WO2009096425A1 - Neovascularization-promoting factor - Google Patents

Abstract

Disclosed is a neovascularization promoter comprising a polypeptide selected from the following polypeptides (A) and (B): (A) a polypeptide comprising an amino acid sequence selected from the amino acid sequences depicted in SEQ ID NOs:2, 4, 6, 8 and 10; and (B) a polypeptide which comprises an amino acid sequence having the deletion, substitution or addition of one or several amino acid residues in the amino acid sequence for the polypeptide (A), and which has a neovascularization-promoting activity. A therapeutic agent comprising a variant 1, 2, 3, 4 or 5 can be used suitably, for example, for the treatment of diseases for which a neovascularization-promoting activity is required.

Description

Angiogenesis-promoting factor

The present invention relates to an angiogenesis promoter. More specifically, the present invention relates to an angiogenesis promoter comprising a specific splicing variant of vasohibin, and a pharmaceutical composition for treating a disease requiring an angiogenesis promoting action, containing the promoter.

Angiogenesis refers to a phenomenon in which new blood vessels are formed by migration, proliferation and lumen formation of vascular endothelial cells from existing venules and capillaries in animal tissues or organs. Such a phenomenon occurs not only in the morphogenesis period or growth period of animals, but also in the healing of damage to tissues, the repair process of inflammation, and the menstrual cycle. Angiogenesis is controlled, for example, by the balance of expression of an endogenous factor such as VEGF that promotes angiogenesis and a factor that inhibits angiogenesis such as thrombospondin. On the other hand, there are also diseases accompanied by abnormal angiogenesis due to an imbalance of the angiogenesis regulation mechanism. Therefore, in recent years, application to the treatment of various diseases is expected by controlling those angiogenesis regulatory factors.

As factors that control angiogenesis, FGF, VEGF, HGF, and the like are known. For example, in peripheral vascular diseases such as ischemic heart disease such as myocardial infarction, obstructive arteriosclerosis, and Buerger's disease, Treatments that promote angiogenesis using factors have been reported (see Non-Patent Documents 1 to 3). On the other hand, in diabetics and malignant tumors, vascular proliferation causes serious symptoms, and thus a treatment for suppressing angiogenesis using the above-described factors has been reported (see Non-Patent Document 4).

In addition, in the non-patent document 5 and the patent document 1, the present inventors have disclosed AK022567 protein and splicing variants BC051856 protein, BC053836 protein, BC028194 protein, and AY833422 as novel angiogenesis inhibitory factors. Reporting protein.
Motokuni Aoki et al., Japanese Clinical Practice, Vol. 64, No. 4, April 2006 Makino Hiroshi et al., Japanese Clinical Practice, Vol. 63, No. 3, March 2005 Makino Hiroshi et al., Japanese Clinical Practice, Volume 61, Special Issue 4, 2003 Masafumi Shibuya, Biochemistry, Vol. 76, No. 12, 2004 Shibuya T. Et al., Arterioscler Thromb Vasc Biol., Vol. 2, No. 5, pp. 1051-1057, 2006 WO2006 / 073052 pamphlet

An object of the present invention is to provide an angiogenesis promoter comprising a splicing variant of AK022567 protein represented by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10, and a disease requiring an angiogenesis control action containing the promoter. The present invention relates to a therapeutic pharmaceutical composition.

The present inventors have so far clarified that Vasohibin acts as an anti-angiogenic factor in a situation where it coexists with an anti-angiogenic factor such as VEGF. However, the specific splicing variant of Vasohibin, that is, the splicing variant represented by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 is present in a hypoxic state that is also a disease state such as cancer or cerebrovascular disorder. In some cases, it was surprisingly found that it has an angiogenesis-promoting effect and the present invention has been completed.

That is, the present invention
[1] An angiogenesis promoter comprising the following polypeptide (A) or (B):
(A) A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted or added An angiogenesis-promoting agent comprising a vector comprising a polynucleotide encoding a polypeptide of the following (A) or (B):
(A) A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted or added An angiogenesis-promoting agent comprising a vector comprising the following polypeptide (3) or (D):
(C) the nucleotide sequence represented by the 351st A to the 1223rd G of SEQ ID NO: 1, the nucleotide sequence represented by the 311st A to the 1246th G of SEQ ID NO: 3, the 327th of the SEQ ID NO: 5 A nucleotide sequence selected from the group consisting of the nucleotide sequence represented by A from the 836th to A, the nucleotide sequence represented by the 300th A to the 770th A of SEQ ID NO: 7, and the nucleotide sequence represented by the SEQ ID NO: 9 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence of polynucleotides (D) and (C) comprising a polynucleotide that hybridizes under stringent conditions and encodes a polypeptide having an angiogenesis promoting action The angiogenesis promoter according to [2] or [3] above, wherein the polynucleotide [4] vector is a viral vector,
[5] The angiogenesis promoter according to [4], wherein the viral vector is an adenoviral vector,
[6] The angiogenesis promoter according to any one of [1] to [5] above, which is administered intravenously,
[7] A pharmaceutical composition for treating a disease requiring an angiogenesis promoting action, comprising the promoter according to any one of [1] to [6],
[8] Use of the polypeptide defined in [1] for the manufacture of a therapeutic agent for a disease requiring angiogenesis promoting action,
[9] Use of the polynucleotide defined in [2] or [3] above for the manufacture of a therapeutic agent for a disease requiring angiogenesis promoting action,
[10] A method for treating a disease requiring an angiogenesis promoting action, comprising a step of administering the angiogenesis promoting agent according to any one of [1] to [6].
[11] The polypeptide as defined in [1] above for use in the treatment or prevention of diseases requiring angiogenesis promoting action, and [12] for use in the treatment or prevention of diseases requiring angiogenesis promoting action. And [2] or [3].

