JP2000325087A - Tsa2306 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain TSA2306 gene which codes for a polypeptide which has a specific amino acid sequence, and has a blood vessel neogenesis-suppressing activity and an antitumor activity, is useful for a probe for detecting a gene, a blood vessel neogenesis-suppressing agent, an antitumor agent, and so on, and is specific for the liver and the kidney. SOLUTION: This is new TSA 2306 gene which contains a polynucleotide coding for the polypeptide having the amino acid sequence represented by the formula, or a strand complementary to the polynucleotide, or a polynucleotide having a homology at 95% or more to the polynucleotide coding for the polypeptide having the amino acid sequence represented by the formula, or a strand complementary to the polynucleotide, and codes for a protein having a homology to angiopoetin 2 having a blood vessel neogenesis-suppressing activity or an anititumor activity, and is useful for a probe for detecting a gene, a blood vessel neogenesis-suppressing agent, an antitumor agent, and so on. This gene is obtained by preparing a human normal liver cDNA library, followed by screening the obtained library with an appropriate probe having a partial sequence or an antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel gene, more specifically, angiopoietin 2 (expressed specifically in liver and kidney, the expression product of which is known to suppress angiogenesis). angiopoietin 2: Science, Jul. 4 , 277,
55-60 (1997)) and angiopoietin 1 (angiopoietin 1: Cell, De
c. 27, 87 (7), 1161-1169 (1996)) and 33% homology, respectively, and a novel liver / kidney-specific gene thought to act as an angiogenesis inhibitor or antitumor factor About.

[0002] The present invention also relates to novel polypeptides (recombinant proteins or expression products) encoded by the gene of the present invention and specific antibodies thereof, fragments of the gene of the present invention (polynucleotide fragments as probes and primers), The present invention also relates to the gene expression vector of the present invention and a transformant having the vector.

Further, the present invention provides a pharmaceutical composition (gene therapeutic agent) containing the gene of the present invention or a fragment thereof as an active ingredient, and a pharmaceutical composition containing an expression product of the gene of the present invention (the above polypeptide) as an active ingredient, particularly It also relates to angiogenesis inhibitors and antitumor agents.

[0004]

2. Description of the Related Art Conventionally, cancer has been treated mainly by chemotherapeutic agents alone or in combination, but these have not always produced satisfactory results. In recent years, cytokines such as interferon (IFN) and interleukin (IL) -2 have been used for the treatment of some cancer types. In recent years, drug delivery systems (DD) have been developed to reduce the side effects of cancer chemotherapeutic agents and to improve the efficacy of drugs on target tissues.
S) has been developed, and a cancer treatment method with enhanced target organ selectivity by use of S) has been attempted. The DDS modifies the drug with an antibody or other ligand that binds to a cell surface marker such as a protein highly expressed in cancer cells and surrounding tissues, does not adversely affect normal tissues, and positively promotes cancer tissues. It uses a formulation designed to deliver a drug selectively and selectively.

[0005] On the other hand, in the field of cancer treatment, angiogenesis has recently attracted attention. Angiogenesis means that in the process of cancer growth, blood vessels inside the cancer tissue develop simultaneously with the growth of the cancer. Active growth of cancer cells, growth of cancer tissues, and metastasis of cancer require the establishment of new blood vessels, which are organs that supply nutrients and oxygen to cancer tissues and excrete metabolic waste products. Is essential, and in this regard, the growth of cancerous tissue is highly dependent on angiogenesis. Therefore, it is considered that by suppressing this angiogenesis, the growth of cancer tissue, cancer metastasis, and the like can be suppressed. Therefore, development of a new treatment method for cancer targeting the angiogenesis, or such treatment method and conventional cancer treatment The development of combination therapy with the law is being studied in the art.

[0006]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a gene encoding a novel protein having homology to angiopoietin 2 which suppresses the above-mentioned angiogenesis.

[0007] With the above object, the present inventors have repeatedly searched for genes derived from various human tissues. As a result, the present inventors have succeeded in isolating and identifying a new liver / kidney-specific gene that meets the purpose. Was completed.

[0008]

That is, according to the present invention,
A gene comprising the polynucleotide of the following (a) or (b), particularly a human gene, is provided. (A) a polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or a complementary chain thereof; (b) a polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 Polynucleotides with 95% homology or their complements.

Further, according to the present invention, there is provided a gene encoding the following polypeptide (a) or (b). (A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence comprising one or more amino acids deleted, substituted or added in the amino acid sequence of (a), and an angiogenesis inhibitory activity Or a polypeptide having antitumor activity.

Further, according to the present invention, there is provided a gene comprising the following polynucleotide (a) or (b). (A) a nucleotide sequence represented by SEQ ID NO: 2, and (b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide having the nucleotide sequence of (a).

[0011] Specific examples of the above genes according to the present invention include:
A polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, that is, the polynucleotide having the base sequence represented by SEQ ID NO: 2 is encompassed.

Further, according to the present invention, there are provided the following recombinant expression vectors, host cells, polynucleotides, gene therapeutics, polynucleotide probes, polypeptides (gene expression products), pharmaceutical compositions and antibodies. You. (1) A recombinant expression vector having any of the above genes. (2) A host cell having the above recombinant expression vector. (3) at least one of the nucleotide sequences represented by SEQ ID NO: 2
A polynucleotide comprising 0, more preferably at least 30, contiguous nucleotide sequences. (4) A gene therapy agent comprising the polynucleotide or a vector having the same as an active ingredient. (5) A polynucleotide probe consisting of the base sequence represented by SEQ ID NO: 2. (6) at least one of the nucleotide sequences represented by SEQ ID NO: 2
A polynucleotide probe comprising 0, more preferably at least 30, contiguous nucleotide sequences. (7) The following polypeptide (a) or (b): (A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, (b) an amino acid sequence comprising one or more amino acids deleted, substituted or added in the amino acid sequence of (a), and an angiogenesis inhibitory activity Or a polypeptide having antitumor activity. (8) A gene expression product obtained by expressing the gene of the present invention. (9) A pharmaceutical composition, particularly an angiogenesis inhibitor and an antitumor agent, containing the polypeptide (the gene expression product) as an active ingredient. (10) An antibody or a fragment thereof having specific binding property to the polypeptide (the gene expression product).

The probes shown in the above (5) and (6) are useful, for example, as specific probes for detecting the gene of the present invention.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, abbreviations for amino acids, peptides, base sequences, nucleic acids and the like are referred to as IUP
AC-IUB regulations [IUPAC-IUB Communication on Bio
logical Nomenclature, Eur.J. Biochem., 138 : 9 (19
84)], “Guidelines for the preparation of specifications containing base sequences or amino acid sequences” (edited by the Patent Office) and commonly used symbols in the art.

