JP5154438B2 - Lymphangiogenesis inhibitory factor - Google Patents

Description

  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 effect 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 administration 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 cancer tissue. On the other hand, many cancer cells secrete VEGF-C. VEGF-C or VEGF-D acts on VEGFR3 expressed in existing lymphatic endothelial cells to regenerate 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 the lymph vessels, resulting in lymph node metastasis of the cancer cells. Recently, it has also been found that cancer cells that have metastasized to lymphatic vessels induce lymphangiogenesis and angiogenesis and further distant metastasis.

  On the other hand, focusing on the action of VEGF-C / VEGFR3 or VEGF-D / VEGFR3, a technique for suppressing lymphangiogenesis in the cornea of a patient by using a composition containing a VEGFR3 inhibitor is disclosed (Patent Literature) 1). In addition, since N-oxide anthranilamide derivatives and cyanoanthranilamide derivatives show excellent effects as VEGFR kinase inhibitors, they are reported to be effective inhibitors of lymphangiogenesis (see Patent Documents 2 and 3). ).

In addition, angiopoietin / Tie, PDGF-BB / PDGFR-β, FGF2 / FGFR3, HGF / Met, etc. have been reported as lymphangiogenesis-related molecules other than VEGF-C / VEGFR3 and VEGF-D / VEGFR3. There are no reports on lymphangiogenesis inhibitors related to the related molecules.
JP 2005-525352 A JP 2004-532281 A JP 2004-528378 A

Summary of invention

The present invention
[1] A pharmaceutical composition for treating a disease requiring a lymphangiogenesis-inhibiting action selected from the group consisting of lymphangitis, lymphadenitis, lymphangioma, nevus, and pulmonary lymphangioleiomyomatosis, containing bathohibin and (2) containing a vector comprising the polynucleotide encoding Vasohibin, lymphangitis, lymphadenitis, lymphangioma, lymphoma selected from the group consisting of nevi, and pulmonary lymphangioleiomyomatosis Pharmaceutical composition for treatment of diseases requiring angiogenesis inhibitory action
About the.

FIG. 1 shows inhibition of lymphangiogenesis induced by bFGF by Vasohibin. FIG. 2 shows suppression by vasohibin of lymphangiogenesis induced by VEGF-A. FIG. 3 shows inhibition by vasohibin of lymphangiogenesis induced by VEGF-C. FIG. 4 shows suppression by lymphovascularization of lymphangiogenesis induced by PDGF-BB.

Detailed Description of the Invention

  The present invention relates to a lymphangiogenesis inhibitor comprising a substance other than a VEGFR3 inhibitor, a pharmaceutical composition for treating a disease requiring a lymphangiogenesis inhibitory action, containing the inhibitor, and a therapeutic drug for a disease requiring a lymphangiogenesis inhibitory action The present invention relates to a method for treating a disease requiring a lymphangiogenesis-inhibiting action, comprising the use of vasohibin for producing a composition and a step of administering the inhibitor.

  With the inhibitor of the present invention, lymphangiogenesis can be suppressed.

  We have heretofore revealed that Vasohibin acts as a negative feedback regulator in angiogenesis. However, 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 contains an inhibitor comprising vasohibin (embodiment 1) and a polynucleotide encoding vasohibin. And a suppressor comprising the vector (embodiment 2), and is 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, thereby causing angiogenesis. It is a substance that suppresses 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. On the other hand, vasohibin is also considered to act on lymphatic endothelial cells and suppress lymphangiogenesis similarly in lymphatic vessels. Therefore, it is possible to suppress lymphangiogenesis by administering vasohibin protein or vasohibin gene from outside the body. Is estimated to be possible.

  Examples of vasohibin in the present invention include vasohibin 1 and vasohibin 2. As disclosed in WO02 / 090546, WO2006 / 073052, etc., Vasohibin 1 and Vasohibin 2 are different genes existing on different chromosomes, but the amino acid sequences of the proteins encoded by these genes are 58% homologous. Both have an inhibitory activity on angiogenesis. Vasohibin 1 is a protein encoded by the KIAA1036 polynucleotide consisting of the base sequence represented by the 386th A to 1480th C of SEQ ID NO: 1, and the KIAA1036 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2. Vasohibin 2 is a protein encoded by the AY833422 polynucleotide comprising the nucleotide sequence represented by the first A to the 1068th G of SEQ ID NO: 3, and the AY833422 polypeptide comprising the amino acid sequence represented by SEQ ID NO: 4 I mean.