Angiogenesis can be promoted by the promoter of the present invention.

FIG. 1 shows a photograph (FIG. 1A) of an operation performed when angiogenesis is evaluated in a hypoxic angiogenesis model in a subcutaneous mouse (FIG. 1A), an enlarged photograph of the treatment site (FIG. 1B), a photograph of each section (FIG. 1C), and blood vessels FIG. 2 shows vascular amplification by neoplasia (FIG. 1D). FIG. 2 is a view showing expression of variant 5 (FIG. 2A) and a view showing cells derived from variant 5 (FIG. 2B) at an angiogenesis site in a hypoxic angiogenesis model under the mouse. FIG. 3 is a diagram showing blood vessel amplification by angiogenesis in a mouse hypoxic angiogenesis model in which human Vasohibin-1 or variant 5 is highly expressed. FIG. 4 is a view showing blood vessel amplification by angiogenesis in a hypoxic angiogenesis model of a variant 5 gene deficient mouse [variant 5 (+/−): hetero deficient mouse, variant 5 (− / −): homo deficient mouse]. It is. FIG. 5 shows a comparison of the expression level of vasohibin in a variant 5 gene-introduced cell (FIG. 5A), a result of an endothelial cell proliferation assay between a parent cell and a variant 5 gene-introduced cell group (bulk) (FIG. 5B), It is a figure which shows the result (FIG. 5C) of the endothelial cell proliferation assay of an expression cell.

The present invention has a great feature in using a protein represented by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10 in promoting angiogenesis. As an angiogenesis promoter of the present invention, SEQ ID NO: An accelerator consisting of a polypeptide represented by an amino acid sequence selected from the group consisting of 2, 4, 6, 8, and 10 (Aspect 1) and an amino acid selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, and 10 A promoter comprising a polynucleotide comprising a polynucleotide encoding a polypeptide represented by a sequence, that is, a vector comprising a polynucleotide represented by a base sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, and 9. (Aspect 2) is exemplified, and both are used from the viewpoint of promoting angiogenesis. The polypeptide represented by the amino acid sequence of SEQ ID NO: 2 is AK022567 protein (variant 1) reported as a variant of Vasohibin in WO2006 / 073052. The polypeptides represented by the amino acid sequences of SEQ ID NOs: 4, 6, 8, and 10 are the four splicing variants of variant 1 described in WO2006 / 073052, and are reported as variants 2, 3, 4, and 5, respectively. Yes. These variants are already known to be highly homologous to Vasohibin. 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 angiogenesis. Therefore, Variants 1 to 5 were expected to have an excellent effect on angiogenesis suppression because of their high homology with Vasohibin. Surprisingly, it was surprising that polymorphs of the variants were added to hypoxic mouse angiogenesis models. It is found that angiogenesis is promoted when nucleotides are administered from the tail vein and expressed, and it is thought that angiogenesis can be promoted more strongly by administering such a variant or the gene from outside the body. It was.

Vasohibin includes vasohibin 1 and vasohibin 2, and as disclosed in WO02 / 090546, WO2006 / 073052, etc., vasohibin 1 and vasohibin 2 are different genes existing on different chromosomes. The amino acid sequence of the protein encoded by the gene has a homology of 58%, and both have an inhibitory activity on angiogenesis. Protein encoded by the polypeptide represented by the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, or 10 and the polynucleotide represented by the base sequence of SEQ ID NO: 1, 3, 5, 7, or 9 in the present invention Is included in the variant of Vasohibin 2.

The polypeptide in the present invention includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8 or 10 (hereinafter also referred to as variant 1, 2, 3, 4, 5 respectively), 1 in the amino acid sequence. Alternatively, polypeptides having an amino acid sequence in which several amino acids are deleted, substituted or added, and having an angiogenesis-promoting action, and derivatives thereof, and salts thereof are exemplified.

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.