[0015] One specific example of the gene of the present invention is a PCR named "TSA2306" shown in Examples described later.
Mention may be made of those deduced from the DNA sequence of the product. Its base sequence is as shown in SEQ ID NO: 3. The gene is a novel liver / kidney-specific protein (TSA2306) having a 460 amino acid sequence shown in SEQ ID NO: 1.
A protein represented by SEQ ID NO: 2
It is a human cDNA containing a polynucleotide sequence of 380 bases in length as an open reading frame, and has a total length of 1461 bases.

TSA2306 encoded by the gene of the present invention
The protein was obtained from the FASTA program (Person WR, et a
l., Proc. Natl. Acad. Sci., USA., 85 , 2444-2448 (1
988)), the results of a GenBank / EMBL database search showed that human angiopoietin 2
And angiopoietin 1 (see the above literature).

Thus, the human angiopoietin 2
Is known to inhibit angiogenesis (see the above-mentioned literature). Therefore, the expression product of the gene of the present invention having homology with human angiopoietin 2 functions as a suppressor of angiogenesis, Can be expected to act in a restraining manner. This is also evident from the results of transcript expression tests in cancer cell lines and cancer tissues in Examples described later.

By the way, the formation of blood vessels (angiogenesis) in cancer tissues is characterized by the continuous formation of new blood vessels that support growth, and in this respect, is distinguished from general histological angiogenesis. (Folkman, Nat.Me
d., 1 , 27-31 (1995). Thus, factors that specifically affect angiogenesis are unlikely to have deleterious effects on normal and healthy organs or tissues. Thus, it is known that angiogenesis is essential for malignant transformation of many cancer cells, suggesting that the progression of cancer is largely related to the suppression of angiogenesis and a change in the expression level of promoters. (J. Natl. Cancer Inst., Sep. 61 (3)
639-643 (1978)). Factors that specifically affect angiogenesis can also target other types of angiogenic systems such as those found in inflamed, regenerated or injured tissue. Furthermore, since angiogenesis is also performed in uterine tissue, factors that specifically affect angiogenesis include:
It also affects such uterine tissues, and can be expected to have a therapeutic effect on diseases such as uterine fibroids.

The gene of the present invention is specifically expressed in normal liver / kidney tissues and is not expressed in highly malignant liver or kidney cancers, and thus is implicated in the above-mentioned neovascularization. It is considered to have a function.

The gene of the present invention can be used to produce an expression product of the gene or an antibody that binds to the expression product, and the antibody thus obtained can be used, for example, for cancer diagnosis.

According to the provision of the gene of the present invention, the above-mentioned angiogenesis and the progress of cancer can be suppressed by synthesizing and utilizing a sense fragment of the gene and their expression products.

The gene of the present invention can use these and fragments thereof as probes, and thus can diagnose cancer. The present invention also provides such a kit for cancer diagnosis.

In the present specification, the term "gene" is used to include not only double-stranded DNA but also single-stranded DNAs such as a sense strand and an antisense strand constituting the same. Its length is not limited at all. Therefore, unless otherwise specified, the gene (DNA) of the present invention includes a double-stranded DNA containing human genomic DNA and a single-stranded DNA containing cDNA (sense strand) and a single-stranded DNA having a sequence complementary to the sense strand. Both DNA (antisense strand) and fragments thereof are included.

The gene (DNA) of the present invention may further comprise a leader sequence,
It can include coding regions, exons, introns. Polynucleotides include RNA and DNA. The DNA includes cDNA, genomic DNA, and synthetic DNA. Polypeptides include fragments, homologs, derivatives and variants thereof. The variants include naturally occurring allelic variants, non-naturally occurring variants, deletions, substitutions, variants having an amino acid sequence modified by addition and insertion, and modified amino acid sequences having the same function. Are included.

Incidentally, these amino acid sequence alterations (mutations, etc.) may occur in nature, for example, due to mutations or post-translational modifications, but naturally-occurring genes (for example, genes of the present invention) are used. You can also do this artificially.

The above mutant may be at least 70%, preferably 80%, more preferably at least 70%,
It can be 95%, even more preferably, 97% homologous. In addition, the polypeptides, including mutants, homologs, and the like, have structural features that are commonly conserved, and biological activities of the gene expression product of the present invention, for example, angiogenesis inhibitory activity or tumor progression inhibitory activity ( (Antitumor activity). Note that the homology of polypeptides can be determined by using sequence analysis software such as FAST.
Method using program A (Clustal, V., Methods Mo
l. Biol., 25 , 307-318 (1994)) or SWISSPLO
It can be analyzed by TS.

The gene encoding the above mutant is silent or conserved for amino acid substitution. That is, the amino acid residue encoded by the nucleotide sequence does not change.

Conservatively substituted amino acid residues, ie, those substitutions of the original amino acid residue with other amino acid residues that would preserve the activity of the polypeptide having the original amino acid residue. Possible amino acid residues are as follows for each amino acid residue (original amino acid residue): Original amino acid residue Conservatively substituted amino acid residue Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn or Gln Ile Leu or Val Leu Ile or Val Lys Arg, Aln, or Glu Met Leu or Ile Phe Met, Leu or Tyr Ser Thr Thr Ser Ser Trp Tyr Tyr Trp or Phe Val Ile Or Leu Also, a cysteine residue (Cys) is replaced with another amino acid residue such as a serine residue (Ser) or an alanine residue (Al
a), valine residue (Val) and the like.

[0029] Substitutions which are generally considered to give a favorable change in the properties of the desired polypeptide are for example: a) substitution of hydrophilic residues such as Ser or Thr for hydrophobic residues such as Leu, Ile, Phe, Val or Ala, b) substitution of Cys or Pro for various other amino acids, c) electricity Amino acid residues having a positive side chain, such as Ly
s, Arg, His, electronegative residues, such as Val or Asp
D) substitution of a residue with a large side chain, for example Phe, for an amino acid residue without a side chain such as Gly.

The provision of the gene of the present invention and its gene product is useful for elucidating, grasping, diagnosing various cancers such as liver cancer and kidney cancer.
Provides extremely useful information and means for prevention and treatment.
The gene of the present invention can also be suitably used in the development of a novel drug that induces the expression of the gene of the present invention, which is used for the treatment of the various cancers described above. Further, detection of the expression of the gene of the present invention or its expression product in an individual or tissue, and detection of mutation (deletion or point mutation) or abnormal expression of the gene are suitably used in elucidation and diagnosis of the above-mentioned various cancers. it can.

The gene of the present invention is specifically SEQ ID NO: 1.
A gene comprising the nucleotide sequence of SEQ ID NO: 2 encoding the amino acid sequence represented by: for example, a gene having the nucleotide sequence of SEQ ID NO: 3 (TSA2306 gene), or the nucleotide sequence represented by SEQ ID NO: 2 or Although shown as a gene consisting of a polynucleotide containing a complementary strand, it is not particularly limited thereto. For example, the gene of the present invention may be a gene encoding an amino acid sequence having a certain modification in the specific amino acid sequence, a gene encoding an amino acid sequence having a certain homology with the specific amino acid sequence, or a gene encoding such a gene. It may be a gene having a nucleotide sequence having a certain homology with the nucleotide sequence.

