JP4757194B2 - Vascular endothelial growth inhibitory gene - Google Patents

Description

  The present invention encodes a novel polypeptide having vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from VEGF promoter, and / or angiogenesis inhibitory activity, and polypeptide thereof Relates to polynucleotides.

  By inhibiting angiogenesis, rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer (eg, solid cancer) can be treated (Non-Patent Documents 1 and 2).

  Angiostatin and the like (Patent Documents 1 and 2) are known as proteins that suppress angiogenesis. However, its angiogenesis inhibitory activity is not sufficient for use in the treatment of diseases. Moreover, since it is necessary to maintain a constant blood concentration, a large amount of protein preparation or high expression of a gene is required.

  Further, unlike vascular endothelial cells, gene mutation induction is rare in vascular endothelial cells, and therefore, treatments such as gene therapy with angiogenesis inhibitory factors targeting endothelial cells can be expected to have a sustained effect and a repetitive effect. However, on the other hand, in order to bring about a therapeutic effect, it has been understood that it is necessary to maintain the blood concentration of an angiogenesis inhibitor at an effective amount or more.

Under the state of the art, to treat diseases caused by angiogenesis-inhibiting factors (eg, rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and / or cancer (eg, solid cancer)) It is necessary to isolate a novel gene encoding a protein having an angiogenesis inhibitory activity different from the conventionally known angiostatin.
JP2000-325087 JP 2003-250549 A Cell, 1994 Oct21; 79 (2): 315-28. Nature Medicine, 1995 Jan; 1 (1): 27-31

  The present invention includes vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, transcription inhibitory activity from AP1 promoter, VEGF activity inhibitory activity, transcription inhibitory activity from NFκB promoter, and It is an object of the present invention to provide a novel polypeptide having angiogenesis-inhibiting activity and a polynucleotide encoding the polypeptide. The present invention also relates to a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer (for example, solid cancer) containing these polypeptides and / or polypeptides. It is an object to provide a pharmaceutical composition for treatment, and a method for treating these diseases.

  The present invention was completed by isolating a nucleic acid encoding a vascular endothelial growth inhibitor by using a novel screening method.

Accordingly, the present invention provides the following.
1. Less than:
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or 3 or a fragment sequence thereof;
(B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof;
(C) a polynucleotide encoding a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 or 4; ,;
(D) a polynucleotide that hybridizes under stringent conditions to any one polynucleotide of (a) to (c); and (e) any one polynucleotide of (a) to (c) or a A polynucleotide comprising a base sequence having at least 70% identity to a complementary sequence;
A polynucleotide selected from the group consisting of vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, AP1 promoter A polynucleotide that encodes a peptide having an activity selected from the group consisting of transcription repression activity, VEGF activity repression activity, transcription repression activity from NFκB promoter, and angiogenesis repression activity.
2. The polynucleotide according to claim 1, which has the base sequence set forth in SEQ ID NO: 1 or 3.
3. The polynucleotide of claim 1, which encodes the amino acid sequence of SEQ ID NO: 2 or 4.
4). Less than:
(A) a polynucleotide having the base sequence of SEQ ID NO: 5 or 7 or a fragment sequence thereof;
(B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 6 or 8, or a fragment thereof;
(C) a polynucleotide encoding a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 6 or 8; ,;
(D) a polynucleotide that hybridizes under stringent conditions to any one polynucleotide of (a) to (c); and (e) any one polynucleotide of (a) to (c) or a A polynucleotide comprising a base sequence having at least 70% identity to a complementary sequence;
A polynucleotide selected from the group consisting of vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, AP1 promoter A polynucleotide encoding a peptide having an activity selected from the group consisting of transcription repression activity, VEGF activity repression activity, transcription repression activity from NFκB promoter, and angiogenesis repression activity.
5. The polynucleotide of Claim 4 which has the base sequence of SEQ ID NO: 5 or 7.
6). The polynucleotide according to claim 4, which encodes the amino acid sequence set forth in SEQ ID NO: 6 or 8.
7). The polynucleotide according to claim 1 or 4, which encodes a peptide having an anti-angiogenic activity.
8). The polynucleotide according to claim 1 or 4, which encodes a peptide having vascular endothelial cell growth inhibitory activity.
9. A pharmaceutical composition for inhibiting angiogenesis, comprising the polynucleotide according to claim 1 or 4.
10. A pharmaceutical composition for inhibiting vascular endothelial cell proliferation, comprising the polynucleotide according to claim 1 or 4.
11. A pharmaceutical composition for the treatment of a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer, comprising the polynucleotide of claim 1 or 4.
12 A pharmaceutical composition for the treatment of a disease selected from the group consisting of chronic inflammation, diabetic retinopathy, and cancer, comprising the polynucleotide of claim 1 or 4.
13. A method for inhibiting angiogenesis of a tissue, comprising:
(1) providing a tissue that suppresses angiogenesis; and (2) introducing a nucleic acid comprising the polynucleotide of claim 1 or 4 into the vascular endothelial cell;
Including the method.
14 14. The method of claim 13, wherein the inhibition of angiogenesis is performed in vivo or in vitro.
15. 14. The method of claim 13, wherein the tissue is in a condition comprising a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer.
16. A method for inhibiting vascular endothelial cell proliferation comprising:
(1) providing a vascular endothelial cell; and (2) introducing a nucleic acid comprising the polynucleotide of claim 1 or 4 into the vascular endothelial cell;
Including the method.
17. A plasmid comprising the polynucleotide according to claim 1 or 4.
18. A gene transfer vector comprising the polynucleotide according to claim 1 or 4.
19. A method for inhibiting vascular endothelial cell proliferation comprising:
(1) providing vascular endothelial cells; and (2) contacting the vascular endothelial cells with the gene transfer vector according to claim 18;
Including the method.
20. A polypeptide encoded by the polynucleotide of claim 1 or 4.
21. Less than:
(A) a polypeptide encoded by the base sequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof;
(B) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof,
(C) a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 2 or 4, and (d) ( a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of the polypeptide of any one of a) to (c),
A polypeptide selected from the group consisting of vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from VEGF promoter, and angiogenesis inhibitor A polypeptide having an activity selected from the group consisting of activities.
22. The polypeptide of claim 21, which is encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or 3.
23. The polypeptide according to claim 21, which has the amino acid sequence set forth in SEQ ID NO: 2 or 4.
24. Less than:
(A) a polypeptide encoded by the base sequence set forth in SEQ ID NO: 5 or 7 or a fragment thereof;
(B) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 6 or 8, or a fragment thereof,
(C) a variant polypeptide having at least one mutation selected from the group consisting of substitution, addition and deletion in the amino acid sequence of SEQ ID NO: 6 or 8, and (d) ( a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of the polypeptide of any one of a) to (c),
A polypeptide selected from the group consisting of vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from VEGF promoter, and angiogenesis inhibitor A polypeptide having an activity selected from the group consisting of activities.
25. The polypeptide according to claim 24, which is encoded by the nucleotide sequence set forth in SEQ ID NO: 5 or 7.
26. 25. A polypeptide according to claim 24 having the amino acid sequence set forth in SEQ ID NO: 6 or 8.
27. The polypeptide according to claim 21 or 24, which has an angiogenesis inhibitory activity.
28. The polypeptide according to claim 21 or 24, which has a vascular endothelial cell growth inhibitory activity.
29. A pharmaceutical composition for inhibiting angiogenesis, comprising the polypeptide of claim 21 or 24.
30. A pharmaceutical composition for inhibiting vascular endothelial cell proliferation, comprising the polypeptide according to claim 21 or 24.
31. 25. A pharmaceutical composition for the treatment of a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer, comprising the polypeptide of claim 21 or 24.
32. 25. A pharmaceutical composition for the treatment of a disease selected from the group consisting of chronic inflammation, diabetic retinopathy, and cancer, comprising the polypeptide of claim 21 or 24.
33. A method for inhibiting angiogenesis of a tissue, comprising:
(1) providing angiogenic tissue; and (2) introducing a nucleic acid comprising the polypeptide of claim 21 or 24 into the vascular endothelial cell;
Including the method.
34. 34. The method of claim 33, wherein the angiogenesis inhibition is performed in vivo or in vitro.
35. 34. The method of claim 33, wherein the tissue is in a condition comprising a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer.
36. A method for inhibiting vascular endothelial cell proliferation comprising:
(1) providing vascular endothelial cells; and (2) contacting the vascular endothelial cells with the polypeptide of claim 21 or 24,
Including the method.

  According to the present invention, vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, transcription inhibitory activity from AP1 promoter, VEGF activity inhibitory activity, transcription inhibitory activity from NFκB promoter and A polypeptide having at least one activity of angiogenesis inhibitory activity and a polynucleotide encoding the polypeptide are provided.

  Further, according to the present invention, a method for treating a disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer (eg, solid cancer), and pharmaceutical composition for treatment Things are also provided.

FIG. 1 is a scheme for the isolation of the nucleic acid of the present invention.
FIG. 2 shows the results of MTS assay and c-fos promoter assay of clones pCMV9 and pCMV15 isolated in the present invention.
FIG. 3 shows the results of inserts of clones pCMV9 and pCMV15 isolated in the present invention and in vitro translation assay.
[FIG. 4] FIG. 4 shows the nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the gene contained in pCMV9.
[FIG. 5] FIG. 5 shows the nucleic acid sequence (SEQ ID NO: 5) and amino acid sequence (SEQ ID NO: 6) of the gene contained in pCMV15.
[FIG. 6] FIG. 6 schematically shows motifs contained in the amino acid sequences of genes contained in pCMV9 and pCMV15.
[FIG. 7] FIG. 7 shows the results showing the activity of suppressing E2F promoter and AP1 promoter by the genes contained in pCMV9 and pCMV15.
FIG. 8 is a view showing the results of MTS assay of clones pCMV15, pCMV15-2, and pCMV15-3 isolated in the present invention.
FIG. 9 shows the results of c-fos promoter assay for clones pCMV15, pCMV15-2, and pCMV15-3 isolated in the present invention.
[FIG. 10] FIG. 10 is a diagram showing the results of an assay for the suppression activity of transcription activation via NFκB elements of clones pCMV15, pCMV15-2, and pCMV15-3 isolated in the present invention.
[FIG. 11] FIG. 11 is a diagram showing the results of an assay for the suppression activity of transcription activation via AP-1 enhancer of clones pCMV15, pCMV15-2, and pCMV15-3 isolated in the present invention.
Description of sequence listing

SEQ ID NO: 1 Nucleic acid sequence (pCMV9) encoding a novel peptide having angiogenesis inhibitory activity
SEQ ID NO: 2: Amino acid sequence of a novel peptide having antiangiogenic activity SEQ ID NO: 3: EST sequence corresponding to pCMV9 (EST-BD095420)
SEQ ID NO: 4: Amino acid sequence encoded by EST sequence (EST-BD095420) corresponding to pCMV9 SEQ ID NO: 5: Nucleic acid sequence (pCMV15) encoding a novel peptide having angiogenesis inhibitory activity
SEQ ID NO: 6: Novel peptide having angiogenesis inhibitory activity SEQ ID NO: 7: EST sequence corresponding to pCMV15 (EST-HSM805282)
SEQ ID NO: 8: amino acid sequence encoded by EST sequence (EST-HSM805282) corresponding to pCMV15 SEQ ID NO: 9: 3492 bp nucleic acid sequence including upstream of pCMV15 (pCMV15-2)
SEQ ID NO: 10: Amino acid sequence of peptide encoded by pCMV15-2 SEQ ID NO: 11: 4413 bp nucleic acid sequence including upstream of pCMV15 (pCMV15-3)
SEQ ID NO: 12: amino acid sequence of the peptide encoded by pCMV15-3

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles or adjectives (eg, “a”, “an”, “the”, etc., in the English language) also include the plural concept unless otherwise stated. is there. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  As used herein, “homology” of genes (eg, nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity of two or more gene sequences with each other. In the present specification, the identity of sequences (nucleic acid sequences, amino acid sequences, etc.) refers to the degree of two or more comparable sequences that are identical to each other (individual nucleic acids, amino acids, etc.). Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% are identical, the genes are homologous. In the present specification, “similarity” of genes (for example, nucleic acid sequences, amino acid sequences, etc.) refers to two or more gene sequences when the conservative substitution is regarded as positive (identical) in the above homology. The degree of identity with each other. Thus, when there is a conservative substitution, homology and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, homology and similarity indicate the same numerical value.

  In this specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using FASTA, which is a sequence analysis tool, and using default parameters.

  As used herein, “fragment” refers to a polypeptide or polynucleotide having a sequence length of 1 to n−1 with respect to a full-length polypeptide or polynucleotide (length is n). The length of the fragment can be appropriately changed according to the purpose. For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10, Examples include 15, 20, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) are also suitable as lower limits. obtain. In the case of polynucleotides, examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides. Non-integer lengths (eg, 11 etc.) may also be appropriate as a lower limit. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute, and the upper limit is not limited as long as they have the same function. Alternatively, the above-mentioned number as the lower limit is intended to include the upper and lower numbers (or, for example, up and down 10%) of the number. In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value. The length of a fragment useful herein can be determined by whether or not at least one of the functions of a full-length protein that serves as a reference for the fragment is retained.

  As used herein, an “isolated” biological factor (eg, a nucleic acid or protein, etc.) refers to other biological factors within the cells of the organism in which it is naturally occurring (eg, In the case of a nucleic acid, it is substantially separated from a nucleic acid containing a non-nucleic acid factor and a nucleic acid sequence other than the target nucleic acid; in the case of a protein, a protein containing a non-protein factor and an amino acid sequence other than the target protein) Or it is purified. “Isolated” nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

  As used herein, a “purified” biological agent (such as a nucleic acid or protein) refers to a product from which at least part of the factors naturally associated with the biological agent has been removed. Thus, typically the purity of the biological agent in a purified biological agent is higher (ie, enriched) than the state in which the biological agent is normally present.

  The terms “purified” and “isolated” as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably Means that at least 98% by weight of the same type of biological agent is present.

  In the present specification, “purified” and “cloned” are used interchangeably. “Purification” and “cloning” refer to the state of a substance or nucleic acid, and to make the presence of the nucleic acid high, preferably , Which is substantially free from other types of substances or nucleic acids. As used herein in the context of “purification” and “cloning”, “substantially free of” other types of substances or nucleic acids is the absence of those other types of substances or nucleic acids. Even if it exists or exists, it refers to a state that does not affect the target substance or nucleic acid. Thus, in a more preferred state, the purified nucleic acid or nucleic acid composition contains only certain nucleic acids.

  As used herein, “gene transfer vector” and “gene vector” are used interchangeably. “Gene transfer vector” and “gene vector” refer to a vector capable of transferring a target polynucleotide sequence into a target cell. “Gene transfer vector” and “gene vector” include, but are not limited to, “virus envelope vector” and “liposome vector”.

  As used herein, “virus envelope vector” refers to a vector in which a foreign gene is encapsulated in a virus envelope, or a vector in which a foreign gene is encapsulated in a component containing a protein derived from the virus envelope. The virus used for the preparation of the gene transfer vector may be a wild type virus or a recombinant virus.

  In the present invention, the viruses used for the preparation of the virus envelope or virus envelope-derived protein include retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyavirus Viruses belonging to the family selected from the group consisting of Family, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, and Hepadnaviridae are included, but are not limited thereto. Preferably, a virus belonging to the Paramyxoviridae family is used, more preferably HVJ (Sendai virus).