In addition, a derivative of the above polypeptide can be prepared by a method known in the art. 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 polynucleotide in the present invention is a polynucleotide comprising the base sequence of SEQ ID NO: 1, 3, 5, 7 or 9, more specifically, the base represented by 351st A to 1223rd G of SEQ ID NO: 1. Sequence, base sequence represented by 311st A to 1246th G of SEQ ID NO: 3, base sequence represented by 327th A to 836th A of SEQ ID NO: 5, 300th A of SEQ ID NO: 7 770 to a polynucleotide comprising the nucleotide sequence represented by A or SEQ ID NO: 9, and a polynucleotide capable of hybridizing under stringent conditions with the polynucleotide or the complementary strand thereof. And the polynucleotide which codes polypeptide which has an angiogenesis promotion effect is illustrated.

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 to 2-fold concentration of SSC (Saline Sodium Citrate: 150 mM sodium chloride, 15 mM sodium citrate) solution. It refers to a polynucleotide which can be identified by the. 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.

As used herein, “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, a polynucleotide having at least 60% or more, preferably 80% or more, more preferably 95% or more homology with the polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 as such a polynucleotide. 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 the polypeptide or DNA encoding the 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.

The promoter of aspect 1 is substantially constituted by the variant 1, 2, 3, 4 or 5, and the promoter of aspect 2 contains a polynucleotide encoding the variant 1, 2, 3, 4 or 5. It is comprised by the vector formed.

The vector is preferably composed of a promoter, a ribosome binding sequence, a gene encoding variants 1, 2, 3, 4 or 5 and a transcription termination sequence, as well as being capable of autonomous replication in a host cell. 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 promoter of the present invention is preferably administered intravenously because it exerts an angiogenesis promoting action at the site where Variant 1, 2, 3, 4 or 5 is delivered via circulating blood.

The present invention also provides a pharmaceutical composition for treating a disease requiring an angiogenesis promoting action, that is, a therapeutic agent, comprising the promoter of the present invention.

In the present invention, the disease requiring an angiogenesis-promoting action for treatment is not particularly limited as long as it has a therapeutic effect by promoting angiogenesis. For example, obstructive peripheral vascular disease, ischemic heart disease And obstructive arteriosclerosis, Buerger's disease, cerebrovascular disorder, intermittent claudication and the like. Especially, application of the therapeutic agent of this invention is anticipated for obstructive arteriosclerosis.

Examples of the therapeutic agent of the present invention include those prepared by combining the promoter of the present invention with a known pharmaceutical carrier. In addition, as the therapeutic agent of the present invention, variants 1, 2, 3, 4 or 5 can be used for other components that can be used in the same application as the variant, for example, components having an action of promoting known angiogenesis, such as VEGF Can also be blended.

In the production of the therapeutic agent of the present invention, the promoter of the present invention is usually pharmaceutically used as long as it can produce a dosage form in which the variant can reach a site requiring an angiogenesis promoting action through circulating blood. It is carried out by blending with an acceptable liquid or solid carrier, and if necessary, adding a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, etc. , Solid agents such as granules, powders, powders, capsules, etc., and liquids such as normal solutions, suspensions, and emulsions. 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. In this specification, the site | part which requires an angiogenesis promotion effect means the onset site | part of the disease which requires the said angiogenesis promotion effect | action.

The therapeutic agents in various preparation forms as described above can be appropriately produced by conventional methods using known pharmaceutical carriers and the like. In addition, the content of the promoter 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 promoter 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 as long as the variant can be delivered via circulating blood, and can be administered by, for example, 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 according to the preparation form, administration method, purpose of use, and age, weight, and symptoms of the patient who is the administration target of the therapeutic agent, 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 the use of Variant 1, 2, 3, 4 or 5 and the use of a polynucleotide encoding the variant for the manufacture of a therapeutic agent for a disease requiring angiogenesis-promoting action. In addition, the present invention provides Variant 1, 2, 3, 4 or 5 and a polynucleotide encoding the variant for use in the treatment or prevention of diseases requiring angiogenesis-promoting action.

The present invention also provides a method for treating a disease requiring an angiogenesis promoting action, comprising the step of administering Variant 1, 2, 3, 4 or 5 to a subject.

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

In the present specification, the effective amount means an angiogenesis-promoting action when variant 1, 2, 3, 4 or 5 is administered to the subject as compared to a subject not administered with the variant. The amount of the variant that exhibits 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 angiogenesis-promoting action of the present invention, an effective amount of variant 1, 2, 3, 4 or 5 may be administered to the subject as it is, and the therapeutic agent as described above, etc. It may be administered as a pharmaceutical. 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 that is a target of the therapeutic agent of the present invention can be treated, and for example, an effect of treating a disease requiring an angiogenesis promoting action can be exhibited.

An angiogenesis-promoting agent comprising a vector comprising a polynucleotide encoding variant 1, 2, 3, 4 or 5 of the present invention is used for gene therapy in a patient with a disease requiring angiogenesis-promoting action. Can do.

As a method for introducing the promoter comprising the vector of the present invention into a patient, an in vivo method in which the promoter is directly introduced into the body, and taking out certain cells from humans 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, an in vivo method is preferable.