  Further, as disclosed in WO07 / 037245, the specific sequence of Vasohibin 1, that is, the amino acid sequence from Met at position 77 to Val at position 365 in SEQ ID NO: 2 has higher angiogenesis inhibitory activity, Vasohibin in the present invention preferably includes the amino acid sequence or a polynucleotide encoding the amino acid sequence.

  The polynucleotide in the present invention includes a KIAA1036 polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 1, an AY833422 polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 3, and the above-described polynucleotide or a complementary strand thereof and a stringent Examples are polynucleotides that can hybridize under various conditions.

  The “polynucleotide capable of hybridizing under stringent conditions” as used herein refers to a polynucleotide fragment as a probe and a commonly used method such as colony hybridization, plaque hybridization or Southern blotting. A polynucleotide obtained by using a hybridization method or the like, specifically, using a membrane on which a polynucleotide derived from a colony or plaque is immobilized, in the presence of 0.7 to 1.0 M NaCl, After hybridization at 0 ° C., the membrane is washed at 65 ° C. using a 0.1-2 fold concentration of SSC (Saline Sodium Citrate: 150 mM sodium chloride, 15 mM sodium citrate) solution. It means more identification can polynucleotide. Hybridization, Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Molecular Biology (1994) (Wiley-Interscience), DNA Cloning 1: A Practical Approach Core Techniques, Second Edition (1995) (Oxford University Press) and the like. Here, the sequence consisting only of adenine (A) or thymine (T) is preferably excluded from sequences that hybridize under stringent conditions.

  In the present specification, the “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, 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 AY833422 polynucleotide represented by SEQ ID NO: 3. The polynucleotide which has the above homology can be mentioned. In this specification, the homology is determined by, for example, using a search program BLAST using an algorithm developed by Altschul et al. (The Journal of Molecular Biology, 215, 403-410 (1990)). 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 AY832022 polypeptide based on the amino acid sequence. Chemical synthesis of DNA can be performed using a Shimadzu DNA synthesizer using the thiophosphite method, a Perkin Elmer DNA synthesizer model 392 using the phosphoramidite method, or the like.

  Examples of the polypeptide in the present invention include KIAA1036 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2, AY833422 polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 4, and one or several amino acid residues in the amino acid sequence And polypeptides having deletions, additions, insertions or substitutions thereof, and derivatives thereof, and salts thereof.

  In the present specification, the “polypeptide derivative” means, for example, acetylation, palmitoylation, myristylation, amidation, acrylation, dansylation, biotinylation, phosphorylation, succinylation, anilideation of a polypeptide, Benzyloxycarbonylation, formylation, nitration, sulfonation, aldehyde formation, cyclization, glycosylation, monomethylation, dimethylation, trimethylation, guanidylation, amidination, maleylation, trifluoroacetylation, carbamylation, tri Nitrophenylated, nitrotroponylated, polyethyleneglycolated or acetoacetylated derivatives. Among these, N-terminal acetylation, C-terminal amidation, and C-terminal methylation impart resistance to exopeptidase that degrades the polypeptide from the terminal, and also in vivo by glycosylation or polyethylene glycolation. It is preferable because stability is expected to be high.

  As used herein, “salt” refers to any pharmacologically acceptable salt (including inorganic and organic salts) of a polypeptide or a derivative thereof, such as sodium of the polypeptide or a derivative thereof. Salt, potassium salt, calcium salt, magnesium salt, ammonium salt, hydrochloride, sulfate, nitrate, phosphate, organic acid salt (acetate, citrate, maleate, malate, oxalate, lactate Succinate, fumarate, propionate, formate, benzoate, picrate, benzenesulfonate, etc.). Of these, 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.

  Moreover, the derivative | guide_body of the said polypeptide can be produced by a well-known method in the said field | area. Moreover, the salt of the said polypeptide can also be easily produced by those skilled in the art by any method known in the art.

  The inhibitor of aspect 1 is substantially constituted by the vasohibin, whereas the inhibitor of aspect 2 is constituted by a vector containing a polynucleotide encoding the vasohibin.

  The vector is preferably capable of autonomously replicating in a host cell, and at the same time, 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 vectors described below.