A certain homology to the above specific amino acid sequence or base sequence is, for example, at least 70% or more.
Preferably, the homology can be 90% or more, more preferably 95% or more, and still more preferably 97% or more. The present invention includes homologs (gene homologs and protein homologs) having such homology.

[0033] The gene of the present invention also includes "a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 (modified amino acid sequence)". Genes that do ". Here, the degree of “deletion, substitution or addition of amino acids” and their positions are determined by modifying the polypeptide of the amino acid sequence with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (TSA2306
It is not particularly limited as long as it has the same biological function as the protein). Here, “similar functions” include, for example, angiogenesis inhibitory activity or tumor progression inhibitory activity.
(Antitumor activity). Further, a gene encoding a polypeptide consisting of the modified amino acid sequence,
The TSA2306 gene of the present invention, which encodes a polypeptide having an amino acid sequence before modification, may be detected by its use.

A homologue of the TSA2306 gene of the present invention (and a homologue of the gene product) has sequence homology to the TSA2306 gene of the present invention (or a gene product thereof) and has a common structural feature and gene expression pattern. A series of related genes (and their expression products) that are recognized as one gene family based on gender, similarity in biological functions as described above, and the like. This naturally includes alleles (alleles) of the TSA2306 gene of the present invention.

As an artificial means for altering (mutating) the amino acid sequence, for example, cytospecific mutagenesis [Methods in Enzymology, 154 , 350, 36]
7-382 (1987); id. 100, 468 (1983); Nucleic Acids Re
s., 12 , 9441 (1984); Genetic engineering techniques such as the Seismic Chemistry Laboratory Course 1 "Genetic Research Method II", edited by The Japanese Biochemical Society, p105 (1986)], the phosphate triester method and the phosphate amidite method. Chemical synthesis means (J. Am. Chem. Soc., 89 , 4801).
91, 3350 (1969); Science, 150 , 178 (196
8); Tetrahedron Lett., 22 , 1859 (1981); 24 , 245
(1983)] and methods for combining them.
More specifically, the synthesis of DNA can be performed by a chemical synthesis using a phosphoramidite method or a triester method, or can be performed on a commercially available automatic oligonucleotide synthesizer. The double-stranded fragment synthesizes a complementary strand,
It can also be obtained from chemically synthesized single stranded products by annealing the strands together under appropriate conditions or adding a complementary strand using a DNA polymerase with the appropriate primer sequences.

As a specific embodiment of the gene of the present invention, a gene having the nucleotide sequence shown in SEQ ID NO: 3 can be exemplified. The coding region in this nucleotide sequence (SEQ ID NO: 2
Shows an example of one combination of codons indicating each amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. The gene of the present invention is not limited to a gene having such a specific base sequence, and may have a base sequence selected by combining arbitrary codons for each amino acid residue. Codons can be selected according to a conventional method, for example, by considering the codon usage of the host to be used [Ncleic Acids Res., 9 , 43 (1981)].

As described above, the gene of the present invention
Also included are those consisting of a base sequence having a certain homology to the base sequence shown in SEQ ID NO: 2.

The above-mentioned homology is at least 70% identity, preferably at least 90% identity, more preferably at least 95% identity with the nucleotide sequence shown in SEQ ID NO: 2.
And their complementary strand polynucleotides.

Examples of such a gene having homology include, for example, stringent conditions at 50 ° C. in 0.2 × SSC containing 0.1% SDS or at 60 ° C. in 1 × SSC containing 0.1% SDS. A gene having a nucleotide sequence that hybridizes with the DNA having the nucleotide sequence shown in SEQ ID NO: 2 can also be exemplified.

The gene of the present invention can be prepared based on the sequence information on the specific examples of the gene of the present invention disclosed by the present invention.
It can be easily produced and obtained by general genetic engineering techniques [Molecular Cloning 2d Ed, Cold Spring H
arbor Lab. Press (1989); see Seismic Chemistry Laboratory Course "Gene Research Methods I, II, III", edited by The Biochemical Society of Japan (1986), etc.).

Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed according to a conventional method, and the desired library is prepared from the library using an appropriate probe or antibody specific to the gene of the present invention. can be carried out by selecting clones [Proc. Natl. Acad. Sci. , USA., 7
8 , 6613 (1981); Science, 222 , 778 (1983) and the like].

In the above, the origin of the cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Separation of total RNA therefrom, separation and purification of mRNA, acquisition of cDNA, and cloning thereof can all be carried out according to conventional methods. In addition, cDNA libraries are commercially available, and in the present invention, such cDNA libraries, for example, Clontech Lab.
Various cDNA libraries commercially available from c.) can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used.

More specifically, for example, a method for selecting a corresponding cDNA clone from a protein produced by cDNA by immunoscreening using an antibody specific to the protein, and selectively binding to a target DNA sequence Examples include plaque hybridization and colony hybridization using a probe, and combinations thereof.

As the probe used herein, DNA or the like chemically synthesized based on information on the nucleotide sequence of the gene of the present invention can be generally used. Good use. In addition, sense primers and antisense primers set based on the nucleotide sequence information of the gene of the present invention can be used as screening probes.

The nucleotide sequence used as the probe is a partial nucleotide sequence corresponding to SEQ ID NO: 2, and has at least 10 continuous bases, preferably 20 continuous bases, more preferably 30 continuous bases. Bases, most preferably having 50 consecutive bases. Further, a positive clone having the oligonucleotide sequence represented by SEQ ID NO: 2 itself can be used as a probe.

When obtaining the gene of the present invention, the PCR method
[Science,230, 1350 (1985)]
The width method can be suitably used. Especially from library to full length
If it is difficult to obtain cDNA of RACE,
[Rapid amplification of cDNA ends; experimental medicine,12
(6), 35 (1994)], especially the 5'-RACE method [M.A.
n, etal., Proc. Natl. Acad. Sci., USA.,8, 8998 (198
8)] is suitable.

The primers used when employing the PCR method can be appropriately set based on the sequence information of the gene of the present invention revealed by the present invention, and can be synthesized according to a conventional method. The isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNA fragments obtained above can be prepared by a conventional method, for example, the dideoxy method [Proc.
Natl. Acad. Sci., USA., 74 , 5463 (1977)) or the Maxam-Gilbert method (Methods in Enzymology, 65 , 499 (1977)).
980)], or simply using a commercially available sequencing kit or the like.

According to the gene of the present invention obtained as described above, for example, the presence or absence of the expression of the gene of the present invention in an individual or various tissues can be determined by utilizing the nucleotide sequence of a part or all of the gene. Can be detected.