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. In addition, direct injection of tissues with lesions is also preferred. Taking a tissue with lesions using any available in the art such as nuclear magnetic resonance imaging or computer tomography, for example, administering a promoter comprising the vector of the present invention by stereotaxic injection it can.

When the promoter comprising the vector of the present invention is used as a gene therapy vector, the promoter can take various dosage forms suitable for the above administration forms. For example, when an injection containing DNA encoding variant 1, 2, 3, 4 or 5 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 variant 1, 2, 3, 4 or 5 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 is adjusted as appropriate. In general patients (with a body weight of 60 kg), the weight of DNA encoding the variant is generally about 0.01 to 2000 mg, preferably 0.1 to 100 mg.

Hereinafter, a method for preparing variants 1 to 5, a method for preparing a gene therapy vector, and the like are specifically described.

(1) Production Method of Variants 1 to 5 Variants 1 to 5 are obtained from Molecular Cloning: A Laboratory Manual, Second Edition (1989) (Cold Spring Harbor Laboratory Press), Current Protocols in Biomolecule 19 (Current Protocols in Molecule 19). For example, the gene of variant 1, 2, 3, 4 or 5 can be expressed in a host cell and produced by the following method.

Based on the full-length DNA encoding the protein of variant 1, 2, 3, 4 or 5, if necessary, a DNA fragment having an appropriate length including 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. A transformant producing Variant 1, 2, 3, 4 or 5 can be obtained by introducing the expression plasmid into a host cell suitable for the expression vector.

As the host cell, any prokaryotic cell, animal cell, insect cell, etc. can be used as long as it can express the target gene. The expression vector can be autonomously replicated in the host cell or can be integrated into the chromosome, and contains a promoter at a position suitable for transcription of the gene encoding variant 1, 2, 3, 4 or 5. What is used is used.

(I) When a prokaryote is used as a host The expression vector for variants 1, 2, 3, 4 or 5 is capable of autonomous replication in prokaryotes, and at the same time, a promoter, a ribosome binding sequence, and a gene encoding the variant It is preferably composed of a transcription termination sequence. A gene that controls the promoter may also be included.

Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (Roche Diagnostics), Bluescript II SK (+), pBluescript II SK (-) (Stratagene), pSTV28, pUC118, pUC19 (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 (A ricultural Biological Chemistry, 48, 669 (1984)), pLSA1 (Agricultural Biological Chemistry, 53, 277 (1989)), pGEL1 (Proceeding of the National Acer 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, U S4686191, US4939094, US5160735), etc. can be illustrated.

As the promoter, any promoter can be used so 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.

It is also preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start 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 variants 1 to 5, but it is preferable to place the transcription termination sequence immediately below the structural gene.

Examples of host cells include prokaryotic organisms such as Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, and Pseudomonas, and Escherichia. E. coli strain XL1-Blue, XL2-Blue, DH1, MC1000, KY3276, W1485, JM109, HB101, 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 an 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) 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. wear.

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 murine leukemia virus Long terminal repeat promoters of (Moloney Murine Leuleemia Virus), retroviral promoters, HSP promoters, SRα promoters, promoters of metallothioneins, and the like. An enhancer of hCMV IE gene may be used together with a promoter.

Animal cells used as hosts 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); derived from hamster Cells CHO-K1 (Chinese hamster ovary cells, ATCC: CCL-61) and BHK-21 (C-13) (Sicilian hamster offspring cells, ATCC: CCL-10); rat-derived cell line PC12 (adrenal brown cells) Tumors, ATCC: CRL-1721) and YB2 / 0 (rat myeloma cells, ATCC: CRL-1662).

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), and 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 thereof include baculovirus (Vaculovirus) Autographa california nuclear polyhydrovirus (AcMNPV) Bac-N-Blue DNA that infects insects. 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 Manual, 1989. , Current Protocols in Molecular Biology (1994) (Wiley-Interscience), Biotechnology, 6, 47 (1988).

Variants 1 and 2 are obtained by adding an expression vector containing a target gene to an insect cell culture medium and baculovirus DNA for infection of insect cells, and infecting insect cells with a recombinantly produced virus expressing the target gene. 3, 4 or 5 can be expressed.

Examples of insect cells used as a 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 having an expression plasmid incorporating DNA encoding variant 1, 2, 3, 4 or 5 is a cell such as E. coli or animal cells, The protein can be produced by culturing according to a suitable normal culture method, 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 variant 1, 2, 3, 4 or 5 is bred, and the variant encoded by the plasmid is produced and accumulated in the animal. Variants 1, 2, 3, 4 or 5 can be prepared by recovering the protein from the individual animal. 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 carrying a gene encoding variant 1, 2, 3, 4 or 5 is cultured in a medium, and the variant encoded by the plasmid is used as a culture solution. Variants 1, 2, 3, 4 or 5 can be produced by producing and accumulating and recovering the protein from the culture solution.