  The vector can be prepared according to a known method, for example, according to the method disclosed in WO02 / 090546, 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.

  In the present invention, the disease requiring a lymphangiogenesis-inhibiting action for treatment is not particularly limited as long as it has a therapeutic effect by inhibiting lymphangiogenesis. For example, lymph node metastasis of malignant tumor, lymph Vasculitis, lymphadenitis, lymphangioma, nevus, pulmonary lymphangioleiomyomatosis, corneal transplant, caboji sarcoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma and the like are exemplified. In particular, lymphangitis, lymphadenitis, lymphangioma, nevus, and pulmonary lymphangioleiomyomatosis are not expected to have a therapeutic effect even when angiogenesis is suppressed, and therefore the therapeutic agent of the present invention is expected to be applied. The

  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. In addition, as the therapeutic agent of the present invention, 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 optionally, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer. Add excipients, binders, disintegrants, lubricants, etc. to form solids such as tablets, granules, powders, powders and capsules, and liquids such as normal liquids, suspensions and emulsions. be able to. Moreover, it can also be used as a dried product which can be made liquid by adding an appropriate carrier before use, and other external preparations. The pharmaceutical carrier can be selected depending on 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 prepared by conventional methods using known pharmaceutical carriers and the like. In addition, the content of the inhibitor of the present invention in such a 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 something. 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. There is also no particular limitation on the administration method, and for example, it can be administered by internal use, external use or injection. When the therapeutic agent of the present invention is administered by injection, it can be administered, for example, intravenously, intramuscularly, subcutaneously, intradermally, and when administered externally, for example, as a topical agent such as a suppository, its suitable administration It may be administered by a method.

  The dosage of the therapeutic agent of the present invention is appropriately set depending on 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. Further, the administration may be performed once or divided into several times within one day within a desired dose range. The administration period is also arbitrary.

  The present invention also provides a method for treating a disease requiring a lymphangiogenesis-inhibiting action, comprising administering Vasohibin to a subject.

  In the present specification, the subject is preferably a human who needs a lymphangiogenesis inhibitory action, but may be a pet animal or the like.

  In the present specification, the effective amount is the amount of vasohibin that exerts an inhibitory action on lymphangiogenesis when vasohibin is administered to the subject as compared to a subject not administered vasohibin. The specific effective amount is appropriately set according to the administration form, administration method, purpose of use, age, weight, symptom, etc. of the subject, and is not constant.

  In the method for treating a disease requiring lymphangiogenesis-inhibiting action of the present invention, an effective amount of Vasohibin may be administered to the subject as it is, or may be administered as a medicament such as the above therapeutic agent. . Moreover, there is no limitation also in the administration method, For example, what is necessary is just to administer by oral administration, injection, etc. similarly to said pharmaceutical.

  According to the treatment method of the present invention, a disease targeted by the therapeutic agent of the present invention can be treated, and for example, an effect of treating a disease caused by lymphangiogenesis can be exhibited. .

  The lymphangiogenesis inhibitor comprising a vector comprising a polynucleotide encoding vasohibin of the present invention can be used for gene therapy in a patient having a disease caused by lymphangiogenesis.

  As a method for introducing the inhibitor comprising the vector of the present invention into a patient, an in vivo method in which the inhibitor is directly introduced into the body, and taking out certain cells from a human and introducing DNA into the cells outside the body, There is an ex vivo method for returning cells into the body [Nikkei Science, April, 20-45 (1994), Monthly Pharmaceutical Affairs, 36, 23-48 (1994), Experimental Medicine Extra Number, 12, 15 (1994)]. In the present invention, the in vivo method is preferable.

  When administered by the in vivo method, it is administered by an appropriate 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. In addition, direct injection of tissues with lesions is also preferred. Using any available in the art such as nuclear magnetic resonance imaging or computed tomography, the tissue in which the lesion is seen can be imaged, and for example, the inhibitor comprising the vector of the present invention can be administered by stereotaxic injection it can.

