇ — 5 ⁇ 1—4 (egg) Cell, manufactured by Invitrogen Corporation) and the like.
  • any method that can be introduced into a host can be used.
  • the calcium phosphate method JP-A-2-22705
  • the lipofuxion method Proceedings of the National Academy of) Sciences U3 ⁇ 4A, 84, 741 a (1987)
  • CELLFECTIN reagent Invitrogen
  • the electoral position method As in the case of animal cells, the electoral position method (Cytotechnology, 3, 133 (1990)) can be used.
  • Transformant possessing expression plasmid incorporating DNA encoding nosohibin In the case of cells such as Escherichia coli and animal cells, the protein is produced by culturing according to a normal culture method suitable for various hosts. The protein can be produced by accumulating and recovering the protein from the transformant or culture medium. When the transformant is an individual animal or plant individual, it is bred or cultivated according to a normal growth method suitable for various hosts, the protein is produced and accumulated, and the protein is collected from the animal individual or plant individual. Thus, the protein can be produced.
  • the host is an animal individual, for example, a non-human transgenic animal carrying a gene encoding vasohibin is bred, and vasohibin encoded by the plasmid is produced and accumulated in the animal, It is possible to produce Vasohibin by recovering the protein from Examples of production / accumulation locations in individual animals include the milk, saliva, and eggs of the animals.
  • the host is a prokaryotic organism such as Escherichia coli
  • a transformant carrying a gene encoding vasohibin is cultured in a medium, and vasohibin encoded by the plasmid is produced and accumulated in a culture solution.
  • the ability to produce Vasohibin is measured.
  • the method of culturing a transformant of vasohibin in a medium can be performed according to a usual method used for culturing a host.
  • any medium that contains a carbon source, a nitrogen source, an inorganic salt, and the like that can be assimilated by the organism and that can efficiently culture the transformant can be used.
  • Either a medium or a synthetic medium may be used.
  • any microorganism that can be assimilated by each microorganism may be used, such as glucose, slatose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing substances, and peptone, meat extract, yeast extract. , Corn steep liquor, caseincaro hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and their digests, etc.
  • inorganic salt monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. Culturing is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • such a medium is preferably a YT medium containing, for example, butatotryptone, yeast etact and sodium chloride!
  • the culture time is 15 to 40 ° C, and the culture time is usually 5 hours to 7 days.
  • the pH is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. Further, antibiotics such as ampicillin and tetracycline may be added to the medium as needed during the culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium.
  • the culture medium for culturing the cell is RPMI1640 medium (The Journal of the American Med). ical Association, 199, 519 (1967)), MEM medium (Science, 130, 432 (1959)), D-MEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)) or a medium obtained by adding fetal calf serum (FCS) or the like to these media.
  • RPMI1640 medium The Journal of the American Med). ical Association, 199, 519 (1967)
  • MEM medium Science, 130, 432 (1959)
  • D-MEM medium Virology, 8, 396 (1959)
  • 199 medium Proceedings of the Society for the Biological Medicine, 73, 1 (1950)
  • FCS fetal calf serum
  • the culture is usually carried out under conditions of pH 6-8, 25-40 ° C, 5% CO, etc .;! -7 days.
  • antibiotics such as kanamycin, penicillin, streptomycin and the like may be added to the medium as needed during culture.
  • the culture medium to be cultured is a commonly used TNM-FH medium (Pharmingen), Sf-900II SFM medium (Invitrogen), ExCell400, ExCell405. (Manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)) and the like can be used.
  • Nosohibin can be produced by culturing the transformant and isolating and purifying Vasohibin from the culture medium. Isolation / purification of vasohibin can be carried out by conventional methods well known in the art. For example, the isolation / purification method of enzymes and the purification method of glycosyltransferase of Sandler et al. (Methods in Enzymology, 83 , 458).
  • the culture solution obtained by culturing the transformant as described above is separated into cells or cells and a medium by, for example, centrifugation.
  • the collected cells or cells are washed with an appropriate buffer such as STE solution, and then the cells or cells are sonicated by ultrasonic, French press, Mantongaurin homogenizer, dynomill, etc. Crush and obtain a cell-free solution by centrifugation or filtration.
  • the buffer used for purification may contain an appropriate amount of a surfactant, for example, sodium lauryl sulfate (SDS), sodium N-lauroyl sarcosine (sarcosyl) and the like! / But! /
  • a surfactant for example, sodium lauryl sulfate (SDS), sodium N-lauroyl sarcosine (sarcosyl) and the like! / But! /
  • Separation and purification of the target protein contained in the obtained crude purified product can be performed by combining various separation and purification methods known per se. These known methods include, for example, solvent extraction, salting out with ammonium sulfate, dialysis, precipitation with organic solvents, etc.