The method of culturing the transformant of variant 1, 2, 3, 4 or 5 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 prokaryote 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 cultivating a transformant transformed with an expression vector using the lac promoter, cultivate a transformant transformed with isopropyl-β-D-thiogalactopyranoside or the like with an expression vector using the trp promoter. When doing so, indoleacrylic acid or the like may be added to the medium.

When the variant 1, 2, 3, 4 or 5 production transformant is an animal cell, the culture medium for culturing the cell is a commonly used RPMI 1640 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 (Processings of the Society for Biologic Medicine, 73, 1 (1950) Or a medium obtained by adding fetal calf serum (FCS) or the like to these mediums.

Culturing is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 25 to 40 ° C., and 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). Grace's Insect Medium (Nature, 195, 788 (1962)), etc. can be used.

(V) Production method Variant 1, 2, 3, 4 or 5 can be produced by culturing a transformant and isolating and purifying the variant from the culture solution. Methods for isolating and purifying variants can be performed by conventional methods well known and commonly used in the art. For example, methods for isolating and purifying enzymes and methods for purifying glycosyltransferases of Sandler et al. (Methods in Enzymology, 83, 458). ) Can be used.

When Variant 1, 2, 3, 4 or 5 is produced / accumulated as a soluble polypeptide, the culture solution obtained by culturing the transformant as described above is treated with cells or cells by a method such as centrifugation. Separate into medium. When variant 1, 2, 3, 4 or 5 is present in the host cell, the collected cells or cells are washed with an appropriate buffer solution such as STE solution, and then ultrasonic, French press, Manton Gaurin homogenizer, dynomill The cells or cells can be crushed with a method and the like, and obtained as a cell-free solution by centrifugation or filtration.

The buffer used for the separation / purification of variants 1, 2, 3, 4 or 5 may contain an appropriate amount of a surfactant, such as sodium lauryl sulfate (SDS) and sodium N-lauroyl sarcosine (sarcosyl). 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 known separation and purification methods. 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), ion exchange chromatography, cation chromatography using lysine such as S-Sepharose FF (GE Healthcare), hydrophobic such as butyl sepharose Examples include various chromatographic methods such as affinity chromatography and affinity chromatography, and various electrophoresis methods such as SDS-polyacrylamide gel electrophoresis and isoelectric focusing. Affinity chromatography can also be performed by using an antibody against variant 1, 2, 3, 4 or 5.

When variants 1, 2, 3, 4 or 5 are produced / accumulated as insoluble polypeptides, the cells or cells are isolated in the same manner as described above, and after disruption by an appropriate method, the fractions containing the polypeptides are 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.

Variant 1, 2, 3, 4 or 5 can also be produced as a fusion protein with other proteins and purified using affinity chromatography using a substance having an affinity for the fused protein (Yamakawa) Akio, Experimental Medicine, 13, 469-474 (1995)). Examples of the additional protein used for the fusion protein include protein A, FLAG and the like (Processings of the National Academy of Sciences, USA, 86, 8227 (1989), Genes Development, 4, 1288 (1990), Japanese Patent Laid-Open No. Hei 5- 336963, JP-A-6-83021). When protein A is used, it can be purified by producing a fusion protein of variant 1, 2, 3, 4 or 5 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 variant 1, 2, 3, 4 or 5 and FLAG and performing affinity chromatography using an anti-FLAG antibody.

In addition to the method using the above transformant, Variant 1, 2, 3, 4 or 5 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)).

Variants 1, 2, 3, 4 or 5 are based on their amino acid sequences, such as chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonyl method), and commercially available methods. Peptide synthesizers, 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) It can be chemically synthesized with a peptide synthesis apparatus such as

(2) Gene therapy vector production method The gene therapy vector production method, cell expression method and the like are the same as the expression vectors described in "Methods for producing variants 1-5" above.

Since expression vectors are safe and have low toxicity, they can be administered to, for example, mammals (eg, humans, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). When used for gene therapy, it is preferable to use a highly safe DNA or RNA viral vector or plasmid vector that can express a protein in mammalian cells including humans. Preferred viral vectors for gene therapy include adenovirus, adeno-associated virus (AAV), retrovirus, pox virus, herpes virus, herpes simplex virus, lentivirus (HIV), Sendai virus, Epstein-Barr virus (EBV), vaccinia Examples thereof include vectors derived from viruses, polioviruses, synbisviruses, SV40 and the like. 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 variant 1, 2, 3, 4 or 5, it becomes a secreted protein, and it is not always necessary to administer locally, and the protein produced and secreted in the cell is far away. Acts on target organs and produces lymphangiogenesis inhibition. 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.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples. As a genetic manipulation method, unless otherwise specified, the method described in Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory) was used.