  When the inhibitor comprising the vector of the present invention is used as a gene therapy vector, the inhibitor can be in various dosage forms suitable for the above administration forms. For example, when an injection containing DNA encoding vasohibin which is an active ingredient is used, the injection can be prepared by a conventional method. The base used for the gene therapy agent is not particularly limited as long as it is a base 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, lactose, 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, and the like can be mentioned. In addition, according to a conventional method, a solution using an osmotic pressure adjusting agent, a pH adjusting agent, a vegetable oil such as sesame oil or soybean oil, or a surfactant such as lecithin or a nonionic surfactant in these bases. An injection may be prepared as a suspension or dispersion. These injections can be made into preparations for use and dissolution by operations such as pulverization 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. In general, the weight of DNA encoding Vasohibin is about 0.01 to 2000 mg, preferably 0.1 to 100 mg.

  Hereinafter, a method for producing Vasohibin, a method for producing a gene therapy vector, and the like will be specifically described.

(1) Preparation method of vasohibin Vasohibin is described in Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Biotechnology (19), Current Protocols in Molecule 19 (Cold Spring Harbor Laboratory Press). For example, the gene for vasohibin can be expressed in a host cell and produced by the following method.

  Based on the full-length DNA encoding the protein of vasohibin, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared. In addition, a base-substituted DNA is prepared so that the base sequence of the protein-encoding portion becomes an optimal codon for host expression. The DNA is useful for improving the production rate of the protein. A 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 host cell can be used as long as it can express the target gene, such as a prokaryotic cell, animal cell, or insect cell. As 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.

(I) When a prokaryotic organism is used as a host The expression vector for vasohibin protein is capable of autonomous replication in prokaryotes, and at the same time is composed of a promoter, a ribosome binding sequence, a gene encoding vasohibin, and a transcription termination sequence. Is preferred. A gene that controls the promoter may also be included.

  Examples of the expression vector include pBTrp2, pBTac1, pBTac2 (Roche Diagnostics), Bluescript II SK (+), pBluescript II SK (-) (Stratagene), pSTV28, pUC118, Takara Shuzo. ), PKK233-2 (Amersham Biosciences), pSE280, pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pGEX (Manufactured by Pharmacia), pET system (manufactured by Novagen), pMAL-c2 (manufactured by New England Biolabs), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 (Agri (Ulural Biological Chemistry, 48, 669 (1984)), pLSA1 (Agricultural Biological Chemistry, 53, 277 (1989)), pGEL1 (Proceeding of the National Academy 306). 172, 2392 (1990)), pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM B-6798), pPA1 (Japanese Patent Laid-Open No. 63-233798). , PTerm2 (JP-A-3-22979, US4686191, US4) 39094, US5160735) and the like can be exemplified.

  Any promoter can be used as long as it can be expressed in a host cell such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, SPO1 promoter, SPO2 promoter, penP promoter and the like can be mentioned. 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 letI promoter can also be used.

  Moreover, it is preferable to use 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 expression of the vasohibin gene, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.

  Examples of host cells include prokaryotes such as Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, and the like. E. coli strain XL1-Blue, XL2-Blue, DH1, MC1000, KY3276, W1485, JM109, HB101, No. 49 strains, W3110 strain, NY49 strain, BL21 (DE3) strain, BL21 (DE3) pLysS strain, HMS174 (DE3) strain, HMS174 (DE3) pLysS strain, etc. are known as Serratia sp. ficaria strain, S. fontico strain, S. liquidfaciens, S. et al. marcescens strains, etc. as Bacillius subtilis strain, An amyloliquefaciens strain or the like is classified as B. amoniagenes strains, B. p. Immariophilum (ATCC: 14068) strain, B.I. Saccharolyticum (ATCC: 14066) strain and the like are classified as C. glutamicum (ATCC: 13032) strain, C.I. glutamicum (ATCC: 14067) strain, C.I. gulutamicum (ATCC: 13869) strain, C.I. acetoacidophilum (ATCC: 13870) strain and the like are classified as M. Ammoniaphilum (ATCC: 15354) strain and the like are classified as S. genus Pseudomonas. Examples include mephitica strains.

  As a method for introducing the expression plasmid, any method can be used as long as it is a method for introducing DNA into the host cell. For example, electroporation (Nucleic Acids Research, 16, 6127 (1988)), calcium phosphate method ( Proceedings of the National Academy of Sciences, USA, 69, 2110 (1972)), protoplast method (Japanese Patent Laid-Open No. 63-2483942, Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111) (79). Examples of the method are described.