  Examples of the virus envelope-derived protein include, but are not limited to, HVJ F protein, HN protein, NP protein, and M protein.

  As used herein, “liposome vector” refers to a vector in which a foreign gene is encapsulated in a liposome. Lipids used for the preparation of liposome vectors include, for example, neutral phospholipids such as DOPE (dioleoylphosphatidylethanolamine) and phosphatidylcholine, negatively charged phospholipids such as cholesterol, phosphatidylserine and phosphatidic acid, and DC -Positively charged lipids such as but not limited to cholesterol (dimethylaminoethanecarbamoylcholesterol) and DOTAP (dioleoyltrimethylammoniumpropane).

  As used herein, “liposomes” are a type of lipid bilayer membrane. For example, when a phospholipid such as lecithin is suspended in 50% (by weight) or more of water at a temperature higher than the gel-liquid crystal phase transition temperature inherent to the phospholipid, it is composed of a lipid bilayer, and has a closed small phase having an aqueous phase inside. A vesicle is formed. These vesicles are called liposomes. Multilamellar liposomes (MLV) in which several bilayer membranes are stacked in an onion-like manner and membranes are roughly divided into one liposome. The latter can also be prepared by dispersing a suspension of a phospholipid such as phosphatidylcholine by vigorous stirring with a mixer, followed by sonication.

Liposomes with a single membrane are further classified into small unilamellar liposomes (SUV) and large unilamellar liposomes (LUV) according to their particle size. MLV is prepared by adding water to a lipid film to give mechanical vibrations. The SUV is prepared by, for example, sonicating MLV or removing the surfactant from a mixed solution of lipid and surfactant by dialysis or the like. In addition to this, (1) a method of preparing LUV by repeating freeze-thawing of SUV, (2) SUV made of acidic phospholipid is fused in the presence of Ca ²⁺ , and this Ca ²⁺ is EDTA (3) A method of preparing LUV by changing phase while distilling off ether from a lipid ether solution and water emulsion (reverse phase evaporation method) Liposomes; REV) are well known.

  As used herein, “inactivation” refers to a virus that has inactivated its genome. This inactivated virus is replication defective. Preferably, this inactivation is done by UV treatment or treatment with an alkylating agent.

  In the present specification, “HVJ” and “Sendai virus” may be used interchangeably. For example, in the present specification, “HVJ envelope” and “Sendai virus envelope” are used as terms having the same meaning.

  As used herein, “Sendai virus” refers to a virus belonging to the genus Paramyxovirus belonging to the Paramyxoviridae family and having a cell fusion action. Virus particles have an envelope and a polymorphism with a diameter of 150-300 nm. The genome is a minus-strand RNA having a length of about 15500 bases. It has RNA polymerase, is unstable to heat, aggregates almost all kinds of red blood cells, and is hemolytic.

  As used herein, “HAU” refers to the activity of a virus capable of aggregating 0.5% of chicken erythrocytes, and 1 HAU corresponds to approximately 24 million virus particles (Okada, Y. et al., Biken Journal 4, 209-213, 1961).

  In the present specification, “polynucleotide hybridizing under stringent conditions” refers to well-known conditions commonly used in the art. Such a polynucleotide can be obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using a polynucleotide selected from among the polynucleotides of the present invention as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then 0.1 to 2 times the concentration. Means a polynucleotide that can be identified by washing a filter under conditions of 65 ° C. using a SSC (saline-sodium citrate) solution (composition of 1-fold concentration of SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). To do. Hybridization was performed in Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford Universe. Here, the sequence containing only the A sequence or only the T sequence is preferably excluded from the sequences that hybridize under stringent conditions. The “hybridizable polynucleotide” refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specifically, the hybridizable polynucleotide is a polynucleotide having at least 60% homology with the base sequence of DNA encoding a polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% The polynucleotide which has the above homology, More preferably, the polynucleotide which has 95% or more of homology can be mentioned.

As used herein, “highly stringent conditions” are designed to allow hybridization of DNA strands having a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA having significant mismatches. Say conditions. Hybridization stringency is primarily determined by temperature, ionic strength, and the conditions of denaturing agents such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65-68 ° C., or 0.015 M sodium chloride, 0.0015 M citric acid. Sodium, and 50% formamide, 42 ° C. For such highly stringent conditions, see Sambrook et al. , Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N, Y. 1989); and Anderson et al. Nucleic Acid Hybridization: A Practical Approach, IV, IRL Press Limited (Oxford, England). See Limited, Oxford, England. If necessary, more stringent conditions (eg, higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents can be included in the hybridization and wash buffers for the purpose of reducing non-specific hybridization and / or background hybridization. Examples of such other agents include 0.1% bovine serum albumin, 0.1% polyvinyl pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO ₄ or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, but other suitable agents can also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is almost pH independent. Anderson et al. , Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

Factors that affect the stability of the DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by one of ordinary skill in the art to apply these variables and to allow different sequence related DNAs to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
T _m (° C.) = 81.5 + 16.6 (log [Na ⁺ ]) + 0.41 (% G + C) −600 / N−0.72 (% formamide)
Where N is the length of the duplex formed, [Na ⁺ ] is the molar concentration of sodium ions in the hybridization or wash solution, and% G + C is the (guanine + Cytosine) base percentage. For imperfectly matched hybrids, the melting temperature decreases by about 1 ° C. for each 1% mismatch.

  As used herein, “moderately stringent conditions” refers to conditions under which a DNA duplex having a higher degree of base pair mismatch than can be generated under “highly stringent conditions”. . Examples of representative “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65 ° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20 % Formamide, 37-50 ° C. As an example, a “moderately stringent” condition of 50 ° C. in 0.015 M sodium ion tolerates a mismatch of about 21%.

  It will be appreciated by those skilled in the art that there may not be a complete distinction between “highly” stringent conditions and “moderate” stringent conditions herein. For example, at 0.015M sodium ion (no formamide), the melting temperature of a perfectly matched long DNA is about 71 ° C. In washing at 65 ° C. (same ionic strength), this allows about 6% mismatch. In order to capture more distant related sequences, one of ordinary skill in the art can simply decrease the temperature or increase the ionic strength.

For oligonucleotide probes up to about 20 nucleotides, a reasonable estimate of the melting temperature in 1M NaCl is
Tm = (2 ° C. for one AT base) + (4 ° C. for one GC base pair)
Provided by. The sodium ion concentration in 6 × sodium citrate (SSC) is 1 M (see Suggs et al., Developmental Biology Using Purified Genes, page 683, Brown and Fox (ed.) (1981)).

A natural nucleic acid encoding a protein such as a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, 4, 6 or 8 or a variant or fragment thereof is, for example, the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 It is easily separated from a cDNA library having PCR primers and hybridization probes containing some or variants thereof. A nucleic acid encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 or a variant or fragment thereof is essentially 1% bovine serum albumin (BSA); 500 mM sodium phosphate (NaPO ₄ ); 1 mM EDTA; defined by hybridization buffer containing 7% SDS at a temperature of 42 ° C., and essentially 2 × SSC (600 mM NaCl; 60 mM sodium citrate); wash buffer containing 0.1% SDS at 50 ° C. 1% bovine serum albumin (BSA) at a low stringency condition, more preferably essentially at a temperature of 50 ° C .; 500 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; 7% SDS Hybridization buffer, and 1 × essentially 50 ° C. SC (300 mM NaCl; 30 mM sodium citrate); 1% bovine serum albumin (BSA) under low stringent conditions defined by a wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C .; 200 mM sodium phosphate (NaPO ₄ ); 15% formamide; 1 mM EDTA; hybridization buffer containing 7% SDS, and 0.5 × SSC essentially at 65 ° C. (150 mM NaCl; 15 mM sodium citrate); It can hybridize with one or a portion of the sequence shown in SEQ ID NO: 1, 3, 5 or 7 under low stringency conditions defined by a wash buffer containing 1% SDS.

  As used herein, the percentage of “identity”, “homology” and “similarity” of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences that are optimally aligned in a comparison window. . Here, the reference sequence for optimal alignment of the two sequences (a gap may occur if the other sequences contain additions, in the portion of the polynucleotide or polypeptide sequence within the comparison window, Reference sequences herein shall include no additions or deletions (ie, gaps) as compared to the reference). Find the number of match positions by determining the number of positions where the same nucleobase or amino acid residue is found in both sequences, and divide the number of match positions by the total number of positions in the comparison window. Is multiplied by 100 to calculate the percent identity. When used in a search, homology is assessed using an appropriate one from a variety of sequence comparison algorithms and programs well known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85 (8): 2444-2448, Altschul et al., 1990 ,. J. Mol. Biol. 215 (3): 403-410, Thompson et al., 1994, Nucleic Acids Res. Altschul et al., 1990, J. Mol.Biol.215 (3): 403-410, Altschul et al. 1993, Nature Genetics 3: 266-272), but the like, is not intended to be limited to this. In a particularly preferred embodiment, the Basic Local Alignment Tool (BLAST) known in the prior art (eg, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268, Altschul et al., 1990). J. Mol. Biol. 215: 403-410, Altschul et al., 1993, Nature Genetics 3: 266-272, Altschul et al., 1997, Nuc. Acids Res.25: 3389-3402). Used to assess protein and nucleic acid sequence homology. In particular, a comparison or search can be achieved by performing the following operations using five dedicated BLAST programs.

(1) Compare amino acid query sequence with protein sequence database in BLASTP and BLAST3;
(2) Compare nucleotide query sequence with nucleotide sequence database at BLASTN;
(3) Comparison of a conceptual translation product obtained by converting a nucleotide query sequence (both strands) with BLASTX in six reading frames with a protein sequence database;
(4) Compare protein query sequence with TBLASTN to nucleotide sequence database converted in all six reading frames (both strands);
(5) Comparison of nucleotide query sequence converted by TBLASTX in 6 reading frames with nucleotide sequence database converted in 6 reading frames.

  The BLAST program identifies similar segments called “high-score segment pairs” between an amino acid query sequence or nucleic acid query sequence, and preferably a test sequence obtained from a protein sequence database or nucleic acid sequence database. Thus, a homologous sequence is identified. High score segment pairs are preferably identified (ie, aligned) by a scoring matrix well known in the art. Preferably, a BLOSUM62 matrix (Gonnet et al., 1992, Science 256: 1443-1445, Henikoff and Henikoff, 1993, Proteins 17: 49-61) is used as the scoring matrix. Although not as preferred as this matrix, a PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrixes for Detection Distance Relationships: Atlas of Protein Sequence and Strategic Stratecence. thing). The BLAST program evaluates the statistical significance of all identified high score segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specific homology rate. It is preferred to evaluate the statistical significance of high score segment pairs using Karlin's formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268). thing).

(Modification of genes, protein molecules, nucleic acid molecules, etc.)
In certain protein molecules, certain amino acids contained in the sequence can be replaced with other amino acids in the protein structure, such as, for example, the cationic region or the binding site of a substrate molecule, without an apparent reduction or loss of interaction binding capacity. . It is the protein's ability to interact and the nature that defines the biological function of a protein. Thus, specific amino acid substitutions can be made in the amino acid sequence or at the level of its DNA coding sequence, resulting in proteins that still retain their original properties after substitution. Thus, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.

  In designing such modifications, the hydrophobicity index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions in proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ. Mol. Biol. 157 ( 1): 105-132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the protein produced and then defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on their hydrophobicity and charge properties. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1 .8); Glycine (−0.4); Threonine (−0.7); Serine (−0.8); Tryptophan (−0.9); Tyrosine (−1.3); Proline (−1.6) ); Histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9) And arginine (-4.5)).

  It is well known in the art that one amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, an equivalent protein in enzymatic activity). It is. In such amino acid substitution, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient.

  In the art, hydrophilicity index can also be considered in the modified design. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3. 0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline ( Alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−0.5 ± 1); -1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). It is understood that an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent. In such amino acid substitution, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

  In the present specification, the “conservative substitution” refers to a substitution in which the hydrophilicity index and / or the hydrophobicity index of the amino acid substituted with the original amino acid are similar as described above. Examples of conservative substitution include those having a hydrophilicity index or a hydrophobicity index within ± 2, preferably within ± 1, and more preferably within ± 0.5. But not limited to them. Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and Examples include, but are not limited to, isoleucine.

  In the present specification, the “variant” refers to a substance in which a part of the original substance such as a polypeptide or polynucleotide is changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Such variants include one or several substitutions, additions and / or deletions, or one or more substitutions, additions and / or deletions relative to a reference nucleic acid molecule or polypeptide. But are not limited thereto. An allele refers to genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant having an allelic relationship with a certain gene. Such allelic variants usually have the same or very similar sequence as their corresponding alleles, usually have nearly the same biological activity, but rarely have different biological activities. May have. “Species homologue or homolog” means homology (preferably at least 60% homology, more preferably at least 80%, at a certain amino acid level or nucleotide level within a certain species. 85% or higher, 90% or higher, 95% or higher homology). The method for obtaining such species homologues will be apparent from the description herein. “Ortholog” is also called an orthologous gene, which is a gene derived from speciation from a common ancestor with two genes. For example, taking the hemoglobin gene family with multiple gene structures as an example, the human and mouse alpha hemoglobin genes are orthologs, but the human alpha and beta hemoglobin genes are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention.

  As used herein, “conservative (modified) variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essentially identical if the nucleic acid does not encode an amino acid sequence. An array. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent modifications (mutations)” which are one species of conservatively modified mutations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid. In the art, each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan), produces a functionally identical molecule. It is understood that it can be modified. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. Preferably, such modifications can be made to avoid substitution of cysteine, an amino acid that greatly affects the conformation of the polypeptide. Such base sequence modification methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment and other ligation treatments, site-specific base substitution methods using synthetic oligonucleotides (specification) Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other modifications may also be made by methods usually used in the field of molecular biology. it can.

  As used herein, in addition to amino acid substitutions, amino acid additions, deletions, or modifications can also be made to produce functionally equivalent polypeptides. Amino acid substitution means that the original peptide is substituted with one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids. Addition of an amino acid means adding one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain. Deletion of amino acids means deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide. Amino acid modifications include amidation, carboxylation, sulfation, halogenation, shortening, lipidation, phosphorylation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg acetylation), etc. Including, but not limited to. The amino acid to be substituted or added may be a natural amino acid, a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.

  As used herein, the term “peptide analog” or “peptide derivative” refers to a compound that is different from a peptide but is equivalent to at least one chemical or biological function of the peptide. Thus, peptide analogs include those in which one or more amino acid analogs or amino acid derivatives are added or substituted to the original peptide. Peptide analogs have the same function as the original peptide (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similarity in sex). Such peptide analogs can be made using techniques well known in the art. Thus, a peptide analog can be a polymer that includes an amino acid analog.

  Chemically modified polypeptide compositions in which a polypeptide of the invention is bound to a polymer are within the scope of the invention. The polymer can be water soluble and can prevent precipitation of the protein in a water soluble environment (eg, a physiological environment). Suitable aqueous polymers can be selected, for example, from the group consisting of: polyethylene glycol (PEG), monomethoxy polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymer, poly (N-vinyl pyrrolidone) polyethylene glycol, Polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol. The selected polymer is usually modified and has a single reactive group (eg, an active ester for acylation or an aldehyde for alkylation) so that the degree of polymerization can be controlled. The polymer can be of any molecular weight, and the polymer can be branched or unbranched, and mixtures of such polymers can also be used. This chemically modified polymer of the present invention is selected for use by a pharmaceutically acceptable polymer when determined for therapeutic use.