Such detection can be carried out according to a conventional method. For example, RT-PCR [Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego, 2
RNA amplification and Northern blotting analysis [Molecular Cloning, Cold Spring Harbor La.
b. (1989)], in situ RT-PCR [Nucl.
s., 21 , 3159-3166 (1993)] and cell-level measurement using in situ hybridization, NASB
Method A [Nucleic acid sequence-based amplification, N
, 350 , 91-92 (1991)] and various other methods. Preferably, a detection method by RT-PCR can be mentioned.

The primers used in the case of employing the PCR method are not particularly limited as long as they are specific to the gene of the present invention and can specifically amplify only the gene of the present invention. Can be set appropriately based on Usually, primers having a partial sequence of the gene of the present invention having a length of about 10 to 35 nucleotides, preferably about 15 to 30 nucleotides can be mentioned.

As described above, the gene of the present invention also includes a DNA fragment used as a specific primer and / or a specific probe for detecting the TSA2306 gene according to the present invention.

The DNA fragment can be defined as a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of SEQ ID NO: 2. Here, examples of the stringent conditions include ordinary conditions used as primers or probes, and are not particularly limited. For example, 0.2 × S containing 0.1% SDS as described above may be used.
1 × S containing 50% in SC or 0.1% SDS in SC
The condition of 60 ° C. in SC can be exemplified.

The gene of the present invention can be easily and stably produced in a large amount by using the gene of the present invention in ordinary genetic engineering techniques.

Accordingly, the present invention also provides a polypeptide having an amino acid sequence encoded by the gene of the present invention (expression product of the gene of the present invention). Containing vectors,
It also provides a host cell transformed with the vector, a method for producing the polypeptide of the present invention by culturing the host cell, and the like.

Specific examples of the polypeptide of the present invention include a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 (TSA2306 protein). The polypeptide of the present invention includes not only the TSA2306 protein but also , And homologs thereof. Examples of the homologue include a polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and having a function similar to that of the TSA2306 protein. Can be. Specifically, the TS
A gene product of a homologue of the A2306 gene (TSA2306 equivalent gene including alleles) can be mentioned.

Also, homologs of the TSA2306 protein of the present invention include mammals such as horses, sheep, cows, dogs, monkeys, cats and bears having the same activity as the polypeptide having the amino acid sequence represented by SEQ ID NO: 1. , Rats, mice,
Also included are proteins of rodents such as rabbits.

The polypeptide of the present invention can be prepared by a conventional gene recombination technique [for example, Science, 224 , 1431 (1984); Bioche, based on the sequence information of the gene provided by the present invention.
m. Biophys. Res. Comm., 130 , 692 (1985); Proc. Nat.
l. Acad. Sci., USA., 80 , 5990 (1983), etc.].

The production of the polypeptide is more particularly described
A recombinant DNA (expression vector) capable of expressing a gene encoding a desired protein in a host cell is prepared, introduced into a host cell, transformed, and the transformant is cultured. It is carried out by collecting from.

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts widely include commonly used hosts such as Escherichia coli and Bacillus subtilis. Escherichia coli, especially those contained in the Escherichia coli K12 strain can be exemplified. In addition, the eukaryotic host cell, vertebrate, include cells, such as yeast, COS cells as the former, for example, monkey cells [Cell, 23: 17
5 (1981)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl.
ad. Sci., USA., 77 : 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces are preferably used. Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a promoter and an SD (Shine & Synthetic) are used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell. (Dalgarno) An expression plasmid having a base sequence and an initiation codon (eg, ATG) necessary for initiation of protein synthesis can be suitably used. The above-mentioned vector is generally a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pU
C12, pUC13 and the like are often used, but not limited thereto, and various known vectors can be used. Commercially available products of the above-mentioned vectors used in expression systems using Escherichia coli include, for example, pGEX-4T (Amersham P.
harmacia Biotech), pMAL-C2, pMAl-P
2 (New England Biolabs), pET21, pET2
1 / lacq (Invitrogen), pBAD / His (In
vitrogen).

In the case of using a vertebrate cell as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site and a transcription termination sequence located upstream of the gene of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr [Mol. Cell. Biol., 1 : 854 (1981)]. In addition to the above, various known commercial vectors can be used. Commercially available products of such vectors used in expression systems using animal cells include, for example, pEG
FP-N, pEGFP-C (Clontrech), pIND
(Invitrogen), pcDNA3.1 / His (Invitr
ogen) and pFastBa
cHT (GibciBRL), pAcGHLT (Phar
Mingen), pAc5 / V5-His, pMT / V5-
His, pMT / Bip / V5-his (Invitrog
(en company).

As a specific example of an expression vector when a yeast cell is used as a host, for example, pAM82 having a promoter for the acid phosphatase gene [Proc.
Natl. Acad. Sci., USA., 80 : 1 (1983)]. Commercially available expression vectors for yeast cells include, for example, pP
ICZ (Invitrogen), pPICZα (Invitrogen
Company).

[0065] The promoter is not particularly limited. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a bacterium belonging to the genus Bacillus, SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When yeast is used as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like can be suitably used. When an animal cell is used as a host, preferable promoters include an SV40-derived promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, a cytomegalovirus promoter, and an SRα promoter.

As the expression vector for the gene of the present invention, a normal fusion protein expression vector can also be preferably used. Specific examples of the vector include glutathione-S-
Examples include pGEX (Promega) for expression as a fusion protein with transferase (GST).

Examples of the polynucleotide sequence whose coding sequence of the mature polypeptide assists the expression and secretion of the polypeptide from the host cell include a secretory sequence and a leader sequence. Marker sequence (hexahistidine tag, histidine tag), in the case of mammalian cells, hemagglutinin (H
A) ・ Tags can be exemplified.

The method for introducing a desired recombinant DNA (expression vector) into a host cell and the method for transforming the same are not particularly limited, and various general methods can be employed.

The obtained transformant can be cultured according to a conventional method, and the target protein of the present invention encoded by a gene designed as desired by the culture can be cultured inside, outside the cell, or on the cell membrane of the transformant. Is expressed and produced (accumulated, secreted).

As the medium used for the cultivation, various types of media commonly used depending on the host cells used can be appropriately selected and used, and the cultivation can be carried out under conditions suitable for the growth of the host cells.

The recombinant protein of the present invention thus obtained can be subjected, if desired, to various separation procedures utilizing its physical properties, chemical properties and the like [“Biochemical Data Book II”,
1175-1259, 1st edition, 1st printing, June 23, 1980, issued by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274 (198
6); Eur. J. Biochem., 163 , 313 (1987)).

Examples of the method include ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation,
Osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high-performance liquid chromatography (HPL
Various types of liquid chromatography such as C), dialysis, and combinations thereof can be exemplified. Particularly preferred methods include affinity chromatography using a column to which a specific antibody against the protein of the present invention is bound. it can.