(Ii) When animal cells are used as hosts When animal cells are used as hosts, expression vectors include, for example, pcDNA1 / Amp, pcDNA1, pCDM8, pREP4 (Invitrogen), pHM6 (Roche Diagnostics), pKK223. -3, pGEX (manufactured by Amersham Biosciences), pAGE107 (Cytotechnology, 3, 133 (1990)), pAGE103 (The Journal of Biochemistry, 101, 1307 (1987)), pAMo, pAMoAro (hAMPRo) Chemistry, 268, 22782-2787 (1993)), pAS3-3 (Japanese Patent Laid-Open No. 2-22705), etc. can be used. Yes.

  Any promoter can be used as long as it can be expressed in the host. For example, human cytomegalovirus (hCMV) IE (Immediate-early) gene promoter, SV40 early promoter, Moloney strain murine leukemia virus (Long Termine Repeat Promoter) of (Moloney Murine Leulemia Virus), retrovirus promoter, HSP promoter, SRα promoter, promoter of metallothionein, and the like. An enhancer of hCMV IE gene may be used together with a promoter.

  Animal cells used for the host include human-derived cell lines HEK293 (human embryonic kidney cells, ATCC: CRL-1573), Namalwa (Burkitt lymphoma, ATCC: CRL-1432), HeLa (cervical 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 Sp2 / 0-Ag14 (mouse myeloma cells, ATCC: CRL-1581) and NSO (mouse myeloma cells); COS-1 of monkey-derived cell line (African green monkey kidney cells (SV40 transformed cells), ATCC: CRL-1650) and COS-7 (African green monkey kidney cells) (SV40 transformed cells), ATCC: CRL-1651); hamster-derived strain CHO-K1 (Chinese hamster ovary cells, ATCC: CCL-61) and BHK-21 (C-13) (Sicilian hamster offspring cells, ATCC: CCL-10); PC12 (adrenal brown cells) of rat-derived cell line Tumors, ATCC: CRL-1721) and YB2 / 0 (rat myeloma cells, ATCC: CRL-1662) and the like.

  As a method for introducing an expression plasmid, any method can be used as long as it is a method for introducing DNA into a host. For example, electroporation (Cytotechnology, 3, 133, (1990)), calcium phosphate method (Japanese Patent Laid-Open No. 2). 22705), lipofection method (Proceedings of the National Academy of Sciences, USA, 84, 7413 (1987), Virology, 52, 456 (1973)).

(Iii) When insect cells are used as a host When insect cells are used as a host, examples of expression vectors include pVL1392, pVL1393, pBlueBacIII, pFASTBac1 (manufactured by Invitrogen), and examples of infectious viruses include, Examples include baculovirus that infects insects, Autographa california nuclear polyhydrosis virus (AcMNPV) Bac-N-Blue DNA, and the like. Insect cell transformation methods are described, for example, in Baculovirus Expression Vector: A Laboratory Manual (1992) (WH Freeman and Company 198), Molecular Cloning: A Laboratory Mandatory (1998). , Current Protocols in Molecular Biology (1994) (Wiley-Interscience), Biotechnology, 6, 47 (1988).

  An expression vector containing the gene of interest and baculovirus DNA for infection of insect cells are added to the insect cell culture medium, and the virus expressing the gene of interest produced by recombination expresses basohibin by infecting insect cells be able to.

  Examples of insect cells used as the host include Spodoptera frugiperda (Yotoga) -derived strain cells, Trichoplusia ni (Nettle cinnamon) -derived strain cells, and the like. Frugiperda-derived cells include Sf9 (ATCC: CRL-1711, ovarian cells), Sf21 (ovarian cells), and the like. Examples of ni-derived cell lines include High Five, BTI-TN-5B1-4 (egg cells, manufactured by Invitrogen), and the like.

  As a method for introducing an expression plasmid, any method can be used as long as it can be introduced into a host. For example, the calcium phosphate method (JP-A-2-22705), the lipofection method (Proceedings of the National Academy of Sciences USA, 84, 7413 (1987)). In the lipofection method, CELLFECTIN reagent (Invitrogen) can be used. Moreover, the electroporation method (Cytotechnology, 3, 133 (1990)) etc. can be used like an animal cell.