  If the polymer is modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is modified by reductive alkylation, the polymer should have a single reactive aldehyde group. Preferred reactive aldehydes are polyethylene glycol, propionaldehyde (which is water-soluble) or mono-C1-C10 alkoxy or aryloxy derivatives thereof (eg, US Pat. No. 5,252,714). No., which is hereby incorporated by reference in its entirety).

  Pegylation of the polypeptides of the present invention can be performed by any pegylation reaction known in the art, for example as described in the following references: Focus on Growth Factors 3,4- 10 (1992); EP 0 154 316; and EP 0 401 384, each of which is incorporated herein by reference in its entirety. Preferably, this pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or similar reactive water-soluble polymer). A preferred water soluble polymer for pegylation of the polypeptides of the invention is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any form of PEG, where the PEG is another protein (eg, a mono (C1-C10) alkoxy polyethylene. Glycol or mono (C1-C10) aryloxypolyethylene glycol).

  Chemical derivatization of the polypeptides of the present invention may be performed under suitable conditions used to react biologically active substances with activated polymer molecules. Methods for preparing PEGylated polypeptides of the invention generally include the following steps: (a) Polyethylene glycol (eg, PEG) under conditions such that the polypeptide binds to one or more PEG groups. Reacting the polypeptide with a reactive ester or aldehyde derivative) and (b) obtaining the reaction product. It is easy for those skilled in the art to select the optimal reaction conditions or acylation reaction based on known parameters and the desired result.

  Pegylated polypeptides of the invention can generally be used to treat conditions that can be alleviated or modulated by administering a polypeptide described herein, but herein The chemically derivatized polypeptide molecules of the present invention disclosed in (1) may have additional activity, increased or decreased biological activity, or other characteristics (eg, increased) compared to their non-derivatized molecules. Reduced half-life). The polypeptides, fragments, variants and derivatives thereof of the invention can be used alone, in combination or in combination with other pharmaceutical compositions. These cytokines, growth factors, antigens, anti-inflammatory agents and / or chemotherapeutic agents are suitable for treating symptoms.

  Similarly, “polynucleotide analog” or “nucleic acid analog” refers to a compound that is different from a polynucleotide or nucleic acid but is equivalent to at least one chemical or biological function of the polynucleotide or nucleic acid. . Thus, polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.

  As used herein, a nucleic acid molecule may have a portion of its nucleic acid sequence deleted or otherwise as long as the expressed polypeptide has substantially the same activity as the native polypeptide. It may be substituted with a base, or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Alternatively, it may be a nucleic acid molecule that hybridizes under stringent conditions with a gene encoding a polypeptide and encodes a polypeptide having substantially the same function as the polypeptide. Such genes are known in the art and can be used in the present invention.

  Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method or a hybridization method.

  As used herein, “substitution, addition or deletion” of a polypeptide or polynucleotide is a substitution of an amino acid or a substitute thereof, or a nucleotide or a substitute thereof, respectively, with respect to the original polypeptide or polynucleotide. , Adding or removing. Such substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions or deletions may be any number as long as it is one or more, and such number is a function (for example, information on hormones, cytokines) in a variant having the substitution, addition or deletion. As long as the transmission function is maintained, it can be increased. For example, such a number can be one or several, and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.

(Genetic engineering)
The polypeptide having the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8 and fragments and variants thereof used in the present invention can be produced using genetic engineering techniques.

“Recombinant vectors” for prokaryotic cells that can be used in the present invention include pcDNA3 (+), pBluescript-SK (+/−), pGEM-T, pEF-BOS, pEGFP, pHAT, pUC18, pFT-DEST ^™ 42GATEWAY ( Invitrogen) and the like.

  Examples of “recombinant vectors” for animal cells that can be used in the present invention include pcDNAI / Amp, pcDNAI, pCDM8 (all available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGE103 [J. Biochem. , 101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem. , 268, 22782-2787 (1993)], a retroviral expression vector based on murine stem cell virus (MSCV), pEF-BOS, pEGFP, and the like.

  Strong promoters for expression in mammalian cells include, for example, various natural promoters (eg, SV40 early promoter, adenovirus E1A promoter, human cytomegalovirus (CMV) promoter, human elongation factor- 1 (EF-1) promoter, Drosophila minimal heat shock protein 70 (HSP) promoter, human metallothionein (MT) promoter, Rous sarcoma virus (RSV) promoter, human ubiquitin C (UBC) promoter, human actin promoter), and artificial Promoters (eg, SRα promoter (SV40 early promoter and HTLV LTR promoter fusion), CAG promoter (CMV-IE enhancer and chicken actin) Since fusion promoter such as the hybrid) of promoter) is known, by using these known promoter or variants thereof, it can be easily increased the expression level of recombinant expression.

  When using Escherichia coli as a host cell, promoters derived from Escherichia coli and phage, such as trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter, PSE promoter, etc., SPO1 promoter, SPO2 promoter, penP promoter, etc. Can be mentioned. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrp × 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.

  In this specification, any technique may be used for introducing a nucleic acid molecule into a cell, and examples thereof include transformation, transduction, and transfection. Such a technique for introducing a nucleic acid molecule is well known in the art and commonly used. For example, Ausubel F. et al. A. (1988), Current Protocols in Molecular Biology, Wiley, New York, NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, a separate volume of experimental medicine “Gene Transfer & Expression Analysis Experimental Method” Yodosha, 1997, and the like. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known conventional techniques.

  Moreover, as a method for introducing a vector, any of the above-described methods for introducing DNA into cells can be used. For example, transfection, transduction, transformation, etc. (for example, calcium phosphate method, liposome method, DEAE dextran method, electroporation method, method using particle gun (gene gun), etc.).

  When a prokaryotic cell is used in the present invention for genetic manipulation or the like, the prokaryotic cell includes, for example, a prokaryotic cell belonging to the genus Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc. Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1 are exemplified.

  As used herein, animal cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, CHO cells that are Chinese hamster cells, BHK cells, African green monkey kidney cells, and human leukemia cells. HBT5637 (Japanese Patent Laid-Open No. 63-299), human colon cancer cell line, and the like. Mouse myeloma cells such as ps20 and NSO, rat myeloma cells such as YB2 / 0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1 and Africa COS-1, COS-7 as the kidney monkey cell, HCT-15 as the human colon cancer cell line, human neuroblastoma SK-N-SH, SK-N-SH-5Y, mouse neuroblastoma Neuro2A, etc. Is exemplified.

  As used herein, any method for introducing a DNA can be used as a method for introducing a recombinant vector. For example, a calcium chloride method, an electroporation method [Methods. Enzymol. , 194, 182 (1990)], lipofection method, spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)], lithium acetate method [J. Bacteriol. , 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

  As used herein, retroviral infection methods are well known in the art as described, for example, in Current Protocols in Molecular Biology supra (especially Units 9.9-9.14) and the like, eg, trypsinize. Then, the embryonic stem cells are made into a single-cell suspension and then together with the culture supernatant of virus-producing cells (packaging cell line = packaging cell lines). A sufficient amount of infected cells can be obtained by co-culture for 2 hours.

  The transient expression of Cre enzyme used in the method for removing the genome or the locus used in the present specification, DNA mapping on the chromosome, etc. are described in the cell engineering separate experiment protocol series “FISH experiment protocol human genome”. From analysis to chromosome / gene diagnosis "Kenichi Matsubara, Hiroshi Yoshikawa, Shujunsha (Tokyo), etc. are well known in the field.

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or pharmaceutical adjuvants.

  The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

(Description of Preferred Embodiment)
The description of the preferred embodiment is described below, but it should be understood that this embodiment is an illustration of the present invention and that the scope of the present invention is not limited to such a preferred embodiment. It should be understood that those skilled in the art can easily make modifications, changes and the like within the scope of the present invention with reference to the following preferred embodiments.

(Method for producing polypeptide)
Transformants derived from microorganisms, animal cells, etc. that possess recombinant vectors incorporating the DNA encoding the polypeptide of the present invention are cultured according to a normal culture method, and the polypeptide of the present invention is produced and accumulated, By collecting the polypeptide of the present invention from the culture of the present invention, the polypeptide according to the present invention can be produced.

  The method of culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host. A medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism of the present invention. Either a natural medium or a synthetic medium may be used as long as the medium can efficiently culture the transformant.

  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 ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, 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.

  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 or 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 microorganism transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like is used. When a microorganism transformed with an expression vector using the trp promoter is cultured, indole acrylic An acid or the like may be added to the medium. Cells or organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter.

  For example, when animal cells are used, the culture medium for culturing the cells of the present invention includes RPMI1640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122). , 501 (1952)), DMEM medium (Virology, 8, 396 (1959)), 199 medium (Proceedings of the Society for the Biological Medicine, 73, 1 (1950)), or fetal bovine serum or the like was added to these media A medium 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 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin, penicillin, streptomycin, to a culture medium as needed during culture | cultivation.

  In order to isolate or purify the polypeptide of the present invention from the culture of the transformant transformed with the nucleic acid sequence encoding the polypeptide of the present invention, isolation of conventional enzymes well known and commonly used in the art Alternatively, a purification method can be used. For example, when the polypeptide of the present invention is secreted outside the transformant for producing the polypeptide of the present invention, the culture is treated with a technique such as centrifugation to obtain a soluble fraction. Get minutes. From the soluble fraction, anion exchange chromatography using a resin such as a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei), etc. Chromatography method, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography method using resin such as butyl sepharose, phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography A purified sample can be obtained by using a technique such as a chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide of the present invention accumulates in a dissolved state in the cells of the transformant for producing the polypeptide of the present invention, the culture is centrifuged to collect the cells in the culture and wash the cells. After that, the cells are crushed by an ultrasonic crusher, French press, Manton Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Kasei) Anion exchange chromatography using a resin, cation exchange chromatography using a resin such as S-Sepharose FF (Pharmacia), hydrophobic chromatography using a resin such as butyl sepharose, phenyl sepharose A purified sample can be obtained by using a technique such as a chromatography method, a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, an electrophoresis method such as isoelectric focusing.

  When the polypeptide of the present invention is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted and centrifuged from the precipitate fraction obtained by the usual method. After recovering the polypeptide, the insoluble body of the polypeptide is solubilized with a polypeptide denaturant. The solubilized solution is diluted or dialyzed into a dilute solution that does not contain the polypeptide denaturing agent or the concentration of the polypeptide denaturing agent does not denature the polypeptide. Then, a purified sample can be obtained by the same isolation and purification method as described above.

  Further, a conventional protein purification method [J. Evan. Sadler et al .: Methods in Enzymology, 83, 458]. In addition, the polypeptide of the present invention can 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 ( (Experimental Medicine), 13, 469-474 (1995)]. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227-8231 (1989), Genes Develop. , 4, 1288 (1990)], the polypeptide of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G.

  In addition, the polypeptide of the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop. , 4, 1288 (1990)]. In such fusion proteins, in the expression vector, the proteolytic cleavage site joins the fusion moiety to the recombinant protein to allow separation of the recombinant protein from the fusion moiety following purification of the fusion protein. Introduced into the department. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Representative fusion expression vectors include pGEX (Pharmacia Biotech; Smith and Johnson (1988) Gene 67, which fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A to the target recombinant protein, respectively. , 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ).

  Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself of the present invention. The polypeptide of the present invention can be produced by known methods [J. Biomolecular NMR, 6,129-134, Science, 242, 1162-1164, J. Biol. Biochem. , 110, 166-168 (1991)], can be produced using an in vitro transcription / translation system.

  The polypeptide of the present invention can also be produced by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid information. Chemical synthesis can also be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.

  The structural analysis of the purified polypeptide of the present invention can be carried out by a method usually used in protein chemistry, for example, the method described in Protein Structural Analysis for Gene Cloning (Hirano Hisashi, Tokyo Kagaku Dojin, 1993). is there. The physiological activity of the polypeptide of the present invention can be measured according to a known measurement method.

  Production of soluble polypeptides useful in the present invention can also be accomplished by various methods known in the art. For example, a polypeptide can be derived from an intact polypeptide molecule by proteolysis by using a particular endopeptidase in combination with exopeptidase, Edman degradation, or both. This intact polypeptide molecule can be purified from its natural source using conventional methods. Alternatively, intact polypeptides can be produced by recombinant DNA techniques utilizing well known techniques for cDNA, expression vectors and recombinant gene expression.

  Preferably, soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for the entire polypeptide as a starting material. This can be accomplished by conventional chemical synthesis techniques, or by well-known recombinant DNA techniques where only the DNA sequence encoding the desired peptide is expressed in the transformed host. For example, a gene encoding the desired soluble polypeptide can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble polypeptide. The specific DNA sequence encoding the desired peptide can also be derived from the full length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of specific regions from cDNA.

(Method for producing mutant polypeptide)
Deletion, substitution or addition (including fusion) of amino acids of the polypeptide of the present invention can be carried out by site-directed mutagenesis which is a well-known technique. Such deletion, substitution or addition of one or several amino acids is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratories Press (1989), Current Protocols in BioWarm. (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci. USA, 81, 5562 (1984), Science, 224, 1431 (1984), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.

(Synthetic chemistry)
Factors such as peptides, chemical substances, and small molecules in this specification can be synthesized using synthetic chemistry techniques. As such synthetic chemistry techniques, techniques well known in the art can be used. Examples of such well-known techniques include Fiesers 'Reagents for Organic Synthesis (Fiesers' Reagents for Organic Synthesis) Tse-Lok Ho, John Wiley & Sons Inc (2002).

  When the agent of the present invention is used as a compound, it can be used in a salt form. The “salt” is preferably a pharmaceutically acceptable salt, such as a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a salt with a basic or acidic amino acid, and the like. Can be mentioned. Examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, magnesium salt and barium salt, and aluminum salt and ammonium salt. Examples of the salt with an organic base include salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, dietanolamine, triethanolamine, dicyclohexylamine, N, N'-dibenzylethylenediamine and the like. Examples of the salt with inorganic acid include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid and the like. Salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid, gluconic acid, methanesulfonic acid , Salts with p-toluenesulfonic acid, benzenesulfonic acid and the like. Examples of salts with basic amino acids include salts with arginine, lysine, ornithine, and examples of salts with acidic amino acids include salts with aspartic acid, glutamic acid, and the like.

  When the agent of the present invention is used as a compound, it can be used in a hydrate form. As the “hydrate”, a pharmacologically acceptable hydrate is preferable, and a hydrated salt is also included. Specific examples include monohydrate, dihydrate, hexahydrate and the like.

(Immunochemistry)
The production of antibodies that recognize the polypeptides of the present invention is also well known in the art. For example, a polyclonal antibody can be prepared by administering a full-length or partial fragment purified preparation of the obtained polypeptide or a peptide having a partial amino acid sequence of the protein of the present invention to an animal as an antigen. .