In designing a desired gene encoding the polypeptide of the present invention, the TS shown in SEQ ID NO: 2 was used.
The base sequence of the A2306 gene can be used favorably. The gene can be used by appropriately selecting and changing codons indicating each amino acid residue, if desired. Further, in the amino acid sequence encoded by the TSA2306 gene, when a part of the amino acid residue or the amino acid sequence is modified by substitution, deletion, addition, etc., for example, by the various methods described above such as cytospecific mutagenesis It can be carried out.

The polypeptide of the present invention also has the sequence of SEQ ID NO:
According to the amino acid sequence shown in No. 1, it can be produced by a general chemical synthesis method. The method includes a conventional liquid phase method and a solid phase method for peptide synthesis.

The method for peptide synthesis is described in more detail below.
Based on the amino acid sequence information, a so-called stepwise elongation method in which each amino acid is sequentially linked one by one to extend the chain, a fragment consisting of several amino acids is synthesized in advance, and then each fragment is subjected to a coupling reaction. And the fragment condensation method, and the synthesis of the protein of the present invention may be carried out by any of them.

The condensation method employed in the above peptide synthesis also includes
Conventional methods can be followed, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPP
A (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide, N-hydroxy-5-norbornene-
2,3-dicarboximide) method and Woodward method.

The solvent that can be used in each of these methods can also be appropriately selected from well-known general solvents that are used in this kind of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethylsulfoxide (DMSO), hexaphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and a mixed solvent thereof.

In the above peptide synthesis reaction, carboxyl groups in amino acids or peptides not involved in the reaction are generally esterified to form lower alkyl esters such as methyl ester, ethyl ester and tertiary butyl ester such as benzyl ester. And aralkyl esters such as p-methoxybenzyl ester and p-nitrobenzyl ester.

The amino acid having a functional group in the side chain, for example, the hydroxyl group of a tyrosine residue may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, etc. No need to do. Further, for example, a guanidino group of an arginine residue may be a nitro group, a tosyl group, a p-methoxybenzenesulfonyl group, a methylene-2-sulfonyl group, a benzyloxycarbonyl group, an isobornyloxycarbonyl group, an adamantyloxycarbonyl group or the like. Can be protected by various protecting groups.

Deprotection of these protecting groups in the amino acids and peptides having the above protecting groups and the finally obtained protein of the present invention can also be carried out by a conventional method such as a catalytic reduction method, liquid ammonia / sodium, hydrogen fluoride, and the like. , Hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid and the like.

The polypeptide of the present invention thus obtained is
Various methods described above, for example, ion-exchange resin, partition chromatography, gel chromatography, according to methods commonly used in the field of peptide chemistry such as countercurrent distribution method,
Purification can be performed as appropriate.

The polypeptide of the present invention can be suitably used as an immunizing antigen for preparing a specific antibody thereof. By using this antigen, desired antisera (polyclonal antibody) and monoclonal antibody can be obtained. it can.

The method of producing the antibody itself is well understood by those skilled in the art, and the present invention can also follow these conventional methods [for example, “Seismochemistry Laboratory Course“ Immunobiochemical Research Method ”, Japan See Biochemical Society (ed., 1986).

The antibody thus obtained is, for example, TSA2306
It can be advantageously used for protein purification and its measurement or identification by immunological techniques. More specifically, since amplification and enhanced expression of the gene of the present invention have been confirmed in cancer cells, the antibody can be used for diagnosing cancer or determining the degree of malignancy of cancer.

The polypeptide of the present invention is useful in the pharmaceutical field as a drug containing the polypeptide as an active ingredient. Therefore, the present invention also provides a pharmaceutical composition comprising the polypeptide of the present invention as an active ingredient.

The usefulness of the polypeptide of the present invention as the above-mentioned pharmaceutical composition is, as described above, an activity of inhibiting the angiogenesis or tumor progression surrounding the liver / kidney cancer tissue of the liver / kidney-specific polypeptide. (Antitumor activity). Methods for confirming these activities include, for example, the following methods. (1) In vitro method for measuring the activity of vascular endothelial cells to inhibit vascular vascular formation The vascular endothelial cells are cultured on collagen and angiogenesis factor (basic fibroblast growth factor; bFGF,
By adding ndothelial growth factor (VEGF, etc.) to the medium or by co-culture of cancer cells, a capillary-like space is formed in the gel. The substance whose antiangiogenic effect is to be measured (the polypeptide of the present invention, for example, TS
A2306 protein; an expression system using Escherichia coli; obtained by pET21 vector (obtained from Invitrogen)) is added to the culture medium to observe under a microscope whether tube formation is suppressed as compared with the control. Sato Y., et
al., Expt. Cell Res., 204 , 223-229 (1993)), (2) In vivo measurement method For example, an expression vector (pET2
1) and cancer cells stably expressing the candidate protein for inhibiting angiogenesis (in which the candidate gene for inhibiting angiogenesis has been incorporated into an expression vector and forcibly expressed in the cancer cells) are administered, respectively. Or the number of tumor neovessels is compared to controls (Folkman, J., et al., J. Exp. Med.,
133 , 275-288 (1971)).

To express a gene in human cancer cells (gene therapy), an adenovirus vector or the like (Straus, ES, Plenum Press New York, 451-496 (19)
84), Setoguchi, Y., et al., Blood, 84 , 2953-2964 (19
94)). That is, it is conceivable to incorporate the gene of the present invention into an adenovirus vector and directly administer it to the tumor site to determine whether the tumor regresses.

In the pharmaceutical composition of the present invention, the polypeptide as an active ingredient also includes a pharmaceutically acceptable salt thereof. Such salts include those prepared by methods well known in the art, for example, sodium, potassium, lithium,
Non-toxic alkali metal salts such as calcium, magnesium, barium and ammonium, alkaline earth metal salts, ammonium salts and the like are included. The above salts also include non-toxic acid addition salts obtained by reacting the polypeptide of the present invention as an active ingredient with a suitable organic or inorganic acid. Representative non-toxic acid addition salts include, for example, hydrochloride,
Hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate,
Borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate , Maleate, ascorbate, benzenesulfonate and napsylate.

The pharmaceutical compositions of the present invention include those that contain a pharmaceutically effective amount of the polypeptide of the present invention as an active ingredient, together with a suitable nontoxic pharmaceutical preparation carrier or diluent.

Pharmaceutical carriers that can be used in the above-mentioned pharmaceutical composition (pharmaceutical preparation) include fillers, extenders, binders, humectants, disintegrants and the like which are usually used according to the use form of the preparation.
Diluents such as surfactants and lubricants or excipients can be exemplified, and these can be appropriately selected and used according to the dosage unit form of the obtained preparation.

Particularly preferred pharmaceutical preparations of the present invention include various additive components which can be used for ordinary protein preparations, such as stabilizers, bactericides, buffers, isotonic agents, chelating agents,
It can be prepared by appropriately using a regulator, a surfactant and the like.