Production example 1 of variant 5 protein
Single-stranded cDNA was synthesized using 1st Strand cDNA Synthesis Kit (Roche Diagnostics) using total RNA prepared from human umbilical vein endothelial cells using ISOGEN (Nippon Gene) as a template. To this was added a primer pair, 5′-GCAGCCATGAAGCCAACCGT-3 ′ and 5′-CCATACCGGCCAGCAAGAG-3 ′, and RT-PCR reaction was performed. The synthesized DNA fragment was cloned into the plasmid pCR2.1TOPO, and the base sequence of the insert was confirmed by a sequencing reaction. Next, using this plasmid as a template, PCR reaction was performed using the primer pair 5′-TAGCGGCCCCCACCCATGACCGGCTCCCGCG-3 ′ and 5′-TGTCTAGACTCAATTCGGATTTGATAGCCCCA-3 ′, and the resulting DNA fragment was cloned into the plasmid p3xFLAG-CMV14. A FLAG tag was connected to the 3 ′ end, and this DNA fragment was further inserted into an expression vector pFastBac1 (manufactured by Invitrogen) for baculovirus system. For expression in insect cells, a Bac-To-Bac Baculovirus Expression System (manufactured by Invitrogen) was used, and the operation was performed according to the attached manual. That is, E. coli DH10Bac was transformed with the constructed plasmid to obtain recombinant Bacmid DNA. Next, this Bacmid DNA was introduced into Sf9 cells with CELLFECTIN reagent (Invitrogen) to obtain a recombinant baculovirus solution. The virus solution 0.5 mL was adjusted to a concentration of 1.5 × 10 ⁶ cells / mL, and then used at a rate of infecting 50 mL of Sf9 cells. The cells were cultured for 76 hours after infection, and the cells were collected by centrifugation.

Confirmation of the expression of the variant 5 protein in insect cells was performed by Western blot using this Sf9 cell extract and an HRP-labeled anti-FLAG monoclonal antibody (manufactured by Sigma). The cell extract was prepared by washing the cells 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 M EGTA, 1 mM DTT, 0 After suspending in 50 mM Tris-HCl (pH 7.4) containing 0.1 mM NP40, ultrasonic treatment (15 seconds) with MICROCON (HEART SYSTEMS) Then, the mixture was centrifuged at 14500 × g for 20 minutes, and an lysate solution was prepared by adding an equal amount of Lysis solution to the supernatant. For protein purification, the lysate solution was added to an anti-FLAG antibody column equilibrated with Tris buffered saline (TBS) containing 0.05% Tween 20, and then washed with the same solution to obtain an eluate (0.02% By eluting with 6 mM HCl containing Tween 20, pH 2.2 (0.5 mL / fraction) and immediately neutralizing with 1 M Tris-HCl, pH 8.0 (7 μL for 0.5 mL of eluate) went. Variant 5 protein bands were confirmed by staining with Bio-Safe Coomassie (manufactured by Bio-Rad) after SDS-PAGE of each fraction.

Test Example 1 [Angiogenesis induction model by hypoxia in mouse subcutaneous]
According to the method already reported by Topper et al. (Blood 105, p.1068-1077, 2005), the validity of using an angiogenesis model with hypoxia in mice subcutaneously for expression and functional analysis of variant 5 protein in vivo is evaluated. did.

That is, two back portions of an anesthetized C57BL6 mouse were incised in parallel, and the skin was peeled off and a silicon sheet was buried over a region of 2.5 cm × 1.25 cm and sutured (FIG. 1A). After that, the peeled skin became hypoxic after 7 days due to the blockage of blood vessels by the silicon sheet and necrosis occurred from the central part (FIG. 1B). Angiogenesis was occurring toward the area. The area from the end of the necrotic tissue to 8 mm was divided into 4 pieces each of 2 mm, and sections were prepared in each region (FIG. 1C), and the blood vessel area was measured by immunostaining with anti-CD31 antibody. The region of ˜4 mm has the largest blood vessel area (FIG. 1D), indicating that the neovascularization is most activated in this region, and it was found that angiogenesis can be evaluated by such an angiogenesis induction model.

Test Example 2 [Expression site of variant 5 protein in vivo]
The expression of variant 5 protein in the hypoxic model subcutaneous tissue shown in Test Example 1 was examined by immunostaining. When stained with anti-variant 5 antibody, anti-CD31 (vascular endothelial cell marker) antibody, and anti-CD11b antibody in an area within 2 mm from the necrotic end, the stained image of variant 5 protein is a marker of bone marrow-derived cells such as monocytes and macrophages It was in good agreement with the stained image of CD11b (FIG. 2A).

Next, bone marrow cells of a mouse (GFP mouse) in which green fluorescent protein (GFP) was systemically expressed were collected and purified by density gradient centrifugation using Ficoll-paque PLUS (manufactured by GE Healthcare). Transplantation was performed at a rate of 5 × 10 ⁶ per wild-type mouse irradiated with gamma rays (9 Gy). The transplanted mice were bred for 6 weeks to replace bone marrow cells with cells derived from GFP mice. Subcutaneous dorsal skin of this bone marrow transplanted mouse was hypoxic in the same manner as in Test Example 1, and when immunostaining of variant 5 protein was performed in an area within 2 mm from the necrotic end, the stained image is considered to be derived from bone marrow cells. It was consistent with GFP stained cells (FIG. 2B). From the above results, it was suggested that variant 5 protein was expressed from bone marrow-derived cells at an in vivo angiogenesis site.