  When producing antibodies, rabbits, goats, rats, mice, hamsters, etc. can be used as animals to be administered. The dose of the antigen is preferably 50 to 100 μg per animal. In the case of using a peptide, it is desirable to use a peptide covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin. The peptide used as an antigen can be synthesized with a peptide synthesizer. The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. After each administration, blood was collected from the fundus venous plexus on the 3rd day, and the serum reacted with the antigen used for immunization. -Confirmation with A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].

  Serum can be obtained from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and polyclonal antibodies can be separated and purified from the serum using well-known techniques. The production of monoclonal antibodies is also well known in the art. For the preparation of antibody-producing cells, first, a rat whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the polypeptide of the present invention used for immunization was used as a source of antibody-producing cells. A hybridoma is prepared by fusion with myeloma cells. Thereafter, a hybridoma that specifically reacts with the partial fragment polypeptide of the polypeptide of the present invention is selected by enzyme immunoassay or the like. The monoclonal antibody produced from the hybridoma thus obtained can be used for various purposes.

  Such an antibody can be used, for example, in the immunological detection method of the polypeptide of the present invention. As an immunological detection method of the polypeptide of the present invention using the antibody of the present invention, a microtiter plate is used. Examples of the ELISA method, fluorescent antibody method, Western blot method, and immunohistochemical staining method that can be used.

It can also be used in an immunological quantification method of the polypeptide of the present invention. As a method for quantifying the polypeptide of the present invention, sandwich ELISA method using two kinds of monoclonal antibodies having different epitopes among antibodies that react with the polypeptide of the present invention in a liquid phase, labeled with a radioisotope such as ¹²⁵ I Examples thereof include a radioimmunoassay method using the protein of the present invention and an antibody that recognizes the protein of the present invention.

  Methods for quantifying the polypeptide mRNA of the present invention are also well known in the art. For example, the expression level of the DNA encoding the polypeptide of the present invention can be quantified at the mRNA level by the Northern hybridization method or the PCR method using the oligonucleotide prepared from the polynucleotide or the DNA of the present invention. Such techniques are well known in the art and are also described in the literature listed herein.

  These polynucleotides can be obtained and the nucleotide sequence of these polynucleotides can be determined by any method known in the art. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (see, eg, Kutmeier et al., BioTechniques 17: 242 (1994)). In short, this includes the synthesis of overlapping nucleotides containing portions of the antibody-encoding sequence, annealing and ligation of those oligonucleotides, and then amplification of this ligated oligonucleotide by PCR. Including.

  Polynucleotide encoding the antibody can be made from nucleic acid from a suitable source. A clone containing a nucleic acid encoding an antibody is not available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or can be synthesized by a suitable source (eg, Antibody cDNA library or cDNA library generated from any tissue or cell that expresses the antibody (eg, a hybridoma cell selected for expression of the antibody of the invention), or a nucleic acid isolated therefrom (preferably Poly A + RNA)), for example, by PCR amplification using synthetic primers hybridizable to the 3 ′ and 5 ′ ends of the sequence to identify cDNA clones from a cDNA library encoding the antibody, or Use oligonucleotide probes specific for that particular gene sequence It can be obtained by cloning. Amplified nucleic acids generated by PCR can be cloned into replicable cloning vectors using any method well known in the art.

  Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, the nucleotide sequence of the antibody can be determined using methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (See, for example, Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al. Which are both incorporated herein by reference in their entirety))). Are have been operated, for example, substitution of amino acids, to generate antibodies having a different amino acid sequences to generate deletions, and / or insertion.

  In certain embodiments, the heavy chain variable domain and / or light chain variable domain amino acid sequence is determined by methods well known in the art for identification of complementarity determining region (CDR) sequences (eg, sequence hypervariables). To determine the sex region, it can be examined (by comparison with known amino acid sequences of other heavy and light chain variable regions). Using conventional recombinant DNA technology, one or more CDRs can be inserted into the framework region as described above (eg, into the human framework region to humanize non-human antibodies). . This framework region can be naturally occurring or can be a consensus framework region, and preferably can be a human framework region (see, eg, Chothia et al., J. Mol. Biol.278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions can be made within the framework regions, and preferably the amino acid substitution improves the binding of the antibody to its antigen. In addition, such methods also provide for amino acid substitutions or deletions of one or more variable region cysteine residues involved in intrachain disulfide bonds to produce antibody molecules that lack one or more intrachain disulfide bonds. Can be used to make. Other changes to the polynucleotide are encompassed by the present invention and in the art.

  In addition, a technique developed for the production of “chimeric antibodies” (Morrison) by splicing a gene from an appropriate antigen-specific mouse antibody molecule with a gene from a human antibody molecule of appropriate biological activity. Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)). As described above, a chimeric antibody is a molecule in which different portions are derived from different animal species, and such molecules have variable regions derived from mouse mAb and human immunoglobulin constant regions (eg, humanized antibodies). .

  When producing single chain antibodies, known techniques described for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Natl. Acad.Sci.USA 85: 5879-5883 (1988); and Ward et al., Nature 334: 544-54 (1989)) can be utilized. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

(Method of producing antibody)
The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, by chemical synthesis, or preferably by recombinant expression techniques.

  Recombinant expression of an antibody of the invention, or a fragment, derivative or analog thereof (eg, a heavy or light chain of an antibody of the invention or a single chain antibody of the invention) comprises expression comprising a polynucleotide encoding the antibody. Requires vector construction. Once a polynucleotide encoding an antibody molecule of the invention or a heavy or light chain of an antibody, or a portion thereof (preferably containing a heavy or light chain variable domain) is obtained, production of the antibody molecule Vectors for can be generated by recombinant DNA techniques using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing a nucleotide sequence encoding an antibody are described herein. Methods well known to those skilled in the art can be used for the construction of expression vectors containing sequences encoding the antibody and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention relates to a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a variable domain of a heavy or light chain, operably linked to a promoter. I will provide a. Such vectors can comprise a nucleotide sequence encoding the constant region of the antibody molecule (see, eg, PCT publication WO86 / 05807; PCT publication WO89 / 01036; and US Pat. No. 5,122,464), The variable domain of the antibody can then be cloned into such a vector for the entire expression of the heavy or light chain.

  The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies of the present invention. Accordingly, the present invention includes host cells comprising a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment for the expression of a double chain antibody, vectors encoding both heavy and light chains are co-expressed in a host cell for expression of the entire immunoglobulin molecule, as detailed below. Can be done.

  In a related aspect of the invention, a pharmaceutical composition (eg, a vaccine composition) is provided for prophylactic or therapeutic applications. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the invention and an immunostimulatory agent (eg, an adjuvant).

The antibodies of the invention (eg, monoclonal antibodies) can be used to isolate the polypeptides of the invention, etc. by standard techniques (eg, affinity chromatography or immunoprecipitation). An antibody specific for a factor can facilitate the purification of the natural factor from the cell and the recombinantly produced factor expressed in the host cell. In addition, such antibodies can be used to detect proteins of the invention (eg, in cell lysates or cell supernatants) to assess the amount and pattern of expression of the proteins of the invention. Such antibodies can be used to diagnostically monitor protein levels in tissues as part of a clinical trial procedure, for example, to determine the effectiveness of a given treatment regimen. Detection can be facilitated by linking (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of such fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of luminescent materials include luciferase, luciferin, and aequorin; and, ¹²⁵ I examples of suitable radioactive material ^{131 I, 35} include S or ³ H but not limited to.

  Another aspect of the invention relates to a method of inducing an immune response against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response in the mammal. This amount depends on the animal species, animal size, etc., but can be determined by one skilled in the art.

(screening)
As used herein, “screening” refers to selecting a target such as a target organism or substance having a specific property from a large number of populations by a specific operation / evaluation method. For screening, an agent (eg, antibody), polypeptide or nucleic acid molecule of the invention can be used. For the screening, a system using an actual substance such as in vitro or in vivo may be used, or a library generated using an in silico (computer system) system may be used. In the present invention, it is understood that compounds obtained by screening having a desired activity are also encompassed within the scope of the present invention. The present invention also contemplates providing a drug by computer modeling based on the disclosure of the present invention.

  In one embodiment, the invention screens for candidate or test compounds that bind to or modulate the activity of the protein of the invention or polypeptide of the invention, or biologically active portion thereof. An assay is provided. Test compounds of the present invention are obtained using any of a number of approaches in combinatorial library methods known in the art, including: biological libraries; spatial access Possible parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; “one bead one compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, but the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12). 145).

  Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994) J. MoI. Med. Chem 37: 2678; Cho et al. (1993) Science 2611: 1303; Carrell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell et al. (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) J. MoI. Med. Chem 37: 1233.

  The library of compounds can be in solution (eg, Houghten (1992) BioTechniques 13: 412-421), or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386-390; Devlin (1990) Science 249: 404-406; Cwillla et al. (1990) Proc. Natl. Acad. ci.U.S.A.87: 6378~6382; Felici (1991) J Mol Biol 222: 301~310; Ladner above) can be shown in.

(Pharmaceutical composition)
The polynucleotide of the present invention, an expression vector and a gene transfer vector containing the polynucleotide, an antisense nucleic acid, a polypeptide, and an antibody against the polypeptide are used for suppressing vascular endothelial cell growth, for suppressing transcription from the c-fos promoter, For transcriptional repression from the E2F promoter, for transcriptional repression from the AP1 promoter, for VEGF activity inhibition and for angiogenesis inhibition, and rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and It can also be used as a component of a pharmaceutical composition for the treatment, prevention, diagnosis or prognosis of cancer.

  In the present specification, the “effective amount” of a drug refers to an amount capable of exerting the intended drug effect of the drug. In the present specification, among such effective amounts, the minimum concentration may be referred to as the minimum effective amount. Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art or can be determined as appropriate by those skilled in the art. In order to determine such an effective amount, an animal model or the like can be used in addition to actual administration. The present invention is also useful in determining such effective amounts.

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, but are not limited to: antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents , Fillers, bulking agents, buffering agents, delivery vehicles, excipients and / or agricultural or pharmaceutical adjuvants.

  The type and amount of the drug used in the treatment method of the present invention is based on the information obtained by the method of the present invention (for example, information on the disease), the purpose of use, the target disease (type, severity, etc.), A person skilled in the art can easily determine the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

  In the present invention, once an analysis result of a specific sugar chain structure and a disease level are correlated with organisms, cultured cells, tissues, etc. of similar types (for example, mice against humans), the corresponding sugar Those skilled in the art will readily understand that the analysis results of the chain structure can be correlated with the disease level. Such matters are described and supported by, for example, the Animal Culture Cell Manual, edited by Seno et al., Kyoritsu Shuppan, 1993, etc., all of which are incorporated herein by reference.

(Gene therapy)
For a general review of methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 11 (5): 155-215 (1993). Commonly known recombinant DNA techniques used in gene therapy are described in Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene TransferMand Transfer and ExplorMorL. , Stockton Press, NY (1990).

  Nucleic acid constructs used for gene therapy can be administered either locally or systemically using well-known gene transfer vectors. Where such a nucleic acid construct includes a coding sequence for a protein, expression of the protein can be induced by use of an endogenous mammalian promoter or a heterologous promoter. The expression of the coding sequence can be constitutive or regulated.

  When various well-known gene transfer vectors are used as a composition for gene therapy, administration of the vector is performed by locally administering a vector suspension suspended in PBS (phosphate buffered saline) or saline. This can be done by direct injection (eg, in cancer tissue, liver, muscle, brain, etc.) or by administration into blood vessels (eg, intraarterial, intravenous, and portal vein).

  In one embodiment, the gene transfer vector is generally used in a unit dose injectable form (solution, suspension or emulsion) of the gene transfer vector (ie, a pharmaceutically acceptable carrier (ie, used). Non-toxic to recipients at different dosages and concentrations, and compatible with the other ingredients of the formulation). For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to gene transfer vectors.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphoric acid, citric acid, succinic acid, acetic acid, and other organic acids or their salts An antioxidant such as ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide (eg, polyarginine or tripeptide); a protein (eg, serum albumin, gelatin, or an immunoglobulin); a hydrophilic polymer ( Amino acids (eg, glycine, glutamic acid, aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (including cellulose or derivatives thereof, glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg mannitol) Other sorbitol); counterions (such as sodium); and / or nonionic surfactants (e.g., may include polysorbates, poloxamers), or PEG.

  A pharmaceutical composition comprising a gene transfer vector can typically be stored as an aqueous solution in unit or multi-dose containers, such as sealed ampoules or vials.

  A pharmaceutical composition comprising a gene transfer vector is a clinical condition of an individual patient (eg, a condition to be prevented or treated), in a manner consistent with good medical practice, of the composition comprising the gene transfer vector. It is formulated and administered taking into account the delivery site, target tissue, method of administration, dosing schedule and other factors known to those of skill in the art.

  For example, when an HVJ (Sendai virus) envelope vector is administered to a mouse, an envelope equivalent to 20 to 20,000 HAU, preferably equivalent to 60 to 6,000 HAU, more preferably equivalent to 200 to 2,000 HAU per mouse. A vector is administered. The amount of the foreign gene contained in the administered envelope vector is 0.1 to 100 μg, preferably 0.3 to 30 μg, more preferably 1 to 10 μg per mouse.

  When an HVJ (Sendai virus) envelope vector is administered to a human, it corresponds to 400 to 400,000 HAU, preferably 1,200 to 120,000 HAU, more preferably 4,000 to 40,000 HAU per subject. A considerable envelope vector is administered. The amount of the foreign gene contained in the administered envelope vector is 2 to 2,000 μg, preferably 6 to 600 μg, more preferably 20 to 200 μg per subject.

  It is also possible to place the polypeptide of the present invention into a gene transfer vector and deliver the polypeptide to a desired site in the same manner as used in the above gene therapy.

(General techniques used in this specification)
Unless otherwise specifically indicated, the techniques used herein are within the scope of the art, unless otherwise indicated, glycoscience, microfluidics, microfabrication, organic chemistry, biochemistry, genetic engineering, Well-known conventional techniques in molecular biology, microbiology, genetics and related fields are used. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein.

  For microfabrication, see, for example, Campbell, S .; A. (1996). The Science and Engineering of Microelectronic Fabrication, Oxford University Press; V. (1996). Micromicroarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, M .; J. et al. (1997). Fundamentals of Microfabrication, CRC1 5 Press; Rai-Chudhury, P .; (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, etc., which are incorporated by reference in the relevant portions herein.

  Molecular biological techniques, biochemical techniques, microbiological techniques, and glycoscience techniques used in this specification are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J. et al. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Techniques, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Related parts (which may be all) are incorporated by reference.

(Prescription)
The invention also provides a method of treating and / or preventing a disease or disorder (eg, an infection) by administering an effective amount of a therapeutic agent to a subject. By therapeutic agent is meant a composition of the invention in combination with a pharmaceutically acceptable carrier type (eg, a sterile carrier).

  The therapeutic agent is considered to be a medical practice standard (GMP = good) taking into account the clinical condition of the individual patient (especially the side effects of the therapeutic agent alone treatment), the site of delivery, the method of administration, the dosage regimen and other factors known to those skilled in the art Formulate and dose in a manner consistent with medical practice. Therefore, the target “effective amount” in the present specification is determined by taking such consideration into consideration.