Examples of the stabilizer include human serum albumin, ordinary L-amino acids, saccharides, and cellulose derivatives. These can be used alone or in combination with a surfactant or the like, if necessary.
According to the combination with a surfactant or the like, there are cases where the stability of the active ingredient can be further improved.

The L-amino acid is not particularly limited, and may be, for example, glycine, cysteine, glutamic acid or the like.

The above-mentioned sugars are not particularly restricted but include, for example, monosaccharides such as glucose, mannose, galactose and fructose; sugar alcohols such as mannitol, inositol and xylitol; disaccharides such as sucrose, maltose and lactose; dextran; Polysaccharides such as propyl starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof can be used.

The surfactant is not particularly limited.
Both ionic and non-ionic surfactants can be used. As specific examples, for example, polyoxyethylene glycol sorbitan alkyl ester type, polyoxyethylene alkyl ether type, sorbitan monoacyl ester type, fatty acid glyceride type and the like can be used.

The cellulose derivative is not particularly limited, and examples thereof include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose and sodium carboxymethylcellulose.

The amount of the additive component such as the above-mentioned saccharides can be appropriately determined by referring to the case where such additives are usually used. In general, the saccharide is at least about 0.0001 mg, preferably 0.01 to 10 mg per 1 μg of the active ingredient.
It is appropriate to select from the range of about mg. The surfactant is usually selected from the range of about 0.00001 mg or more, preferably about 0.0001 to 0.01 mg per 1 μg of the active ingredient. Human serum albumin as a stabilizing agent is 0.000 per 1 μg of the active ingredient.
It is suitably selected from a range of about 1 mg or more, preferably about 0.001 to 0.1 mg. Amino acids as stabilizers are used in an amount of 0.001 to 10 per 1 μg of the active ingredient.
It is appropriate to select from the range of about mg. The amount of the cellulose derivative added was 0.0
0001 mg or more, preferably 0.001 to 0.1
It is appropriate to select from the range of about mg.

The amount of the active ingredient contained in the pharmaceutical preparation of the present invention is appropriately selected from a wide range, but is usually about 0.00000.
It is appropriate that the content is in the range of about 1 to 70% by weight, preferably about 0.0001 to 5% by weight.

The pharmaceutical preparation of the present invention may further contain, for example, a buffer, an isotonic agent, a chelating agent and the like. Here, boric acid, phosphoric acid, acetic acid, citric acid, ε-aminocaproic acid, glutamic acid and / or
Or salts corresponding thereto (for example, alkali metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt and alkaline earth metal salts) and the like. Examples of the tonicity agent include sodium chloride, potassium chloride, sugars, glycerin and the like. Examples of the chelating agent include sodium edetate and citric acid. The amounts of these additives can be the same as those usually used.

The pharmaceutical preparation of the present invention can be prepared and used as a solution preparation, or it can be lyophilized and stored. Such a lyophilized product can be used after dissolving it in a buffer solution containing water, fresh saline, or the like at the time of use to prepare an appropriate concentration.

The dosage unit form of the pharmaceutical preparation of the present invention includes
Various forms can be selected according to the purpose of treatment. Representative examples include solid dosage forms such as tablets, pills, powders, powders, granules, capsules and the like, and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These various forms, oral preparations, parenteral preparations,
It is classified into nasal, vaginal, suppository, sublingual, ointment and the like. Each of the above forms can be prepared, molded or prepared according to a usual method.

For example, in the case of molding into a tablet form, as the pharmaceutical preparation carrier, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc. Excipients; water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone and other binders; sodium carboxymethylcellulose, calcium carboxymethylcellulose, low-substituted hydroxypropyl Disintegrants such as cellulose, dried starch, sodium alginate, powdered agar, powdered laminaran, sodium hydrogen carbonate, calcium carbonate; polyoxyethylene sorbitan fatty acid ester , Sodium lauryl sulfate, surfactants such as stearic acid monoglyceride; disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil; quaternary ammonium bases, absorption promoters such as sodium lauryl sulfate; glycerin, starch and the like Humectants; adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; and lubricants such as purified talc, stearates, boric acid powder, polyethylene glycol and the like.

Further, the tablets may be tablets coated with a usual coating as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, and double tablets or multilayer tablets. Can also.

In the case of molding into a pill form, as a pharmaceutical preparation carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc; gum arabic powder, tragacanth powder, gelatin, ethanol, etc. And disintegrating agents such as laminaran and agar.

Capsules can be prepared by mixing the active ingredient of the present invention with the various pharmaceutical preparation carriers exemplified above in a usual manner.
It can be prepared by filling in hard gelatin capsules, soft capsules and the like.

Liquid dosage forms for oral administration include pharmaceutically acceptable solutions, emulsions, suspensions, syrups, elixirs and the like containing conventional inert diluents, for example water, and further include wetting agents, Auxiliaries such as emulsions and suspensions can be included. These can be prepared according to a conventional method.

In preparing liquid dosage forms for parenteral administration, such as sterile aqueous to non-aqueous solutions, emulsions and suspensions, diluents such as water, ethyl alcohol, propylene glycol, polyethylene glycol,
Ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and vegetable oils such as olive oil can be used. Injectable organic esters, such as ethyl oleate, can also be blended. Further, ordinary solubilizers, buffers, wetting agents, emulsifiers, suspending agents, preservatives, dispersants and the like can also be added.

The above various forms of the pharmaceutical preparations are sterilized according to a conventional method. The sterilization treatment can be performed by, for example, a filtration operation of passing through a bacteria retaining filter, blending of a bactericide, irradiation treatment, heat treatment, and the like. They can also be prepared in the form of sterile solid compositions which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.

For shaping in the form of suppositories or preparations for vaginal administration, carriers for pharmaceutical preparations such as polyethylene glycol, cocoa butter, higher alcohols, higher alcohol esters, gelatin and semisynthetic glyceride can be used.

When shaping into the form of an ointment such as a paste, cream or gel, diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, propylene glycol, polyethylene glycol,
Silicone, bentonite and vegetable oils such as olive oil can be used.

A composition for nasal or sublingual administration can be prepared by a conventional method using well-known standard excipients.

The pharmaceutical agent of the present invention may contain a coloring agent, a preservative, a fragrance, a flavor, a sweetener, and other pharmaceuticals, if necessary.

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient age, gender and other conditions, degree of disease, and the like. For example, tablets, pills,
Solutions, suspensions, emulsions, granules and capsules are administered orally, injections are administered intravenously alone or in admixture with normal replenishers such as glucose and amino acids, and if necessary intramuscularly, alone. Administered intradermally, subcutaneously or intraperitoneally,
Suppositories are administered rectally, vaginal agents are administered intravaginally, nasal agents are administered intranasally, sublingual agents are administered intraorally, and ointments are administered topically transdermally.