  • nosohibin When nosohibin is produced / accumulated as an insoluble polypeptide, cells or cells are separated in the same manner as described above, and disrupted by an appropriate method, and then a fraction containing the polypeptide is collected.
  • the collected sample is solubilized with a solubilizing agent such as a surfactant such as sodium lauryl sulfate (SDS) or sodium N-lauroyl sarcosine (sarkosyl).
  • SDS sodium lauryl sulfate
  • sarkosyl sodium lauryl sulfate
  • the solubilized solution does not contain a solubilizer! / Or contains almost no solubilities! /
  • a purified sample can be obtained by a purification method.
  • Vasohibin can also be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance with affinity for the fused protein (Akio Yamakawa) , Experimental Medicine, 13, 469-474 (1995)).
  • additional proteins used for fusion proteins include protein A and FLAG (Proceedings of the National Academy of Sciences, USA, 86, 8227 (1989), Genes
  • protein A When protein A is used, it can be purified by producing a fusion protein of vasohibin and protein A and performing affinity chromatography using immunoglobulin G. When using a FLAG peptide, it is necessary to produce a fusion protein of vasohibin and FLAG and purify it by affinity chromatography using an anti-FLAG antibody.
  • Vasohibin can also be produced using an in vitro transcription / translation system according to a known method (Journal of Biomolec ular NMR, 6, 129-134 (1995), Science, 242, 1162-1164 (1988), The Journal of Biochemistry, 110, 166-168 (1991)).
  • Vasohibin is sold based on its amino acid sequence, such as chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (tbutyloxycarbonyl method).
  • Peptide synthesis equipment such as APEX396 (manufactured by Advanced Chemtech), 433 A (manufactured by Applied Biosystems), PS3 (manufactured by Protein Technologies), 9050 (manufactured by Perceptive), PSSM-8 (manufactured by Shimadzu) It can be chemically synthesized using any of these peptide synthesizers.
  • the method for producing a gene therapy vector, the method for expression in cells, etc. are the same as the expression vector described in the above-mentioned “Method for producing Vasohibin”.
  • the expression vector is safe and has low toxicity, it is administered to, for example, mammals (eg, humans, rats, mice, rabbits, hidges, pigs, rushes, cats, inu, monkeys, etc.). Is possible. When used for gene therapy, it is preferable to use a DNA or RNA virus vector or plasmid vector that can express a protein in mammalian cells including humans and is highly safe.
  • Viral vectors preferred for gene therapy include adenovirus, adeno-associated winores (AAV), retrovirus, boxwinores, herpes virus, simple herpes virus, lentivirus (HIV), Sendai virus, Epstein Examples include vernorevirus (EBV), vaccinia virus, poliovirus, symbis virus, and SV40.
  • Preferred plasmids for gene therapy include pCAGGS (Gene, 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Suragene) and the like.
  • Nosohibin protein was produced and purified by baculovirus / insect cell expression system.
  • PFASTBacl Invitrogen Corp.
  • a vector for insect cell expression encodes a cDNA encoding a 3xFLAG sequence at the end of KIAA1036 (region consisting of amino acids 1 to 365 of SEQ ID NO: 2) of Vasohibin 1
  • the plasmid pF ASTBacl036 for insect cell expression was constructed.
  • Bac-To-Bac Baculovirus Expression Systems (Invitrogen) was used, and the operation was also followed. That is, the constructed pFASTBacl036 plasmid was transformed into DHlOBac E.
  • BacmidDNA was introduced into Sf9 cells using CELLFECT IN reagent (Invitrogene) to obtain recombinant baculovirus.
  • CELLFECT IN reagent Invitrogene
  • 0.5 mL of this virus solution was used at a rate of infecting 50 mL of Sf9 cells at a concentration of 1.5 ⁇ 10 6 cells / mL.
  • the cells were cultured for 96 hours after infection, and the cells were collected by centrifugation.
  • the expression of FLAG-added vasohibin protein by insect cells was confirmed by Western blotting using this Sf9 cell extract and HRP-labeled anti-FLAG M2 antibody (Sigma).
  • the cells were washed once with 5 mL of phosphate buffered saline (PBS), and then 5 mL of Lysis solution (0 ⁇ 15M NaCl, 0. ImM EDTA, 0. ImM EGTA, ImM DTT, 0. Suspend in ImM amidinophenyl methansulufonyl fluride hydroc hydride, 0.1% NP-40, 50 mM Tris-HCl, pH 7.4), then sonicate with MICR OCON (HEART SYSTEMS) and ice-cool.
  • PBS phosphate buffered saline
  • the solution was centrifuged 4 times for 15 seconds, centrifuged at 14,500 xg for 20 minutes, and the supernatant was added with an equal volume of Lysis solution to make a lysate solution.
  • Western blot was separated by SDS-PAGE according to Laemli's method, and 0.5 ⁇ L was electrically transferred to a nitrocellulose membrane (Tefco), then blocked in TBS containing 5% skim milk. And reacted with an HRP-labeled anti-FLAG M2 antibody.
  • FGF-2 protein fibroblast growth factor-2, bFGF, BD Biosciences
  • FGF-2 protein fibroblast growth factor-2, bFGF, BD Biosciences
  • 16 Ong VEGF-A in 0 ⁇ S / ig hydrone reagent (IFN Sciences) Protein Sigma
  • 160 ng VEGF-C protein Acris Antibodies
  • 80 ng PDGF-BB protein R & D Systems
  • the therapeutic agent containing vasohibin of the present invention is suitably used, for example, for the treatment of a disease requiring a lymphangiogenesis inhibitory action.
  • SEQ ID NO: 1 in the sequence listing is a KIAA1036 polynucleotide.
  • Sequence number 2 of a sequence table is KIAA1036 polypeptide.
  • Sequence number 3 of a sequence table is AY834202 polynucleotide.
  • Sequence number 4 of a sequence table is AY834202 polypeptide.