  As a general proposition, the total pharmaceutically effective amount of peptide therapeutic administered parenterally per dose is in the range of about 0.1 μg / kg / day to 10 mg / kg / day of patient weight, As noted above, this is left to therapeutic discretion. More preferably, for the cell bioactive agent of the present invention, this dose is at least 1 μg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. is there. For continuous administration, typically the therapeutic agent is injected 1 to 4 times daily at a dosage rate of about 1 μg / kg / hour to about 50 μg / kg / hour or continuous subcutaneous infusion (eg, using a minipump). It is administered by either of the following. Intravenous bag solutions may also be used. The duration of treatment necessary to observe the change and the post-treatment interval at which a response occurs appears to vary depending on the desired effect.

  The therapeutic agent can be oral, rectal, parenteral, intracisternally, intravaginally, intraperitoneally, topically (such as by powder, ointment, gel, instillation, or transdermal patch), oral or oral or It can be administered as a nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation aid. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are oral, rectal, parenteral, intracisternally, intravaginally, intraperitoneally, topically (powder, ointment, gel, infusion, or transdermal patch) Etc.), or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulant or any type of formulation aid. The term “parenteral” as used herein refers to modes of administration including intravenous and intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injections and infusions.

  The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents include suitable polymer materials (eg, semipermeable polymer matrices in the form of molded articles (eg, films or microcapsules)), suitable hydrophobic materials (eg, in acceptable quality oils). Or an ion exchange resin, and poorly soluble derivatives (eg, poorly soluble salts).

  Sustained release matrices include polylactide (US Pat. No. 3,773,919, EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-556 (1983)). ), Poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl. Acetate (Langer et al., Ibid.) Or poly-D-(-)-3-hydroxybutyric acid (EP133,988).

  Sustained release therapeutic agents also include the therapeutic agents of the present invention entrapped in liposomes (generally, Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes in the Disease of Infectious Disease and Cancer, Loop, -See Berstein and Fiddler (eds.), Liss, New York, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP52,322; EP36,676; EP88,046; EP143,949; EP142,641; Japanese Patent Application No. 83-118008; US Pat. No. 4,485,045 and No. 4,544,545 and EP No. 102,324. Usually, liposomes are small (about 200-800 cm) unilamellar type, where the lipid content is greater than about 30 mol% cholesterol, and the selected proportion is adjusted for optimal therapeutic agents.

  In still further embodiments, the therapeutics of the invention are delivered by a pump (Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgary 88: 507. (1980); see Saudek et al., N. Engl. J. Med. 321: 574 (1989)).

  Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

  For parenteral administration, in one embodiment, in general, the therapeutic agent is toxic to the recipient in the desired degree of purity, in a pharmaceutically acceptable carrier, i.e. the dosage and concentration used. It is formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with one that is not and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidation and other compounds known to be harmful to therapeutic agents.

  Generally, formulations are prepared by contacting the therapeutic agent uniformly and intimately with liquid carriers or finely divided solid carriers or both. Next, if necessary, the product is shaped into the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Nonaqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

  The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipient at the dosages and concentrations used, such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts. Buffering agents; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone Hydrophilic polymers such as: amino acids such as glycine, glutamic acid, aspartic acid or arginine; monosaccharides, disaccharides and other carbohydrates including cellulose or derivatives thereof, glucose, mannose or dextrin; chelating agents such as EDTA Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

  The therapeutic agent is typically formulated in such a vehicle at a pH of about 3-8 at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml. It is understood that by using the specific excipients, carriers or stabilizers described above, salts are formed.

  Any drug to be used for therapeutic administration may be in a state free of organisms / viruses other than viruses as an active ingredient, that is, in a sterile state. Aseptic conditions are easily achieved by filtration through sterile filtration membranes (eg, 0.2 micron membranes). In general, the therapeutic agent is placed in a container having a sterile access port, for example, an intravenous solution bag or vial with a stopper puncturable with a hypodermic needle.

  The therapeutic agents are typically stored in unit dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 ml vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous therapeutic agent and the resulting mixture is lyophilized. The lyophilized therapeutic agent is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

  The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the therapeutic agent of the present invention. A notice of the form prescribed by a government agency that regulates the manufacture, use or sale of a medicinal product or biological product may be attached to such a container, and this notice may be attached to the government regarding the manufacture, use or sale for human administration. Represents institutional approval. In addition, the therapeutic agent may be used in combination with other therapeutic compounds.

  The therapeutic agents of the present invention can be administered alone or in combination with other therapeutic agents. The combinations can be administered, for example, either simultaneously as a mixture; simultaneously or concurrently but separately; or over time. This includes the presentation that the combined agents are administered together as a therapeutic mixture, and also the procedure where the combined agents are administered separately but simultaneously, eg, through separate intravenous lines to the same individual . Administration in combination further includes separate administration of one of the compounds or agents given first, followed by the second.

  In certain embodiments, the pharmaceutical compositions of the invention are administered in combination with an anticancer agent.

(Polypeptide form of the protein encoded by pCMV9)
In one aspect, the invention provides a composition for treating, preventing or prognosing chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer comprising a polypeptide of a protein encoded by pCMV9. I will provide a. Here, an amount effective for prevention, treatment or prognosis can be determined by a person skilled in the art using various well-known techniques in consideration of various parameters, and in order to determine such an amount, For example, it can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like.

  In one embodiment, the polypeptide derived from the protein encoded by pCMV9 of the present invention is (a) a polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 or 3 or a fragment thereof; (b) SEQ ID NO: A polypeptide having the amino acid sequence described in 2 or 4, or a fragment thereof; (c) in the amino acid sequence described in SEQ ID NO: 2 or 4, one or more amino acids are selected from the group consisting of substitutions, additions and deletions A variant polypeptide having at least one mutation, encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 1 or 3 having a biological activity. (E) a species homologue poly of the amino acid sequence set forth in SEQ ID NO: 2 or 4 Or (f) a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of the polypeptide of any one of (a) to (e) and having biological activity Including.

  In one preferred embodiment, the number of substitutions, additions and deletions in (c) above may be limited, for example, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less. 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably an activity similar to or substantially identical to the protein encoded by pCMV9, eg, vascular endothelial cell proliferation Repression activity, transcription repression activity from c-fos promoter, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity) As long as the number is large.

  In another preferred embodiment, the allelic variant in (d) above preferably has at least 99% homology with the amino acid sequence shown in SEQ ID NO: 2 or 4.

  In another preferred embodiment, the species homologue can be identified as described herein above and preferably has at least about 30% homology with the amino acid sequence set forth in SEQ ID NO: 2 or 4. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  The species homologue can be identified by searching the amino acid sequence of the peptide encoded by pCMV9 of the present invention as a query sequence in the gene sequence database of the species. Alternatively, it can be identified by screening such a gene library using all or part of the gene encoded by pCMV9 of the present invention as a probe or primer. Such identification methods are well known in the art and are also described in the literature described herein. The species homologue preferably has at least about 30% homology with, for example, the nucleic acid sequence shown in SEQ ID NO: 1 or 3 or the amino acid sequence shown in SEQ ID NO: 2 or 4. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  In another preferred embodiment, the biological activity of the variant polypeptide includes, for example, mutual interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof. Action, vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, transcription inhibitory activity from AP1 promoter, VEGF activity inhibitory activity, transcription inhibitory activity from NFκB promoter, angiogenesis Examples thereof include, but are not limited to, inhibitory activity. These can be measured, for example, by immunological assays, phosphorylation assays, cell proliferation assays, promoter assays, angiogenesis assays, and the like.

  In preferred embodiments, the homology to any one of the polypeptides (a)-(d) above may be at least about 80%, more preferably at least about 90%, and even more preferably at least about 98. %, Most preferably at least about 99%.

  The polypeptide of the present invention usually has at least 3 consecutive amino acid sequences. The amino acid length of the polypeptide of the present invention can be any length as long as it suits the intended use, but preferably a longer sequence can be used. Thus, it may preferably be at least 4 amino acids long, more preferably at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long. More preferably it may be at least 15 amino acids long, and still more preferably at least 20 amino acids long. The lower limit of these amino acid lengths is a number between them (for example, 11, 12, 13, 14, 16, etc.) or more (for example, 21, 22, ..., Etc.). The polypeptide of the present invention includes an interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4, or a fragment thereof, vascular endothelial cell growth inhibitory activity, transcription from the c-fos promoter. As long as it has at least one activity of repression activity, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity, The length may be the same as the full length of the sequence shown in SEQ ID NO: 2 or 4, or may be longer than that.

  In one embodiment, the disease, disorder or condition to be treated is described elsewhere herein, such as rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer. Etc. Preferably, the disease, disorder or condition targeted by the composition of the present invention may be cancer.

  In another embodiment, the peptide derived from the protein encoded by pCMV9 of the present invention is preferably soluble.

(Nucleic acid form of gene encoded by pCMV9)
In one aspect, the invention provides a composition for treating, preventing, or prognosing chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer comprising a nucleic acid molecule that encodes a protein encoded by pCMV9. I will provide a. Here, an amount effective for prevention, treatment or prognosis can be determined by a person skilled in the art using various well-known techniques in consideration of various parameters, and in order to determine such an amount, For example, it can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like.

  In one embodiment, a nucleic acid molecule encoding a protein encoded by pCMV9 used in the present invention, or a fragment or variant thereof has (a) the nucleotide sequence set forth in SEQ ID NO: 1 or 3 or a fragment sequence thereof (B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 2 or 4, or a fragment thereof; (c) in the amino acid sequence set forth in SEQ ID NO: 2 or 4, wherein one or more amino acids are substituted, A polynucleotide having a variant polypeptide having at least one mutation selected from the group consisting of addition and deletion, the variant polypeptide having biological activity; (d) SEQ ID NO: 1 or 3 Is a splice variant or allelic variant of the nucleotide sequence described in (E) a polynucleotide encoding a species homologue of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4; (f) a string in any one of the polynucleotides of (a) to (e) A polynucleotide encoding a polypeptide having a biological activity and hybridizing under a gentle condition; or (g) identity to a polynucleotide of any one of (a) to (e) or a complementary sequence thereof A polynucleotide selected from the group consisting of a polynucleotide comprising a base sequence that is at least 70% and encoding a polypeptide having biological activity.

  In one preferred embodiment, the number of substitutions, additions and deletions in (c) above is limited, for example, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less. It is preferably 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably an activity similar to or substantially identical to the protein encoded by pCMV9, eg, vascular endothelial cell proliferation Repression activity, transcription repression activity from c-fos promoter, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity) As long as the number is large.

  In another preferred embodiment, the biological activity of the variant polypeptide includes, for example, mutual interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 4 or a fragment thereof. Action, interaction with an antibody specific for the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4, or a fragment thereof, vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, E2F promoter Include, but are not limited to, transcriptional repressing activity from ATP, transcriptional repressing activity from the AP1 promoter, VEGF activity repressing activity, transcription repressing activity from the NFκB promoter, and angiogenesis repressing activity. These can be measured, for example, by immunological assays, phosphorylation assays, cell proliferation assays, promoter assays, angiogenesis assays, and the like.

  In another preferred embodiment, the allelic variant described in (d) advantageously has at least 99% homology with the nucleic acid sequence shown in SEQ ID NO 1 or 3.

  When the gene sequence database of the species exists, the species homologue can be identified by searching the database for the nucleic acid sequence of the gene encoded by pCMV9 of the present invention as a query sequence. Alternatively, it can be identified by screening such a gene library using all or part of the nucleic acid sequence of the gene encoded by pCMV9 of the present invention as a probe or primer. Such identification methods are well known in the art and are also described in the literature described herein. The species homologue preferably has, for example, at least about 30% homology with the nucleic acid sequence shown in SEQ ID NO: 1 or 3. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  In a preferred embodiment, the identity to any one of the polynucleotides (a) to (e) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%, even more preferably May be at least about 98%, and most preferably at least about 99%.

  In a preferred embodiment, a nucleic acid molecule encoding a polypeptide encoded by pCMV9 of the present invention or fragments and variants thereof may be at least 8 contiguous nucleotides long. The appropriate nucleotide length of the nucleic acid molecule of the present invention can vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides long, more preferably at least 15 contiguous nucleotides long, and even more preferably at least 20 contiguous nucleotides long. The lower limit of these nucleotide lengths is, in addition to the numbers specifically mentioned, numbers between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more numbers (for example, 21, 22, ... 30, etc.). As long as the nucleic acid molecule of the present invention can be used for an intended use (for example, antisense, RNAi, marker, primer, probe, capable of interacting with a predetermined factor), the upper limit length thereof is It may be the full length of the sequence shown in SEQ ID NO: 1 or 3, or a length exceeding it. Alternatively, when used as a primer, it can usually be at least about 8 nucleotides in length, preferably about 10 nucleotides in length. When used as a probe, it can usually be at least about 15 nucleotides in length, preferably about 17 nucleotides in length.

  In one embodiment, the nucleic acid molecule encoding a polypeptide encoded by pCMV9, or a fragment or variant thereof, comprises the entire range of the nucleic acid sequence of SEQ ID NO: 1 or 3. More preferably, the nucleic acid molecule encoding the polypeptide encoded by pCMV9, or a fragment or variant thereof consists of the entire range of the nucleic acid sequence of SEQ ID NO: 1 or 3.

  In one embodiment, the disease, disorder or condition to be treated is described elsewhere herein, such as rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer. Etc. Preferably, the disease, disorder or condition targeted by the composition of the present invention may be cancer.

(Polypeptide form of the protein encoded by pCMV15)
In one aspect, the present invention provides for treating, preventing or prognosing rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer comprising a polypeptide of the protein encoded by pCMV15. A composition is provided. Here, an amount effective for prevention, treatment or prognosis can be determined by a person skilled in the art using various well-known techniques in consideration of various parameters, and in order to determine such an amount, For example, it can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like.

  In one embodiment, the polypeptide derived from the protein encoded by pCMV15 of the present invention is (a) a polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 5 or 7 or a fragment thereof; (b) SEQ ID NO: A polypeptide having the amino acid sequence described in 6 or 8 or a fragment thereof; (c) in the amino acid sequence described in SEQ ID NO: 6 or 8, one or more amino acids are selected from the group consisting of substitution, addition and deletion A variant polypeptide having at least one mutation, encoded by a splice variant or allelic variant of the base sequence set forth in SEQ ID NO: 5 or 7 having a biological activity. (E) a species homologue of the amino acid sequence set forth in SEQ ID NO: 6 or 8; A polypeptide; or (f) a polypeptide comprising an amino acid sequence having at least 70% identity to the amino acid sequence of any one of the polypeptides (a) to (e) and having biological activity, Including.

  In one preferred embodiment, the number of substitutions, additions and deletions in (c) above may be limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less. 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably an activity similar to or substantially identical to the protein encoded by pCMV15 (eg, vascular endothelial cell proliferation Repression activity, transcription repression activity from c-fos promoter, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity) As long as the number is large.

  In another preferred embodiment, the allelic variant in (d) above preferably has at least 99% homology with the amino acid sequence shown in SEQ ID NO: 6 or 8.