The dosage of the above pharmaceutical preparation is not particularly limited, and the desired therapeutic effect, administration method, treatment period, patient age,
It is appropriately selected from a wide range according to gender and other conditions. In general, usually 1 kg of patient weight per day,
The amount of the active ingredient may be about 0.01 μg to 10 mg, preferably about 0.1 μg to 1 mg. The formulation can be administered once a day, or more than once,
It can be administered in several divided doses.

The liver / kidney-specific polypeptide of the present invention, for example, TSA2306 protein itself has an inhibitory effect on angiogenesis and is useful as an angiogenesis inhibitor, and is also useful as an anticancer agent as a cancer chemotherapeutic agent. It is also useful as a drug, a ligand which is bound to a physical, chemical or biological substance.

That is, the polypeptide of the present invention or a partial fragment thereof is prepared by binding them to an active drug such as an anticancer drug or a cytokine having an anticancer activity, or modifying the active drug with these to prepare a DDS preparation. Can be. By using the DDS preparation, cancer tissue-selective treatment for cancer tissue can be performed.

When the polypeptide of the present invention is bound or combined with a molecule such as a protein or nucleic acid having cytokines having anticancer activity, the expressed product expressed as a fusion protein by genetic recombination technique is used as a drug. Can be provided as The production or expression of the fusion protein is a commonly known technique in this field. That is, the fusion protein
Conventional gene recombination techniques (e.g., Science, 224 , 1431 (1
984); Biochem. Biophys. Res. Comm., 130 , 692 (1985);
Proc. Natl. Acad. Sci. USA, 80 , 5990 (1983)). Alternatively, the protein can be produced by referring to Ohno et al., “Protein Experiment Protocol 1 Functional Analysis, Cell Engineering Separate Volume, Experimental Protocol Series, 1997, Shujunsha”.

The recombinant fusion protein thus obtained may be subjected to various separation operations utilizing its physical properties, chemical properties, etc. [“Biochemical Data Book II”, 1175-125, if desired.
9 pages, 1st edition, 1st printing, June 23, 1980, published by Tokyo Chemical Co., Ltd .; Biochemistry, 25 (25), 8274-8277 (1986);
Eur. J. Biochem., 163 , 313-321 (1987), etc.]. Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography (gel filtration) And various types of liquid chromatography such as adsorption chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography (HPLC), dialysis, and combinations thereof. A particularly preferred example of the above method is affinity chromatography using a column to which a desired protein is bound.

When the pharmaceutical composition of the present invention, particularly the pharmaceutical composition of the present invention as an angiogenesis inhibitor or a therapeutic agent for cancer, is administered to a patient, for example, the fusion protein or its partial peptide as an active ingredient, liver / kidney specific The conjugate of the target polypeptide or a partial peptide thereof and another anticancer agent is administered in the form of a pharmaceutical composition containing the same together with a pharmaceutically acceptable carrier. Here, as the pharmaceutically acceptable carrier, those well-known in the art, for example, an aqueous solution
(Water or physiological buffer) or other solvent or material
(Eg, injectable organic esters such as glycol, glycerol, olive oil) can be used. In addition, as the carrier, for example, an appropriate compound that acts to stabilize or increase the absorption of the complex can be used. Such suitable compounds include, for example, glucose,
Carbohydrates such as sucrose and dextran; antioxidants such as ascorbic acid and glutathione; chelating agents;
Low molecular proteins; stabilizers such as albumin, and the like.

When a complex of the above-mentioned active ingredient and another anti-cancer agent is administered to a patient, the dose of the active ingredient also depends on the amount of another anti-cancer agent used in combination, such as a cytokine or a cancer chemotherapeutic agent. . An effective amount of a complex of the polypeptide of the present invention and a cancer chemotherapeutic agent such as a cytokine for treating such a disease can be appropriately determined by a method well known to those skilled in the art. For example, a cancer chemotherapeutic agent is selected from an amount that is usually administered at a dose of about 0.1 mg / kg to 50 mg / kg per day in the case of 5-FU (5-fluorouracil) for clinical use of this kind. The effective dose of the polypeptide of the present invention (or a partial peptide thereof) to be bound thereto can be easily determined by those skilled in the art. Considering the characteristics of the pharmaceutical composition of the present invention, which has an effect of specifically returning to neovascularization of cancer,
It is anticipated that the dose of the cancer chemotherapeutic agent will be at least more efficacious than conventionally used clinical doses.

As will be shown in the examples below, the gene of the present invention has lost its expression in tissues of liver cancer and kidney cancer. If a gene expression vector is prepared, the expression vector is introduced into a cancer tissue, and the gene of the present invention is forcibly expressed in the tissue, angiogenesis in the cancer tissue is suppressed, and as a result, cancer progresses. This is where cancer metastasis and the like are suppressed. Therefore, the present invention also provides a composition for gene therapy (gene therapeutic agent) having such antitumor activity.

Further, according to the present invention, there are also provided the above-mentioned gene expression vector or gene therapy vector, cells into which the gene of the present invention has been introduced using the vector, and pharmaceutical compositions (gene therapeutic agents) containing these as an active ingredient. .

In the gene therapy using the above-described gene therapy agent, for example, at least one selected from a vector for introducing and expressing the gene of the present invention and a cell into which the gene of the present invention has been introduced by the vector is used to obtain a cancer cell or cancer of a cancer patient. It is performed by administration to a tissue site. Thus, it is possible to suppress angiogenesis in these tissues and suppress cancer cell growth or cancer metastasis. That is, tumor formation, tumor progression and tumor metastasis of the liver and kidney can be suppressed.

Hereinafter, such gene therapy will be described in detail.
In the implementation of the following gene therapy, conventional methods of chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunology can be used unless otherwise specified. These include, for example, Maniatis, T., et a
l., Molecular cloning: A laboratory manual (Cold S
pring Harbor Laboratory, Cold Spring Harbor, New Y
ork (1982)), Sambrook, J., et al., Mole.
cular cloning: A laboratory manual, 2nd Ed. (Cold S
pring Harbor Laboratory, Cold Spring Harbor, New Y
ork (1981)), Ausbel (Ausbel, FM, et al., Curre
nt Protocols in Molecular Biology, John Wiley and S
ons, New York, New York, (1992)), Glover
r, D., DNA Cloning, I and II (Oxford Press) (1985)),
Anand, Techniques for the Analysis of Comp
lex Genomes, (Academic Press (1992)), Guthrie (Guthr
ie, G., et al., Guide to Yeast Genetics and Molecul
ar Biology, (Academic Press) (1991)) and Fink
(Fink, et al., Hum. Gene Ther., 3 , 11-19 (1992).