(Iv) Transformant culture method When the transformant carrying an expression plasmid incorporating DNA encoding vasohibin is a cell such as Escherichia coli or animal cells, the transformant is cultured according to a normal culture method suitable for various hosts. The protein can be produced by producing and accumulating the protein and recovering the protein from the transformant or the culture solution. 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 recovered from the animal individual or plant individual. The protein can be produced.

  When the host is an animal individual, for example, a non-human transgenic animal carrying a gene encoding vasohibin is bred, vasohibin encoded by the plasmid is produced and accumulated in the animal, and the protein is extracted from the animal individual. By collecting, bathohibin can be produced. Examples of the production / accumulation location in an animal individual include milk, saliva, eggs and the like of the animal.

  When the host is a prokaryote such as Escherichia coli, for example, a transformant having a gene encoding vasohibin is cultured in a medium, and vasohibin encoded by the plasmid is produced and accumulated in the culture solution. By recovering the protein, bathohibin can be produced.

  The method for culturing a transformant of vasohibin in a medium can be performed according to a usual method used for culturing a host.

  As a medium for culturing the obtained transformant, a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the organism, and can efficiently culture the transformant. Any of the media may be used.

  Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol can be used.

  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium salts, other nitrogen-containing substances, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the 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. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.

  When the transformant is a prokaryotic organism such as Escherichia coli, such a medium is preferably a YT medium containing bactotryptone, yeast extract and sodium chloride, for example.

  The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when a transformant transformed with an expression vector using the lac promoter is cultured, a transformant transformed with isopropyl-β-D-thiogalactopyranoside or the like and an expression vector using the trp promoter is cultured. When doing so, indoleacrylic acid or the like may be added to the medium.

  When the transformant for producing vasohibin is an animal cell, a culture medium for culturing the cell is generally used RPMI1640 medium (The Journal of the American Medical 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 fetal calf serum ( A medium supplemented with FCS) or the like is used.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and in the presence of 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  When the transformant is an insect cell, the culture medium to be cultured is a commonly used TNM-FH medium (Pharmingen), Sf-900II SFM medium (Invitrogen), ExCell400, ExCell405 (JRH Bio). Sciences, Inc.), Grace's Insect Medium (Nature, 195, 788 (1962)) and the like can be used.

(V) Production Method Vasohibin can be produced by culturing a transformant and isolating and purifying basohibin from the culture solution. The method for isolating and purifying vasohibin can be carried out by conventional methods well known and commonly used in the art. For example, the method for isolating and purifying enzymes and the method for purifying glycosyltransferases of Sandler et al. (Methods in Enzymology, 83, 458). ) Can be used.

  When vasohibin is produced and accumulated as a soluble polypeptide, the culture solution obtained by culturing the transformant as described above is separated into cells or cells and a medium by a method such as centrifugation. When Vasohibin is present in the host cells, the collected cells or cells are washed with an appropriate buffer such as STE solution, and then the cells or cells are crushed with ultrasonic waves, French press, Mantongaurin homogenizer, dynomill, etc. It can be obtained as a cell-free solution by centrifugation or filtration.

  An appropriate amount of a surfactant may be contained in the buffer used for separation and purification of vasohibin, and for example, sodium lauryl sulfate (SDS), sodium N-lauroyl sarcosine (sarcosyl), and the like may be included.

  The method for separating and purifying 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, ultrafiltration, gel filtration, diethylaminoethyl (DEAE) -Sepharose chromatography, DIAION HPA Anion chromatography using lysine such as -75 (Mitsubishi Chemical) or ion exchange chromatography, cation chromatography using lysine such as S-Sepharose FF (Pharmacia), hydrophobicity such as butyl sepharose Examples include various chromatographic methods such as chromatography and affinity chromatography, various electrophoretic methods such as SDS-polyacrylamide gel electrophoresis and isoelectric focusing. Affinity chromatography can also be performed by using an antibody against vasohibin.

  When vasohibin is produced / accumulated as an insoluble polypeptide, the cells or cells are separated in the same manner as described above, and after disruption by an appropriate method, the fraction containing the polypeptide is recovered. The collected sample is solubilized with a solubilizer such as a surfactant such as sodium lauryl sulfate (SDS) or sodium N-lauroyl sarcosine (sarkosyl). The solubilized solution is diluted or dialyzed to a concentration containing little or no solubilizing agent to form the polypeptide into a normal three-dimensional structure, and then purified by the same separation / purification method as described above. Goods can be obtained.