Test Example 3 [Effect of forced expression of Vasohibin-1 and variant 5 protein in vivo]
In order to express human Vasohibin-1 and variant 5 in vivo, adenovirus vectors incorporating the respective cDNAs were prepared and introduced into 293 cell lines to prepare adenoviruses having these genes. In addition, an adenovirus having a β-galactosidase gene was prepared as a negative control. These viruses (10 ⁹ pfu) were injected from the tail vein of mice, and a hypoxic model was prepared in the same manner as in Test Example 1 in the state where β-galactosidase, Vasohibin-1, and variant 5 were expressed in large amounts in the liver. Created. Sections were prepared from 0 to 2, 2 to 4, 4 to 6, and 6 to 8 mm from the necrotic end, and stained with anti-CD31 antibody and anti-α smooth muscle actin (α-SMA) antibody, respectively. As a result, in the mice in which Vasohibin-1 was highly expressed, angiogenesis was strongly suppressed in the originally active region of 0-2, 2-4 mm from the necrotic end. In contrast, in the mice in which variant 5 was highly expressed, the number of mature blood vessels surrounded by α-SMA positive mural cells was significantly reduced in the 0-2 and 2-4 mm regions from the necrotic end, The number of immature blood vessels without mural cells increased in all regions (FIG. 3).

Test Example 4 [Inhibition of Angiogenesis in Variant 5 Gene Deficient Mice]
In a variant 5 gene-deficient mouse (C57BL6 mouse) prepared by US InGenius Targeting Laboratory, a hypoxic model was prepared and angiogenesis was observed in the same manner as in Test Example 1. For negative control, wild type C57BL6 mice were used. In the same manner as in Test Example 3, sections were prepared from 0 to 2, 2 to 4, 4 to 6, and 6 to 8 mm from the necrotic end, and stained with anti-CD31 antibody and anti-α-SMA antibody, respectively. As a result, both the total number of blood vessels and the number of mature blood vessels in variant 5 gene-deficient mice were significantly reduced. Moreover, in the region 2 to 4 mm from the end of the necrotic tissue, the effect was more remarkable in homo-deficient mice than in hetero-deficient mice (FIG. 4). Therefore, it was suggested that variant 5 has an activity of promoting vascular proliferation and maturation.

Test Example 5 [Cell proliferation assay of variant 5 gene-introduced cells]
MS1 cells obtained by immortalizing mouse pancreatic endothelial cells with SV40 large T antigen were selected as cells to which the variant 5 gene was introduced, and purchased from American Type Culture Collection (Manassas, VA, USA). MS1 cells were cultured in a cell culture medium in which 10% fetal bovine serum (FBS, JRH Biosciences, San Antonio, TX, USA) was added to αMEM (manufactured by Invitrogen).

First, in order to improve the expression efficiency, a pCALL2-pcDNA3.1 / Hygro vector was prepared in which the CMV promoter of pcDNA3.1 / Hygro plasmid (manufactured by Invitrogen) was replaced with the chicken β-actin promoter derived from the pCALL2 vector. Next, in order to prepare a variant 5 expression vector, the variant 5 cDNA was inserted into the pCLL2-pcDNA3.1 / Hygro multicloning site obtained above. The obtained variant 5 expression vector was transfected into MS1 cells using Effectene transfection reagent (Qiagen, Valencia, CA, USA) according to the manual attached to the reagent. After gene transfer, MS1 cells were subjected to drug selection with hygromycin (500 μg / mL) (manufactured by Invitrogen), and a variant 5 gene-transferred cell group (bulk) was obtained. The obtained bulk cells were dispensed and cultured in a 96-well plate such that 0.3 cells per well and 100 μL of cell culture medium were contained. The wells in which cell growth was observed were transferred to larger wells and further cultured, and clones of clones with high expression of variant 5 gene (clone 3, clone 11, clone 14) were obtained. As a result of RT-PCR using a primer that specifically amplifies the variant 5 gene, the variant 5 gene-introduced cell group (bulk) expresses the variant 5 gene, and clones 3, 11, 14 Confirmed that the expression of variant 5 gene was stronger than that of bulk cells (FIG. 5A).