  In another preferred embodiment, the species homologue can be identified as described herein above and preferably has at least about 30% homology with the amino acid sequence set forth in SEQ ID NO: 6 or 8. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  The species homologue can be identified by searching the amino acid sequence of the peptide encoded by pCMV15 of the present invention as a query sequence in the gene sequence database of the species. Alternatively, it can be identified by screening such a gene library using all or part of the gene encoded by pCMV15 of the present invention as a probe or primer. Such identification methods are well known in the art and are also described in the literature described herein. The species homologue preferably has at least about 30% homology with, for example, the nucleic acid sequence shown in SEQ ID NO: 5 or 7, or the amino acid sequence shown in SEQ ID NO: 6 or 8. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  In another preferred embodiment, the biological activity of the variant polypeptide includes, for example, mutual interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 8, or a fragment thereof. Action, vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, transcription inhibitory activity from E2F promoter, transcription inhibitory activity from AP1 promoter, VEGF activity inhibitory activity, transcription inhibitory activity from NFκB promoter, angiogenesis Examples thereof include, but are not limited to, inhibitory activity. These can be measured, for example, by immunological assays, phosphorylation assays, cell proliferation assays, promoter assays, angiogenesis assays, and the like.

  In preferred embodiments, the homology to any one of the polypeptides (a)-(d) above may be at least about 80%, more preferably at least about 90%, and even more preferably at least about 98. %, Most preferably at least about 99%.

  The polypeptide of the present invention usually has at least 3 consecutive amino acid sequences. The amino acid length of the polypeptide of the present invention can be any length as long as it suits the intended use, but preferably a longer sequence can be used. Thus, it may preferably be at least 4 amino acids long, more preferably at least 5 amino acids long, at least 6 amino acids long, at least 7 amino acids long, at least 8 amino acids long, at least 9 amino acids long, at least 10 amino acids long. More preferably it may be at least 15 amino acids long, and still more preferably at least 20 amino acids long. The lower limit of these amino acid lengths is a number between them (for example, 11, 12, 13, 14, 16, etc.) or more (for example, 21, 22, ..., Etc.). The polypeptide of the present invention includes an interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 8 or a fragment thereof, vascular endothelial cell growth inhibitory activity, and transcription from the c-fos promoter. As long as it has at least one activity of repression activity, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity, The length may be the same as the full length of the sequence shown in SEQ ID NO: 6 or 8, or may be longer than that.

  In one embodiment, the disease, disorder or condition to be treated is described elsewhere herein, such as rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer. Etc. Preferably, the disease, disorder or condition targeted by the composition of the present invention may be cancer.

  In another embodiment, the peptide derived from the protein encoded by pCMV15 of the present invention is preferably soluble.

(Nucleic acid form of gene encoded by pCMV15)
In one aspect, the present invention is for the treatment, prevention, or prognosis of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer comprising a nucleic acid molecule that encodes a protein encoded by pCMV15. A composition is provided. Here, an amount effective for prevention, treatment or prognosis can be determined by a person skilled in the art using various well-known techniques in consideration of various parameters, and in order to determine such an amount, For example, it can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, and the like.

  In one embodiment, a nucleic acid molecule encoding a protein encoded by pCMV15 used in the present invention, or a fragment or variant thereof has (a) the nucleotide sequence set forth in SEQ ID NO: 5 or 7 or a fragment sequence thereof. (B) a polynucleotide encoding the amino acid sequence set forth in SEQ ID NO: 6 or 8, or a fragment thereof; (c) in the amino acid sequence set forth in SEQ ID NO: 6 or 8, wherein one or more amino acids are substituted, A polynucleotide having a variant polypeptide having at least one mutation selected from the group consisting of addition and deletion, the variant polypeptide having biological activity; (d) SEQ ID NO: 5 or 7 A splice variant or allelic variant of the nucleotide sequence described in (E) a polynucleotide encoding a species homologue of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 8, and (f) any one of the polynucleotides of (a) to (e) A polynucleotide that hybridizes under stringent conditions and encodes a polypeptide having biological activity; or (g) identity to a polynucleotide of any one of (a) to (e) or a complementary sequence thereof A polynucleotide selected from the group consisting of: a polynucleotide consisting of a base sequence that is at least 70% and encoding a polypeptide having biological activity.

  In one preferred embodiment, the number of substitutions, additions and deletions in (c) above is limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less. 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. A smaller number of substitutions, additions and deletions are preferred, but retain biological activity (preferably an activity similar to or substantially identical to the protein encoded by pCMV15 (eg, vascular endothelial cell proliferation Repression activity, transcription repression activity from c-fos promoter, transcription repression activity from E2F promoter, transcription repression activity from AP1 promoter, VEGF activity repression activity, transcription repression activity from NFκB promoter or angiogenesis repression activity) As long as the number is large.

  In another preferred embodiment, the biological activity of the variant polypeptide includes, for example, mutual interaction with an antibody specific for the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6 or 8, or a fragment thereof. Action, interaction with an antibody specific for the polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 or 8 or a fragment thereof, vascular endothelial cell growth inhibitory activity, transcription inhibitory activity from c-fos promoter, E2F promoter Include, but are not limited to, transcriptional repressing activity from ATP, transcriptional repressing activity from the AP1 promoter, VEGF activity repressing activity, transcription repressing activity from the NFκB promoter, and angiogenesis repressing activity. These can be measured, for example, by immunological assays, phosphorylation assays, cell proliferation assays, promoter assays, angiogenesis assays, and the like.

  In another preferred embodiment, the allelic variant described in (d) advantageously has at least 99% homology with the nucleic acid sequence shown in SEQ ID NO: 5 or 7.

  When the gene sequence database of the species exists, the species homologue can be identified by searching the database for the nucleic acid sequence of the gene encoded by pCMV15 of the present invention as a query sequence. Alternatively, it can be identified by screening such a gene library using all or part of the nucleic acid sequence of the gene encoded by pCMV15 of the present invention as a probe or primer. Such identification methods are well known in the art and are also described in the literature described herein. The species homologue preferably has, for example, at least about 30% homology with the nucleic acid sequence shown in SEQ ID NO: 5 or 7. Preferably, the species homologue is at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% can be homologous.

  In a preferred embodiment, the identity to any one of the polynucleotides (a) to (e) above or a complementary sequence thereof may be at least about 80%, more preferably at least about 90%, even more preferably May be at least about 98%, and most preferably at least about 99%.

  In a preferred embodiment, a nucleic acid molecule encoding a polypeptide encoded by pCMV15 of the present invention, or fragments and variants thereof, may be at least 8 contiguous nucleotides long. The appropriate nucleotide length of the nucleic acid molecule of the present invention can vary depending on the intended use of the present invention. More preferably, the nucleic acid molecules of the invention can be at least 10 contiguous nucleotides long, more preferably at least 15 contiguous nucleotides long, and even more preferably at least 20 contiguous nucleotides long. The lower limit of these nucleotide lengths is, in addition to the numbers specifically mentioned, numbers between them (for example, 9, 11, 12, 13, 14, 16, etc.) or more numbers (for example, 21, 22, ... 30, etc.). As long as the nucleic acid molecule of the present invention can be used for an intended use (for example, antisense, RNAi, marker, primer, probe, capable of interacting with a predetermined factor), the upper limit length thereof is It may be the full length of the sequence shown in SEQ ID NO: 5 or 7, or a length exceeding it. Alternatively, when used as a primer, it can usually be at least about 8 nucleotides in length, preferably about 10 nucleotides in length. When used as a probe, it can usually be at least about 15 nucleotides in length, preferably about 17 nucleotides in length.

  In one embodiment, the nucleic acid molecule encoding a polypeptide encoded by pCMV15, or a fragment or variant thereof, comprises the entire range of the nucleic acid sequence of SEQ ID NO: 5 or 7. More preferably, the nucleic acid molecule encoding the polypeptide encoded by pCMV15, or a fragment or variant thereof, consists of the entire range of the nucleic acid sequence of SEQ ID NO: 5 or 7.

  In one embodiment, the disease, disorder or condition to be treated is described elsewhere herein, such as rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer. Etc. Preferably, the disease, disorder or condition targeted by the composition of the present invention may be cancer.

  In further embodiments, the therapeutic agents of the invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation therapy).

  Hereinafter, the present invention will be described in detail with reference to examples and the like, but the present invention is not limited thereto.

(Example 1: Preparation and use of a gene transfer vector in which a foreign gene is encapsulated in a component containing a virus envelope-derived protein)
(Preparation of virus)
The HVJ, Z strain was prepared as previously described (Kaneda, Cell Biology: A Laboratory Handbook, JE Celis (Ed.), Academic Press, Orlando, Florida, vol. 3, pp. 50-57 (1994). )) Purified by differential centrifugation. Purified HVJ was resuspended in balanced salt solution (BSS: 137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.5) and virus titer was determined by measuring absorbance at 540 nm. The optical density at 540 nm corresponds to 15,000 hemagglutinating units (HAU) and correlates with fusion activity.

(Preparation of gene transfer vector)
A lipid mixture of 3.56 mg phosphatidylcholine and 0.44 mg cholesterol was dissolved in chloroform and the lipid solution was evaporated in a rotary evaporator (Uchida et al., J. Cell. Biol. 80: 10-20 (1979). )). The dry lipid mixture was completely dissolved by vortexing in a protein solution (1.6 mg) from 2.0 ml of the above pass-through fraction containing 0.85% NP-40. The solution was then dialyzed against 10 mM phosphate buffer (pH 7.2) containing 0.3 M sucrose and 1 mM KCl to remove NP-40. Dialysis was performed for 6 days with daily buffer changes. The dialyzed solution was agarose beads (Bio-Gel A-50m) equilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and 1 mM KCl (Bio-Rad Laboratories, Hercules, CA, USA). Fractions with an optical density greater than 1.5 at 540 nm were collected as reconstituted fusion particles and fused with nucleic acid-loaded liposomes prepared from 10 mg lipid as described below to prepare gene transfer vectors.

(Luciferase gene expression in transfected cells derived from human HEK293 strain)
In order to confirm the gene transfer activity of the gene transfer vector prepared by the above method, human HEK293 strain and luciferase gene were used as follows.

pCMV-luciferase (7.4 kb) was transferred from pGEM-luc (Promega Corp., Madison, WI, USA) to HindIII in pcDNA3 (5.4 kb) (Invitrogen, San Diego, CA, USA). And by cloning at the BamHI site. A gene transfer vector containing about 40 μg of pCMV-luciferase was constructed as described above and 1 / of this gene transfer vector (about 1.5 × 10 ¹¹ particles / ml, DNA concentration about 40 μg / ml). Ten volumes (100 μl) were incubated with 2 × 10 ⁵ cells from the human 293 cell line (human embryonic kidney: HEK). The same amount of luciferase DNA was introduced into 2 × 10 ⁵ HEK293 cells using HVJ liposomes. Cells were harvested 24 hours after transfection and the luciferase activity assay was confirmed as described elsewhere (Saeki et al., Hum. Gene Ther., 8: 1965-1972 (1997)).

Example 2: Preparation and Use of Inactivated HVJ Envelope Vector with Surfactant
(1: Growth of HVJ)
HVJ can be generally used by inoculating a fertilized egg of a chicken by inoculation with a seed virus. However, cultured cells such as monkeys and humans, and a system for continuously infecting cultured tissues with a virus (cultivating a hydrolase such as trypsin). Any of those that are propagated by using a solution added to the liquid and those that are propagated by infecting a cultured cell with a cloned viral genome and causing persistent infection can be used.

  In this example, HVJ was propagated as follows.

  HVJ (species Z), which has been propagated using SPF (Specific pathogen free) fertilized eggs, separated and purified, is dispensed into a cell storage tube, and 10% DMSO is added and stored in liquid nitrogen And prepared.

  The chicken eggs immediately after fertilization were received and placed in an incubator (SHOWA-FURANKI P-03 type; containing about 300 chicken eggs) and bred for 10 to 14 days under conditions of 36.5 ° C. and humidity of 40% or more. In the dark room, use an ophthalmoscope (light bulb light exits through a window with an aperture of about 1.5 cm) to check embryo survival and air chamber and chorioallantoic membrane. About 5 mm above, the location of the virus injection was marked with a pencil (select the location excluding the thick blood vessels). Seed virus (taken from liquid nitrogen) 500 times with polypeptone solution (mixed with 1% polypeptone, 0.2% NaCl, adjusted to pH 7.2 with 1M NaOH, autoclaved and stored at 4 ° C.) Diluted to 4 ° C. Eggs were sterilized with isodine and alcohol, a small hole was made in the virus injection site, and 0.1 ml of diluted seed virus was injected into the chorioallantoic cavity using a 1 ml syringe with a 26 gauge needle. Dissolved paraffin (melting point: 50-52 ° C.) was placed on the hole using a Pasteur pipette to close it. The eggs were placed in an incubator and bred for 3 days under conditions of 36.5 ° C. and humidity of 40% or more. The inoculated eggs were then placed at 4 ° C. overnight. The next day, the air space of the egg was split with tweezers, a 10 ml syringe with an 18 gauge needle was placed in the chorioallantoic membrane, the chorioallantoic fluid was aspirated, collected in a sterile bottle, and stored at 4 ° C.

(2: Purification of HVJ)
HVJ can be purified by purification methods by centrifugation, column purification methods, or other purification methods known in the art.
(2.1: Purification method by centrifugation)
Briefly, the grown virus solution was collected, and the tissue and cell debris in the culture solution and chorioallantoic fluid were removed by low speed centrifugation. The supernatant was purified by high-speed centrifugation (27,500 × g, 30 minutes) and ultracentrifugation (62,800 × g, 90 minutes) using a sucrose density gradient (30-60% w / v). It should be noted that the virus should be handled as gently as possible during purification and stored at 4 ° C.

  In this example, specifically, HVJ was purified by the following method.

  Approximately 100 ml of HVJ-containing chorioallantoic fluid (collecting HVJ-containing chicken egg chorioallantoic fluid and storing at 4 ° C.) was placed in two 50 ml centrifuge tubes using a wide Komagome pipette (Saeki, Y., and Kaneda, Y: Protein modified liposomes (HVJ-liposomes) for the delivery of genes, volume, and clones, 27th edition, Cell biology; A. laboratory handbook, E. c. ~ 135, 1998), centrifuged in a low speed centrifuge at 3000 rpm for 10 minutes at 4 ° C (brake off) to remove egg tissue debris.

  After centrifugation, the supernatant was dispensed into four 35 ml centrifuge tubes (for high-speed centrifugation) and centrifuged with an angle rotor at 27,000 g for 30 minutes (accelerator and brake on). The supernatant was removed, and about 5 ml of BSS (10 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5.4 mM KCl; autoclaved and stored at 4 ° C.) (can be PBS instead of BSS) was added to the precipitate, It left still at 4 degreeC as it was. Gently pipetted with a Komagome pipette, widened to loosen the precipitate, collected in a single tube, and centrifuged at 27,000 g for 30 minutes in the same manner. Excluding the supernatant, about 10 ml of BSS was added to the precipitate, and the mixture was allowed to stand at 4 ° C. overnight. The precipitate was loosened by gently pipetting with a wide-bottom Komagome pipette, and centrifuged at 3000 rpm for 10 minutes at 4 ° C. (brake off) with a low-speed centrifuge (the brake was off). The supernatant is placed in a new sterile tube and stored as purified virus at 4 ° C.