  Vasohibin can also be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance having affinity for the fused protein (Akio Yamakawa, Experimental Medicine, 13, 469-). 474 (1995)). Examples of the additional protein used for the fusion protein include Protein A, FLAG and the like (Proceedings of the National Academy of Sciences, USA, 86, 8227 (1989), Genes Development, 4, 1288 (1990), Japanese Patent Laid-Open No. Hei 5- 336963, Japanese Patent Laid-Open No. 6-83021). 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 a FLAG peptide is used, it can be purified by producing a fusion protein of vasohibin and FLAG and performing affinity chromatography using an anti-FLAG antibody.

  In addition to the above-described method using a transformant, Vasohibin can also be produced using an in vitro transcription / translation system according to a known method (Journal of Biomolecular NMR, 6, 129-134 ( 1995), Science, 242, 1162-1164 (1988), The Journal of Biochemistry, 110, 166-168 (1991)).

  Vasohibin is based on its amino acid sequence, such as chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method), and commercially available peptide synthesis equipment, for example, Chemical synthesis is performed by peptide synthesis equipment such as APEX396 (manufactured by Advanced Chemtech), 433A (manufactured by Applied Biosystems), PS3 (manufactured by Protein Technologies), 9050 (manufactured by Perceptive), PSSM-8 (manufactured by Shimadzu Corporation). be able to.

(2) Gene therapy vector production method The gene therapy vector production method, cell expression method, and the like are the same as the expression vector described in the above-mentioned "Method for producing Vasohibin".

  Since expression vectors are safe and have low toxicity, they can be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cattle, cats, dogs, monkeys, etc.). 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 has high safety. Preferred viral vectors for gene therapy include adenovirus, adeno-associated virus (AAV), retrovirus, poxvirus, herpes virus, herpes simplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV), vaccinia. A virus, a poliovirus, a symbis virus, SV40 etc. are mentioned. Preferred plasmids for gene therapy include pCAGGS (Gene, 108, 193-200 (1991)), pBK-CMV, pcDNA3.1, pZeoSV (Invitrogen, Stragene) and the like.

  In addition, by adding a signal sequence to DNA encoding Vasohibin, it becomes a secreted protein, and it is not always necessary to administer it locally, but the protein produced and secreted in the cell acts on a distant target organ, causing lymphangiogenesis. Inhibits action. Therefore, administration into normal tissues or normal cells other than pathological tissues is also possible. In addition, when administering to a human, intravenous administration or intramuscular administration is preferable.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following Example. As a genetic manipulation method, unless otherwise specified, a method described in Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory) was used.

Preparation Example 1 of Vasohibin Protein
Vasohibin protein was produced and purified by baculovirus / insect cell expression system. A cDNA encoding a KIAA1036 of Vasohibin 1 (region consisting of amino acids 1 to 365 of SEQ ID NO: 2) with a 3xFLAG sequence added to the C-terminal is inserted into an insect cell expression vector pFASTBac1 (Invitrogen). Insect cell expression plasmid pFASTBac1036 was constructed. For the expression in insect cells, Bac-To-Bac Baculovirus Expression Systems (Invitrogen) was used, and the operation was performed according to the attached manual. That is, the constructed pFASTBac1036 plasmid was transformed into DH10Bac E. coli. E. coli was transformed to obtain recombinant Bacmid DNA. Next, this Bacmid DNA was introduced into Sf9 cells using CELLFECTIN reagent (Invitrogen) to obtain a recombinant baculovirus. 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. Confirmation of the expression of the FLAG-added vasohibin protein by insect cells was performed by Western blotting using this Sf9 cell extract and HRP-labeled anti-FLAG M2 antibody (Sigma). First, the cells were washed once with 5 mL of phosphate buffered saline (PBS) and then 5 mL of Lysis solution (0.15 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.1 mM). After suspending in amiderophenyl methanol fluoride, 0.1% NP-40, 50 mM Tris-HCl, pH 7.4), sonication with MICROCON (made by HEART SYSTEMS) for 15 seconds for 4 seconds Thereafter, the mixture was centrifuged at 14,500 × g for 20 minutes, and an lysate solution was prepared by adding an equal amount of Lysis solution to the supernatant. The Western blot was separated by SDS-PAGE according to the method of Laemli, 0.5 μL was electrically transferred to a nitrocellulose membrane (Tefco), blocked in TBS containing 5% skim milk, and labeled with HRP. And reacted with the anti-FLAG M2 antibody. After washing with TBS containing 0.05% Triton X-100 (Tris-HCl containing 0.15 M NaCl, pH 7.4) three times for 10 minutes, chemiluminescence was performed using ECL-Plus reagent (Amersham Pharmacia), Was detected by exposing to Hyperfilm ECL film (Amersham Pharmacia). The FLAG-added vasohibin protein was purified by adding the lysate solution to an anti-FLAG M2 affinity gel column (Sigma) equilibrated with TBS containing 0.1% NP-40, washing with the same solution, Elution with Gly-HCl (pH 3.5) containing 0.1% NP-40 (1 mL / fraction) and immediately neutralization with 1 M Tris-HCl, pH 8.0 (20 μL for 1 mL of eluate) Went by. A single band of about 50 kDa was confirmed by Western blotting of each fraction or staining with Bio-Safe Coomassie (Bio-Rad) after SDS-PAGE. Further, after transcription to SDS-PAGE and PVDF membrane (BIO-RAD), this band was eluted and the amino acid sequence of the N-terminal part of this polypeptide was analyzed using an amino acid sequencer. Since it matched with the amino acid sequence from the 2nd position to the 10th position, it was confirmed that this protein is a vasohibin protein.