Abstract

L'invention concerne: un inhibiteur de la lymphangiogenèse comprenant de la vasohibine ; un inhibiteur de la lymphangiogenèse comprenant un vecteur portant un polynucléotide codant pour la vasohibine ; une composition pharmaceutique pour le traitement d'une maladie pour laquelle un effet inhibiteur de la lymphangiogenèse est requis, qui comprend l'inhibiteur ; l'utilisation de la vasohibine pour la production d'une composition pharmaceutique pour le traitement d'une maladie pour laquelle un effet inhibiteur de la lymphangiogenèse est requis ; l'utilisation d'un polynucléotide codant pour la vasohibine pour la production d'une composition pharmaceutique pour le traitement d'une maladie pour laquelle un effet inhibiteur de la lymphangiogenèse est requis ; et un procédé pour le traitement d'une maladie pour laquelle un effet inhibiteur de la lymphangiogenèse est requis, qui comprend l'étape consistant à administrer l'inhibiteur. L'inhibiteur peut être utilisé de façon appropriée pour le traitement d'une maladie pour laquelle un effet inhibiteur de la lymphangiogenèse est requis ou similaires.
PCT/JP2007/072838 2006-11-30 2007-11-27 Facteur inhibiteur de la lymphangiogenèse WO2008066032A1 (fr)

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WO2010024293A1 (fr) * 2008-08-27 2010-03-04 塩野義製薬株式会社 Ensemble d'anticorps monoclonaux anti-vasohibines
WO2010074082A1 (fr) * 2008-12-24 2010-07-01 塩野義製薬株式会社 Vasohibine modifiée
CN105194457A (zh) * 2015-09-24 2015-12-30 耿聪 一种用于淋巴管炎的中药制剂及其制备方法

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CN101137390B (zh) * 2005-01-05 2013-03-27 盐野义制药株式会社 新型血管生成抑制因子

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010024293A1 (fr) * 2008-08-27 2010-03-04 塩野義製薬株式会社 Ensemble d'anticorps monoclonaux anti-vasohibines
JP5430573B2 (ja) * 2008-08-27 2014-03-05 塩野義製薬株式会社 抗バソヒビンモノクローナル抗体のセット
WO2010074082A1 (fr) * 2008-12-24 2010-07-01 塩野義製薬株式会社 Vasohibine modifiée
CN105194457A (zh) * 2015-09-24 2015-12-30 耿聪 一种用于淋巴管炎的中药制剂及其制备方法

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