  0.9 ml of BSS was added to 0.1 ml of this virus solution, absorption at 540 nm was measured with a spectrophotometer, and the virus titer was converted to hemagglutination activity (HAU). An absorption value 1 at 540 nm corresponded to approximately 15,000 HAU. HAU is considered to be approximately proportional to the fusion activity. In addition, erythrocyte agglutinating activity may be actually measured using chicken erythrocyte fluid (0.5%) (Animal Cell Utilization Manual, REALIZE INC. (Uchida, Oishi, Furuzawa) P259-268, 1984. See

Furthermore, purification of HVJ using a sucrose density gradient can be performed as necessary. Specifically, the virus suspension is placed in a centrifuge tube overlaid with 60% and 30% sucrose solutions (autoclaved), and density gradient centrifugation is performed at 62,800 × g for 120 minutes. After centrifugation, the band seen on the 60% sucrose solution layer is collected. The collected virus suspension is dialyzed overnight at 4 ° C. using BSS or PBS as an external solution to remove sucrose. If not used immediately, add glycerol (autoclave sterilization) and 0.5M EDTA solution (autoclave sterilization) to the virus suspension to a final concentration of 10% and 2-10 mM, respectively, and gently at −80 ° C. Freeze and finally store in liquid nitrogen (freezing can be done in 10 mM DMSO instead of glycerol and 0.5 M EDTA solution).
(2.2: Purification method by column and ultrafiltration)
Instead of the purification method by centrifugation, purification of HVJ using a column is also applicable to the present invention.

  Briefly, purification was performed using concentration by ultrafiltration with a filter with a molecular weight cut-off of 50,000 (about 10 times) and elution by ion exchange chromatography (0.3 M to 1 M NaCl).

  Specifically, in this example, HVJ was purified by column using the following method.

  After the chorioallantoic fluid was collected, it was filtered through an 80 μm to 10 μm membrane filter. 0.006-0.008% BPL (final concentration) was added to chorioallantoic fluid (4 ° C., 1 hour) to inactivate HVJ. BPL was inactivated by incubating chorioallantoic fluid at 37 ° C. for 2 hours.

  The solution was concentrated about 10 times by tangential flow ultrafiltration using 500 KMWCO (A / G Technology, Needham, Massachusetts). As a buffer, 50 mM NaCl, 1 mM MgCl, 2% mannitol, 20 mM Tris (pH 7.5) was used. The HAU assay gave excellent results with almost 100% HVJ recovery.

  HVJ was purified by column chromatography (buffer solution: 20 mM TrisHCl (pH 7.5), 0.2 to 1 M NaCl) by QSepharoseFF (Amersham Pharmacia Biotech KK, Tokyo). The recovery was 40 to 50%, and the purity was 99% or more.

  The HVJ fraction was concentrated by tangential flow ultrafiltration using 500 KMWCO (A / G Technology).

(3: Inactivation of HVJ)
When inactivation of HVJ was required, it was performed by ultraviolet irradiation or alkylating agent treatment as described below.

(3.1: UV irradiation method)
1 ml of HVJ suspension was placed in a petri dish having a diameter of 30 mm and irradiated with 99 or 198 millijoules / cm ² . Gamma irradiation can also be used (5-20 gray), but complete inactivation does not occur.

(3.2: Treatment with alkylating agent)
Immediately before use, 0.01% β-propiolactone was prepared in 10 mM KH ₂ PO. During the work, it was kept at a low temperature and worked quickly.

  Β-propiolactone was added to a suspension of HVJ immediately after purification to a final concentration of 0.01% and incubated on ice for 60 minutes. Thereafter, it was incubated at 37 ° C. for 2 hours. Dispense 10,000 HAU per tube into an Eppendorf tube, centrifuge at 15,000 rpm for 15 minutes, and store the precipitate at -20 ° C. Regardless of the inactivation method described above, the precipitate can be stored at -20 ° C., and the DNA can be directly incorporated by treatment with a surfactant to prepare a vector.

(4: Creation of HVJ envelope vector)
92 μl of a solution containing 200 to 800 μg of foreign DNA was added to the preserved HVJ and well suspended by pipetting. This solution can be stored at −20 ° C. for at least 3 months. When protamine sulfate was added to DNA before mixing with HVJ, the expression efficiency was enhanced by a factor of 2 or more.

  This mixed solution was placed on ice for 1 minute, 8 μl of octyl glucoside (10%) was added, the tube was shaken on ice for 15 seconds, and left on ice for 45 seconds. The treatment time with the surfactant is preferably 1 to 5 minutes. In addition to octylglucoside, Triton-X100 (t-octylphenoxypolyethoxyethanol), CHAPS (3-[(3-colamidopropyl) -dimethylammonio] -1-propanesulfonic acid), NP-40 (nonylphenoxypoly) Surfactants such as ethoxyethanol may also be used. Preferred final concentrations of Triton-X100, NP-40 and CHAPS are 0.24-0.80%, 0.04-0.12% and 1.2-2.0%, respectively.

  1 ml of cold BSS was added and immediately centrifuged at 15,000 rpm for 15 minutes. 300 μl of PBS or physiological saline was added to the resulting precipitate and suspended by vortexing and pipetting. The suspension can be used directly for gene transfer or can be used for gene transfer after storage at −20 ° C. This HVJ envelope vector maintained comparable gene transfer efficiency after storage for at least 2 months.

(5: Gene transfer into cells using HVJ envelope vector)
(Gene transfer method)
1,000 HAU was placed in an Eppendorf tube (30 μl), and 5 μl of protamine sulfate (1 mg / ml) was added. The medium of BHK-21 cells (200,000 per well and spread in 6 wells the day before) was changed, and 0.5 ml of medium (10% FCS-DMEM) was added per well. To each well, add a mixture of the above-mentioned vector (equivalent to 1,000 HAU) and protamine sulfate, mix the vector and cells well by shaking the plate back and forth, left and right, and in a 5% CO ₂ incubator at 37 ° C. for 10 minutes. I left it alone.

The medium was changed and left at 37 ° C. in a 5% CO ₂ incubator overnight (16 hr to 24 hr) to examine gene expression. In the case of luciferase (pcLuci; luciferase gene having a CMV promoter), the cells were lysed with 0.5 ml of Cell Lysis Buffer (Promega), and the activity in a 20 μl solution was measured using a luciferase assay kit (Promega). In the case of green fluorescent protein (pCMV-GFPE; Promega), it was directly observed with a fluorescence microscope, 5 to 8 fields were observed at 400 magnifications, and the ratio of cells that emitted fluorescence was calculated.

(Example 3: Isolation and analysis of a gene encoding a protein having vascular endothelial growth inhibitory activity from a human heart cDNA library)
By using the present invention, it is possible to isolate a target gene encoding a protein having vascular endothelial growth inhibitory activity. One embodiment of the separation method is schematically shown in FIG.

  In fact, a gene encoding a vascular endothelial growth inhibitory factor was isolated using the gene transfer vector prepared in Example 2 of the present specification. For the gene isolation method exemplified in this example, not only the gene transfer vector prepared in Example 2 of the present specification but also any “virus envelope vector” and “liposome vector” can be used. is there.

A human heart cDNA library (GIBCO BRL; plasmid obtained by ligating human heart-derived cDNA to a plasmid pSPORT having a CMV promoter) introduced into E. coli DH12S. Plasmids were prepared from E. coli. 200 μg of the plasmid was encapsulated in a 10,000 HAU HVJ-E gene transfer vector (the gene transfer vector prepared in Example 2 of the present invention, 3 × 10 ⁹ particles). As host cells, about 5000 cells of human aortic endothelial cells HEAC (Sanko Junyaku) were added to each well of a 96-well macrotiter plate together with the medium, and the cells cultured overnight were used. To each well containing host cells, 1/100 volume of the above HVJ-E was added and allowed to stand at 37 ° C. for 30 minutes, and then the medium was changed.

  The used medium is in a low nutrient state with a serum concentration of 1%, and the culture is performed for 1 week in this state. Under these conditions, HEAC growth was not observed. Two weeks later, a cell proliferation assay was performed by culturing the cells in a growth promotion medium supplemented with growth factors. Cell Titer 96 (Promega) was used as a reagent, and the redox state of mitochondria was regarded as a change in the color of the reagent and used as an index of cell proliferation (MTT assay).

  The darkest well is a well in which cells that have undergone the most active cell growth are present. On the other hand, the lightest well has the cell with the lowest cell proliferating ability. Therefore, the entire microtiter plate was read with a plate reader, and the cell growth was graphed by a computer. From this graph, gene separation was performed using low proliferation activity, that is, human vascular endothelial cell growth inhibitory activity as an index.

  The gene separation was performed by preparing a nucleic acid from cells in the well using a Qeagen DNeasy Tissue Kit. Since the prepared nucleic acid contains plasmid DNA, it is used as competent E. coli. It was introduced into E. coli (DH5α; Takara Shuzo) using the heat shock method.

  This E.I. E. coli was seeded on a plate medium containing ampicillin to form colonies. Plasmid DNA (pDNA) was extracted from each colony, and the presence of the gene fragment in the plasmid was confirmed by restriction enzyme treatment.

  Next, plasmid DNA was purified using Qiagen's Endo Free Plasmid Maxi Kit, the purified plasmid was encapsulated in HVJ-E, and again introduced into HAEC cells, and the same cell proliferation experiment was performed. In this cell proliferation experiment, a plasmid that showed significantly low cell proliferation is a candidate plasmid expected to contain a nucleic acid encoding a vascular endothelial growth inhibitor. As a result, two genes pCMV9 and pCMV15 were separated by secondary screening.

  Next, in order to examine the influence of these genes on cell proliferation in an environment where cell proliferation is promoted by VEGF, MTS assay and c-fos promoter assay were performed using HAEC.

(MTS assay)
The MTS assay was performed as follows.
(1. Encapsulation of plasmid in HVJ envelope vector)
1) Dispensed from HVJ lot and diluted with BSS to 1 × 10 ⁴ HAU / ml.
2) As a 3.5 × 10 ⁴ HAU / 3.5 ml / 10 cm dish, 99 mJ / cm ² UV treatment was performed. (I irradiate with the power ON, ENERGY, 990, START / Dish open)
3) 1 ml (1 × 10 ⁴ HAU) was dispensed into 1.5 ml tubes, centrifuged (15000 rpm, 15 minutes, 4 ° C.), and the supernatant was removed.
4) The pellet was dissolved in 15 μl of a 1 mg / ml protamine sulfate / TE solution (10 mg / ml protamine sulfate solution diluted 10-fold with TE).
5) A plasmid DNA solution corresponding to 200 μg was added.
6) Immediately, 3% Triton X-10 / TE solution of 1/10 volume of the total volume of 4) +5) was added.
7) Lightly mixed and centrifuged (15000 rpm for 15 minutes at 4 ° C.).
8) 1 ml of BSS was added, mixed gently, and centrifuged (15000 rpm, 15 minutes, 4 ° C.).
9) After removing the supernatant, it was resuspended in 300 μl of BSS.
(2. Gene transfer into cells using HVJ envelope vector)
1) The day before, a 96-well plate with 5000 / well of HAEC cells was used.
2) Lightly mixed with the following concentration.

（注意）培地は、ＥＢＭ（Ｃｌｏｎｅｔｅｃｋ）＋５％ＦＢＳ＋抗生剤・抗真菌剤＋増殖因子（ＥＧＦおよび／またはＶＥＧＦ）である。
３）順次培地を交換した。（５０μｌ／ウェル）
４）プレートを遠心（３５００ｒｐｍ １時間３５℃）した。
５）培地を吸引・除去し、新しい培地に交換した。（１００μｌ／ウェル）
６）インキュベートした。
・１ウェルあたり、ＣｅｌｌＴｉｔｅｒ９６ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ Ａｓｓａｙ（Ｐｒｏｍｅｇａ）を２０μｌ添加し、１００μｌの内皮細胞用培地と混合し、古い培地を吸引して除き、ＣｅｌｌＴｉｔｅｒ９６混合溶液を１ウェルあたり１２０μｌ入れてＣＯ_２インキュベーターで１〜５時間インキュベートした。
・プレートリーダーにて４９０ｎｍの吸光度を測定した。

(Caution) The medium is EBM (Clonetec) + 5% FBS + antibiotic / antifungal agent + growth factor (EGF and / or VEGF).
3) The medium was changed sequentially. (50 μl / well)
4) The plate was centrifuged (3500 rpm for 1 hour at 35 ° C.).
5) The medium was aspirated and removed and replaced with a new medium. (100 μl / well)
6) Incubated.
Add 20 μl of CellTiter96 AQueous One Solution Cell Proliferation Assay (Promega) per well, mix with 100 μl of endothelial cell medium, aspirate old medium, add 120 μl of CellTiter96 mixed solution per well and add CO ₂ Incubated for 1-5 hours in incubator.
-Absorbance at 490 nm was measured with a plate reader.

Sample groups (8 wells each) are as follows.
-Positive control for growth promotion (PC) = HGF (hepatocyte growth factor) gene;
-Positive control of growth inhibition (PC) = Angiostatin gene; and-Negative control (NC) = pVAX1 with CMV promoter.

  The results of the MTS assay are shown in the left column of FIG. The Y axis is a relative value when pVAX1 having a CMV promoter is set to “1”. The isolated clones pCMV9 and pCMV15 showed endothelial cell growth inhibitory activity. PCMV15 showed the ability to suppress endothelial cell proliferation comparable to that of the angiostatin gene. Moreover, pCMV9 suppressed endothelial cell proliferation more strongly than the angiostatin gene.

(C-fos promoter assay)
Next, a c-fos promoter assay was performed to show that the endothelial cell growth inhibitory activity of the isolated clones pCMV9 and pCMV15 represses the proliferation signal pathway from the c-fos promoter. Specifically, the gene c-fos-Luci linked with luciferase under the c-fos promoter and the candidate gene were introduced into bovine vascular endothelial cells at a weight ratio of 1: 1 by lipofection, and luciferase activity was measured 24-48 hours later. did. The plasmid c-fos-Luci contains two copies of the 5 'regulatory enhancer region of c-fos (-357 to -276), and the herpes simplex virus thymidine kinase gene promoter (-200 to 70), and the luciferase gene It is a plasmid (Arterioscler Thromb Vasc Biol. 2002: 22: 238-242). The result is shown on the right side of FIG. On the Y axis in FIG. 2, the luciferase activity when pVAX1 was used was “1”, and the relative value was shown in the graph. Both clones pCMV9 and pCMV15 repressed transcription from the c-fos promoter, and the repression ability was stronger than that of the angiostatin gene (right in FIG. 2).

(Characteristics of inserts contained in pCMV9 and pCMV15)
Next, the insert of each clone was confirmed. As a result, as shown in the left of FIG. 3, the inserts of both the pCMV9 and pCMV15 clones were about 2 kb. Upon sequencing, open reading frames were found encoding proteins with estimated molecular weights of 19.96 kD and 51.39 kD, respectively. The nucleic acid sequence and amino acid sequence are shown in FIG. 4 and FIG.