Test example 1
Whether the obtained vasohibin has the ability to suppress lymphangiogenesis is described in J. Org. Immunol. 170: 5704-5711, (2003) and FASAB J. et al. 18: 300-310, (2004). The mouse corneal micropocket assay was used.

  80 ng or 12.5 ng FGF-2 protein (fibroblast growth factor-2, bFGF, BD Biosciences), 160 ng VEGF-A protein (Sigma), 160 ng VEGF in 0.3 μg hydrone reagent (IFN Sciences) -C protein (Acris Antibodies) or 80 ng PDGF-BB protein (R & D Systems) was mixed, 5 ng Vasohibin obtained above was added, and transplanted to the cornea of BALB / C male mice. Seven days later, the eyeball was removed and immobilized with 4% paraformaldehyde / TBS (20 mM Tris-HCl, pH 8.0, 150 mM NaCl) at 4 ° C. overnight. Washing with TBS-T (0.1% Tween20 / TBS) for 5 minutes was performed three times, and blocking reaction was performed with 2% BSA / TBS-T at 4 ° C. for 1 hour. Lymphatic tube staining with LYVE1 antibody was performed at 4 ° C. overnight with a 300-fold diluted anti-mouse LYVE1 antibody (transferred from Dr. Yuichi Oike, Keio University School of Medicine), followed by washing with TBS-T for 20 minutes four times. A 500-fold diluted Anti-Rat IgG-Alexa Fluor 488 (Molecular Probes) was reacted as a secondary antibody for 6 hours at 4 ° C., and washed with TBS-T for 20 minutes four times. The stained specimen thus prepared was photographed with a fluorescence microscope, and an image of LYVE1 staining was quantitatively analyzed with an image analysis software National Institutes of Health (NIH) image. The results are shown in FIGS.

  1 to 4, it was found that lymphangiogenesis induced by bFGF, VEGF-A, VEGF-C and PDGF-BB is significantly suppressed in the presence of vasohibin.

  The therapeutic agent containing vasohibin of the present invention is suitably used, for example, for the treatment of diseases that require lymphangiogenesis inhibition.

Sequence number 1 of a sequence table is KIAA1036 polynucleotide.
Sequence number 2 of a sequence table is KIAA1036 polypeptide.
Sequence number 3 of a sequence table is AY833422 polynucleotide.
Sequence number 4 of a sequence table is AY833422 polypeptide.

Claims (2)

  A pharmaceutical composition for treating a disease requiring a lymphangiogenesis-inhibiting action selected from the group consisting of lymphangitis, lymphadenitis, lymphangioma, nevus, and pulmonary lymphangioleiomyomatosis, comprising vasohibin.   A lymphangiogenesis inhibitory action selected from the group consisting of lymphangitis, lymphadenitis, lymphangioma, nevus, and pulmonary lymphangioleiomyomatosis containing a vector comprising a polynucleotide encoding vasohibin A pharmaceutical composition for treating a necessary disease.

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