Bulk cells, clones 3, 11, and 14 and the parent cell MS1 (mock) were suspended in 10% FBS / αMEM, and 2 × 103 cells were added to each well in a 96-well plate and cultured for 72 hours. . Thereafter, 10 μL of TetraColor ONE (Seikagaku, Tokyo, Japan) was added to each well, and the absorbance at 450 nm was measured using a multiplate reader (Tosoh, Tokyo, Japan). As a result, it was found that the proliferation of bulk cells was promoted with a significant difference of less than 1% compared to MS1 (mock) (FIG. 5B). On the other hand, it was found that the clones 3, 11, and 14 in which the variant 5 gene was highly expressed compared to the bulk cells all suppressed proliferation with a significant difference of less than 1% compared to MS1 (mock) (FIG. 5C). ). From these results, when the expression of vasohibin 2 is weak, the proliferation of endothelial cells (MS1) is promoted, and when the expression of vasohibin 2 is strong, the proliferation of endothelial cells (MS1) is suppressed. Was found to be the opposite of the effect on cell proliferation.

Based on the above results, we investigated the angiogenesis-inhibiting action and angiogenesis-promoting action of Vasohibin. In WO2006 / 073052, variant 1, which is one of the variants of vasohibin 2, shows an angiogenesis-inhibiting action in the rat corneal assay. In the corneal assay, variant 1 is administered directly to the cornea. Variant 1 is considered high concentration. On the other hand, as shown in Test Example 3 above, in the model of angiogenesis model due to hypoxia under the back of mice, Variant 5, which is one of the variants of Vasohibin 2, exhibits angiogenesis promoting action. In No. 3, the variant 5 expressing adenovirus administered intravenously is accumulated in the liver, and it is considered that Vasohibin 2 expressed therein reaches the back of the skin by riding on the bloodstream, and its concentration is considered to be relatively low. That is, when the expression of vasohibin 2 is weak and the concentration of vasohibin 2 is low, the proliferation of endothelial cells is promoted, and as a result, an angiogenesis-promoting effect is exerted. It is presumed that the proliferation of endothelial cells is suppressed, and as a result, an anti-angiogenic action is exerted, and the contradicting physiological actions of Vasohibin 2 can be explained without contradiction.

The therapeutic agent containing the variant 1, 2, 3, 4 or 5 of the present invention is suitably used, for example, for the treatment of a disease requiring an angiogenesis promoting action.

Sequence number 1 of a sequence table is a polynucleotide of AK022567.
Sequence number 2 of a sequence table is polypeptide of AK022567.
Sequence number 3 of a sequence table is a polynucleotide of BC051856.
Sequence number 4 of a sequence table is polypeptide of BC051856.
Sequence number 5 of a sequence table is a polynucleotide of BC053836.
Sequence number 6 of a sequence table is polypeptide of BC053836.
Sequence number 7 of a sequence table is a polynucleotide of BC028194.
Sequence number 8 of a sequence table is polypeptide of BC028194.
Sequence number 9 of a sequence table is a polynucleotide of AY833422.
Sequence number 10 of a sequence table is polypeptide of AY833422.

Claims (12)

An angiogenesis promoter comprising the following polypeptide (A) or (B):
(A) A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted or added A polypeptide comprising an amino acid sequence and having an angiogenesis-promoting action An angiogenesis-promoting agent comprising a vector comprising a polynucleotide encoding the following polypeptide (A) or (B).
(A) A polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted or added A polypeptide comprising an amino acid sequence and having an angiogenesis-promoting action An angiogenesis promoter comprising a vector comprising the following polynucleotide (C) or (D):
(C) the nucleotide sequence represented by the 351st A to the 1223rd G of SEQ ID NO: 1, the nucleotide sequence represented by the 311st A to the 1246th G of SEQ ID NO: 3, the 327th of the SEQ ID NO: 5 A nucleotide sequence selected from the group consisting of the nucleotide sequence represented by A from the 836th to A, the nucleotide sequence represented by the 300th A to the 770th A of SEQ ID NO: 7, and the nucleotide sequence represented by the SEQ ID NO: 9 A polynucleotide comprising a nucleotide sequence complementary to a polynucleotide comprising the nucleotide sequence of polynucleotides (D) and (C) comprising a polynucleotide that hybridizes under stringent conditions and encodes a polypeptide having an angiogenesis promoting action Polynucleotide The angiogenesis promoter according to claim 2 or 3, wherein the vector is a viral vector. The angiogenesis promoter according to claim 4, wherein the viral vector is an adenoviral vector. The angiogenesis promoter according to any one of claims 1 to 5, which is administered intravenously. A pharmaceutical composition for treating a disease requiring an angiogenesis promoting action, comprising the angiogenesis promoting agent according to any one of claims 1 to 6. Use of the polypeptide defined in claim 1 for the manufacture of a therapeutic agent for a disease requiring an angiogenesis promoting action. Use of the polynucleotide defined in claim 2 or 3 for the manufacture of a therapeutic agent for a disease requiring angiogenesis promoting action. A method for treating a disease requiring an angiogenesis-promoting action, comprising a step of administering the angiogenesis-promoting agent according to any one of claims 1 to 6. A polypeptide as defined in claim 1 for use in the treatment or prevention of diseases requiring angiogenesis promoting action. The polynucleotide according to claim 2 or 3 for use in the treatment or prevention of a disease requiring an angiogenesis promoting action.

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