  Next, in order to confirm the gene product, inserts of pCMV9 and pCMV15 were ligated to EcoRI / XhoI sites of the pCMV • SPORT1 expression vector, respectively, and in vitro translation was performed. As a result, as shown in the right of FIG. 3, proteins of about 20 kD and 50 kD were identified for pCMV9 and pCMV15, respectively. The structure of the protein encoded by pCMV9 is shown in FIG. 6A, and the structure of the protein encoded by pCMV15 is shown in FIG. 6B. When the gene sequences were analyzed, both were completely new genes, but their existence was registered in the Genome Project (FIGS. 6A and B). pCMV9 is registered in EST-BD095420 and is located on the short arm of human chromosome 18 (FIG. 6A). We further isolated the full length gene. The nucleotide sequence of our isolated pCMV9 is as shown in SEQ ID NO: 1. In contrast, in the sequence of EST-BD095420 corresponding to pCMV9, the nucleotide residue T (thymine) at position 544 in SEQ ID NO: 1 was changed to C (cytosine). The nucleotide sequence of EST-BD095420 is shown in the sequence listing as SEQ ID NO: 3, and the amino acid sequence as SEQ ID NO: 4. pCMV15 is registered with EST-HSM805282 and is located on human chromosome 15 (FIG. 6B). This time, the present inventors have isolated genes below the third exon. Even from the third exon, the reading frame started with methionine, and the sequence in the vicinity had a Kozak sequence. Therefore, there is a possibility that transcription starts from the third exon. The nucleotide sequence of our isolated pCMV15 is as shown in SEQ ID NO: 5. In contrast, in the sequence of EST-HSM805282 corresponding to pCMV15, the 705th nucleotide residue A (adenine) of SEQ ID NO: 5 is C (cytosine), and the 788th nucleotide residue A (adenine) is G ( In (guanine), the 934th nucleotide residue A (adenine) was changed to G (guanine), and the 991st nucleotide residue G (guanine) was changed to A (adenine). The nucleotide sequence of EST-HSM805282 is shown in the sequence listing as SEQ ID NO: 7 and amino acid sequence as SEQ ID NO: 8.

  FIG. 6 is a schematic diagram showing the position on the genome of a clone isolated according to the present invention. The horizontal line indicates the chromosome, and the shaded square on the line indicates the position of the exon. pCMV9 is located on the p-arm of chromosome 18 (FIG. 6A), and pCMV15 is located on chromosome 5 (FIG. 6B). When the sequence homology was searched, pCMV9 showed homology with SNF7 involved in the intracellular transport system of yeast secreted protein. Therefore, it was shown that pCMV9 may be a protein belonging to a gene family involved in intracellular transport. Several domains were identified in pCMV15. For example, residues 1205-1327 showed homology with the SPRY domain (SPIa and ryanodine receptor). Residues 967 to 1058 and residues 1067 to 1151 showed homology with the FN3 domain (fibronectin type 3 domain). Residues 325-445 showed homology with the SPRY domain (SPIa and ryanodine receptor). Residues 102 to 159 and residues 187 to 278 showed homology with the FN3 domain (fibronectin type 3 domain) (FIG. 6B). The size of the clone pCMV15 obtained by the present inventors on the genome was 55 kDa, but the corresponding known EST sequence had a size of 64 kDa. The positions of exons of the clones of the present invention are indicated by dark shaded squares, and the positions of exons derived from the corresponding known EST sequences are indicated by thin shaded squares.

  Next, as a clue to investigate the mechanism of growth inhibition, a luciferase assay using promoters of E2F and AP-1 which are transcription factors involved in proliferation was performed in the presence of VEGF. Specifically, a vector containing a reporter gene in which a luciferase gene is linked to an E2F promoter, or a vector containing a reporter gene in which a luciferase gene is linked to an AP-1 promoter (AP1 promoter-luciferase, E2F promoter-luciferase, or NFκB promoter— 1 μg of luciferase was introduced into bovine aorta-derived endothelial cells by lipofection together with 0.3 μg of the expression vectors (CMV9, CMV15, HA-CMV9, HA-CMV15, VEGF, or pcDNA3.1 (control)) shown in FIG. Then, luciferase activity (indicated by RLU) was measured. “Control” in FIG. 7 shows the result of transfection of only a vector containing a reporter gene without using an expression vector. In “VEGF”, “pCMV15”, and “pCMV9”, a plasmid in which the reading frame of each clone was linked to the pCMV • SPORT1 expression vector was used. “HA-CMV9” and “HA-CMV15” are genes in which the base sequence of the HA tag of influenza is added to the 5 ′ side (N end) of the pCMV9 and pCMV15 genes.

  As shown in FIG. 7, pCMV9 acted repressively on both promoters. pCMV15 works repressively on the E2F promoter, but when the AP-1 promoter was used, the effect of VEGF could not be suppressed. Moreover, when HA-CMV9 and HA-CMV15 were used, the inhibitory effect was attenuated and the inhibitory effect on the AP1 promoter disappeared. However, the VEGF activity inhibitory effect on the E2F promoter was still detectable.

(Example 4: Angiogenesis inhibitory activity of pCMV9 and pCMV15)
It is investigated whether the luminal formation activity by VEGF is suppressed by recombinant pCMV9 and recombinant pCMV15 using Kurabo Industries's angiogenesis kit (Angiogenesis Kit, KZ-1000, Kurashiki Boseki Co., Ltd.). Recombinant VEGF (a plasmid containing a gene encoding vascular endothelial growth factor) is used as VEGF, and a blank is used as a control as a control.

  Anti-human VEGF-A neutralizing antibody is further added at 0, 250, 500, 1000 ng to a medium in which 10 ng / ml of VEGF-E (NZ-7) is added to a medium dedicated to angiogenesis (Kurashikibo Co., Ltd. KZ-1400). Add to each concentration of / ml and culture.

As an anti-human VEGF-A neutralizing antibody, Anti-VEGF, Human, Mouse-Mono (265503.111) (catalog No. MAB293 manufactured by R & D) can be used. Culture is performed at 37 ° C. in a 5% CO ₂ incubator. The medium is changed daily with one containing the same additives. The culture medium is removed on the 4th, 7th, or 11th day from the start of the culture, and staining is performed according to the following procedure using a luminal staining kit (for CD31 antibody staining: Kurashiki Boseki KZ-1225).

  A primary antibody (mouse anti-human CD31 antibody) stained with CD31 (PECAM-1) is diluted 4,000 times with a blocking solution (Dulbecco phosphate buffer (PBS (-)) containing 1% -BSA) in each well. Add 0.5 ml of the primary antibody solution and incubate for 60 minutes at 37 ° C. After the incubation, wash each well 3 times with 1 ml of blocking solution.

  Next, 0.5 ml of a secondary antibody solution (goat anti-mouse IgG alkaline phosphatase complex) diluted 500-fold with a blocking solution is added to each well, incubated at 37 ° C. for 60 minutes, and then washed three times with 1 ml of distilled water. . Meanwhile, two BCIP / NBT tablets are dissolved in 20 ml of distilled water and filtered through a filter having a pore size of 0.2 μm to prepare a substrate solution. Add 0.5 ml of the prepared BCIP / NBT substrate solution to each well and incubate at 37 ° C. until the lumen is deep purple (usually 5-10 minutes). After completion of the incubation, each well is washed three times with 1 ml of distilled water, and then the washing solution is removed by suction and left to stand for natural drying. After drying, each well is observed under a microscope.

  Observe each well with a 40 × microscope and take a picture of each well. Recombinant VEGF is introduced into HAEC to confirm that tube formation occurs, and that when pCMV9 and / or pCMV15 is used together with recombinant VEGF, tube formation by recombinant VEGF is suppressed.

  Furthermore, in order to quantify the results, the obtained images can be used with angiogenesis quantification software (KSW-5000U, Kurashiki Boseki Co., Ltd.) according to the following method.

(Example 5: Isolation of upstream region of pCMV15)
As shown in Example 4, it was demonstrated that pCMV15 encodes a peptide having antiangiogenic activity. Moreover, since pCMV15 contains a termination codon, pCMV15 contains a nucleic acid sequence corresponding to the carboxy terminus of the protein having angiogenesis inhibitory activity, but does not contain a nucleic acid sequence corresponding to the amino terminus of the protein. Therefore, the upstream region of pCMV15 was isolated and analyzed.

  According to a conventional method, a clone containing the upstream region of pCMV15 was isolated from a human heart cDNA library using pCMV15 as a labeled nucleic acid probe. As a result, clones containing 3492 bp and 4413 bp could be isolated. Each of this clone was named pCMV15-2 (SEQ ID NO: 9) and pCMV15-3 (SEQ ID NO: 11). The nucleic acid contained in pCMV15-2 encodes a polypeptide of 1163 amino acid residues (SEQ ID NO: 10), and the nucleic acid contained in pCMV15-3 encodes a polypeptide of 1470 amino acid residues (SEQ ID NO: 12) To do.

(Example 6: Analysis of pCMV15-2 and pCMV15-3)
(1. Analysis of endothelial cell proliferation inhibitory activity)
The endothelial cell proliferation inhibitory activity of pCMV15, pCMV15-2, and pCMV15-3 was analyzed by the following method.

  The sequences of pCMV15, pCMV15-2, and pCMV15-3 were ligated to the expression vector pcDNA3.1 to produce pcDNA-CMV15, pcDNA-CMV15-2, and pcDNA-CMV15-3. These three expression plasmids and pcDNA3.1, which is a plasmid not containing pCMV15-derived insert, were subjected to the MTS assay described in Example 3 using bovine aorta-derived endothelial cells (BAEC).

The results of the MTS assay are shown in FIG. The Y axis represents absorbance (OD ₄₉₀ ) values. pCMV15 had a clear endothelial cell growth inhibitory activity. However, no inhibitory activity on endothelial cell proliferation was observed in pCMV15-2 and pCMV15-3 including further upstream of pCMV15.

(2. c-fos promoter assay)
In order to show that the endothelial cell growth inhibitory activity of the isolated clones represses the proliferation signal pathway from the c-fos promoter, a c-fos promoter assay was performed. Specifically, c-fos-Luci in which a luciferase gene is linked under the c-fos promoter and a candidate gene expression vector are introduced by lipofection into bovine vascular endothelial cells at a weight ratio of 1: 1, and luciferase is obtained 24 to 48 hours later. Activity was measured. The plasmid c-fos-Luci contains two copies of the 5 'regulatory enhancer region of c-fos (-357 to -276), and the herpes simplex virus thymidine kinase gene promoter (-200 to 70), and the luciferase gene It is a plasmid (Arterioscler Thromb Vasc Biol. 2002: 22: 238-242). The result is shown in FIG. The Y axis in FIG. 9 shows luciferase activity. Clone pCMV15 strongly repressed transcription from the c-fos promoter. On the other hand, in the pCMV15-2 and pCMV15-3 containing the upstream sequence of pCMV15, the c-fos promoter repressing activity was not observed.

(3. Inhibitory activity of transcription activation via NFκB element)
In order to demonstrate that suppression of the growth signal pathway from the c-fos promoter of isolated clones is mediated by the κB element contained in the c-fos promoter, suppression of transcriptional activation via the κB element Activity was assayed. PNFκB-Luc from BD Biosciences (Franklin Lake, NJ) was used for the assay. pNFκB-Luc is a vector for monitoring the activation of the NFκB signal transduction pathway, and includes a firefly (Photinus pyralis) luciferase gene. This vector further includes a TAT-like promoter ( _PTAL ) derived from the herpes simplex virus thymidine kinase (HSV-TK) promoter, and four tandem contiguous NFκB consensus sequences linked to _PTAL . pNFκB-Luc and the candidate gene expression vector were introduced into bovine vascular endothelial cells (BAEC) at a weight ratio of 1: 1 by lipofection, and luciferase activity was measured 24-48 hours later. The result is shown in FIG. The Y axis in FIG. 10 shows luciferase activity.

  Clone pCMV15 strongly repressed transcription from the κB element. In contrast, no inhibitory activity was observed in pCMV15-2 and pCMV15-3 containing the upstream sequence of pCMV15. From this result, it is considered that the suppression of c-fos promoter activity by pCMV15 is caused by suppressing the transcriptional activation via the κB element.

(4. Inhibitory activity of transcriptional activation via AP-1 enhancer)
To demonstrate that the suppression of the growth signal pathway from the c-fos promoter of isolated clones is through the AP-1 element contained in the c-fos promoter, transcription through the AP-1 element. The inhibitory activity of activation was assayed. PAP1-Luc from BD Biosciences (Franklin Lake, NJ) was used for the assay. pAP1-Luc is a vector for monitoring the induction of AP-1 and the stress-activated protein kinase / Jun N-terminal kinase (SAPK / JNK) signaling pathway, and includes the luciferase gene of the firefly (Photinus pyralis). The vector further includes a TAT-like promoter ( _PTAL ) derived from the herpes simplex virus thymidine kinase (HSV-TK) promoter, and four tandem contiguous AP1 enhancer sequences linked to _PTAL . pAP1-Luc and the expression vector of the candidate gene were introduced into bovine vascular endothelial cells (BAEC) at a weight ratio of 1: 1 by lipofection, and luciferase activity was measured 24-48 hours later. The result is shown in FIG. The Y axis in FIG. 11 shows luciferase activity.

  Clone pCMV15 strongly repressed transcription from the AP1 enhancer. In contrast, the inhibitory activity of pCMV15-2 containing the upstream sequence of pCMV15 was lower than that of pCMV15. pCMV15-3 showed no inhibitory activity.

  From this result, it is considered that the suppression of c-fos promoter activity by pCMV15 and pCMV15-2 occurs not only by suppressing the transcriptional activation via the AP1 enhancer but also the κB element.

(Example 7: Treatment of disease using isolated polypeptide and polynucleotide)
It has been reported that systemic administration of angiostatin, a protein having angiogenesis inhibitory activity, suppresses metastasis of Lewis lung carcinoma (Cell, October 1994 21; 79 (2): 315-28). . Therefore, as in the case of angiostatin, metastasis of cancer can be suppressed by administering the polypeptide and polynucleotide of the present invention.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  A disease selected from the group consisting of rheumatoid arthritis, chronic inflammation, diabetic retinopathy, atherosclerosis, and cancer by isolating a novel peptide having angiogenic activity and a gene encoding the peptide Novel pharmaceutical compositions for the treatment, prevention, and prognosis of cancer are provided

Claims (2)

Less than:
(A) a polynucleotide having the base sequence set forth in SEQ ID NO: 1 or a fragment sequence thereof; ( b ) a polynucleotide that hybridizes under a highly stringent condition to a complementary strand of the polynucleotide of (a); and ( c ) (polynucleotide identity against the polynucleotide of a) is the nucleotide sequence which is at least 95%,
A pharmaceutical composition for inhibiting vascular endothelial cell proliferation, comprising a polynucleotide selected from the group consisting of:
Here, the polynucleotide comprises a vascular endothelial cell growth inhibitory activity, a transcription inhibitory activity from the c-fos promoter, a transcription inhibitory activity from the E2F promoter, a transcription inhibitory activity from the AP1 promoter, and a VEGF activity inhibitory activity. A pharmaceutical composition encoding a peptide having an activity selected from. Less than:
(A) a polypeptide encoded by the base sequence set forth in SEQ ID NO: 1 or a fragment thereof; and ( b ) a polypeptide comprising an amino acid sequence having at least 95 % identity to the amino acid sequence of the polypeptide of (a) peptide,
A pharmaceutical composition for inhibiting vascular endothelial cell proliferation, comprising a polypeptide selected from the group consisting of:
Here, the polypeptide comprises a vascular endothelial cell growth inhibitory activity, a transcription inhibitory activity from the c-fos promoter, a transcription inhibitory activity from the E2F promoter, a transcription inhibitory activity from the AP1 promoter, and a VEGF activity inhibitory activity. A pharmaceutical composition having an activity selected from.

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