Gene therapy can be carried out using a gene therapy transfer vector containing all or a part of the gene of the present invention, or a cell into which the gene of the present invention has been introduced using the vector. The gene therapy is, for example, a method of supplying the TSA2306 gene or its function to cells in which the expression of the gene is not observed. Such gene therapy suppresses angiogenesis around the recipient / target cells.

The gene of the present invention or a part of the gene can be introduced into cells using a vector that maintains the gene extrachromosomally. In this case, the gene can be expressed by the cell from an extrachromosomal location. In addition, when the gene portion is introduced and expressed in liver or kidney cancer cells in which the expression of the TSA2306 gene is not observed and its surrounding tissue cells, the gene portion replaces a part of the TSA2306 protein necessary for non-neoplastic growth of the cells. May be coded.

[0127] The gene introduction vector may be one obtained by introducing the gene of the present invention into various vectors already known in the art as described later.

[0128] The introduction of the gene transfer vector into the target cell can be carried out using various cells already known in the art.
Methods for introducing NA, such as electroporation,
It can be easily carried out according to calcium phosphate coprecipitation, virus transduction and the like. The cells transformed by introducing the gene of the present invention can be used as a drug for suppressing angiogenesis or cancer metastasis or as a model system for therapeutic research.

As described above, according to the gene therapy according to the present invention, the introduced gene of the present invention or a fragment thereof increases the amount of the expression product of the gene in a cancer cell tissue or a tissue surrounding the cancer cell tissue, and thus in the gene expression tissue. Angiogenesis can be suppressed. Such gene therapy, compared to normal cells,
It is preferably used in both cancerous and precancerous cell tissues in which the expression of the TSA2306 gene or the TSA2306 protein has been deleted or its level has been reduced.

The gene therapy according to the present invention is performed as follows. That is, first, for example, the presence or absence of TSA2306 gene expression in cells from a tumor tissue of a patient is confirmed. This confirmation can be performed, for example, by first preparing a probe using a part of the TSA2306 gene sequence by a method such as the PCR method, amplifying the DNA, and screening.

In addition, mutations in the TSA2306 gene, for example, mutations such as insertion / deletion of a gene with respect to a wild-type gene and point mutation, are confirmed. The above mutation can be confirmed by various methods, for example, direct DNA sequencing, PCR-SSCP.
(PCR-single-strand conformation polymorphism: Pro
Natl. Acad. Sci., 86 , 2766-2770 (1989)), dot blot analysis, pulse field gel electrophoresis (PFGE) analysis, Southern blot analysis, fluorescence in situ hybridization (FISH; Takahashi, E., et. al., Hum. Gen
e., 86 , 14-16 (1990)), single-stranded conformation (SSC
A; Orita, et al., Proc. Natl. Acad. Sci., 86 , 276-2
770 (1989): Orita, et al., Genomics, 5 , 874-879 (198
9)), RNase protection assay (Finkelstein, J., et a
l., Genomics, 7 , 167-172 (1990)), allele-specific oligonucleotides (ASO; Conner, BJ, et al., Proc.
Acad. Sci., 80 , 278-282 (1983)), allele-specific PCR (Rano, & Kidd, Nucl. Acids Res., 17 , 8392 (1
989)). In these confirmations and diagnoses, DNA obtained from any human or animal tissue
Can also be checked. Suitable methods include methods of extracting blood and extracting DNA from blood cells, or extracting DNA from cells obtained directly from target tissue cells.

The presence or absence of expression of the TSA2306 gene can also be confirmed by screening for a protein. The method can be carried out, for example, by screening a tissue using a monoclonal antibody having immunoreactivity with a target protein. Lack of alloantigen may indicate deletion and mutation of expression of the gene. In addition, methods known in the art, such as a Western blot, an immunohistological assay, and an ELISA assay, can be suitably used for such an immunological assay.

Each of the above methods for confirming gene expression products can be applied to gene expression detection in a target cell or target tissue for examining a desired gene transfer effect in gene therapy described below.

Next, in gene therapy, a virus or plasmid vector containing a copy of the TSA2306 gene linked to an expression control element in a cell from the tumor and peri-tumor tissue of a patient and capable of replicating in the tumor cell and peri-tumor tissue Create Suitable vectors here include US Pat. No. 5,252,479 and PC
The vectors disclosed in T International Publication WO93 / 07282 can be used. Injection of the vector containing the generated TSA2306 gene or a fragment thereof into a patient locally or systemically (in order to reach any tumor cells that can metastasize to other sites) in the tumor site or peri-tumor tissue. I do. If the transformed gene is not permanently incorporated into the chromosomes of various targeted tumor cells, the process can be repeated periodically. The gene therapy method is an in vivo method in which a material for gene transfer is directly administered into the body, and an ex vivo method in which a target cell is once taken out of the patient's body, the gene is introduced outside the body, and then the cell is returned to the body. Both methods can be appropriately selected.

In the production of the vector containing the gene of the present invention, all or a part of the gene to be introduced is easily produced and produced by general genetic engineering techniques based on the nucleotide sequence information of the gene. (Molecular Cloning 2d Ed, Cold Spring Harbor Lab.
Press (1989); Laboratory of Seismic Chemistry "Gene Research Methods I, I
I, III ", edited by The Biochemical Society of Japan (1986)]. As the method, any of the methods described in detail in the section on the artificial modification method of amino acids can be applied mutatis mutandis.

Further, a cDNA library is prepared from an appropriate source in which a desired gene is expressed according to a conventional method.
From the library, all or part of the gene can also be produced by selecting a desired clone using an appropriate probe or antibody specific to the gene of interest (Proc. Natl. Acad. Sci. , USA., 78 , 6613 (198
1); Science, 222, 778 (1983) and the like].

Hereinafter, gene therapy using the sense strand will be described in detail.

According to the present invention, angiogenesis is suppressed by providing a sense drug for producing TSA2306 mRNA in cells in a target cell to express the gene of the present invention, promoting translation, and promoting expression of TSA2306 gene. Gene therapy for cancer by action is provided.

[0139] The gene therapy can be performed, for example, on TSA2306 in a cancer cell tissue without expression of the TSA2306 gene and its surrounding tissue.
This is a method of promoting the expression of a target gene by promoting the transcription or translation of 306 mRNA. For this purpose, a method in which a sense oligonucleotide complementary to the mRNA of the gene is produced and the sense oligonucleotide is supplied to target cells can be preferably employed. By the above-described gene therapy, an effect of promoting the expression function of the TSA2306 gene can be supplied to the cells to suppress angiogenesis in the recipient cells / target cells.

According to the cancer gene therapy using the sense oligonucleotide, the antisense oligonucleotide is incorporated into a vector derived from a retrovirus, an adenovirus, or an AAV, and the resultant is infected to a cancer cell to produce the sense oligonucleotide. By expressing, it is possible to obtain a desired angiogenesis inhibitory effect and, as a result, an antitumor effect.

As described above, when the expression of the TSA2306 protein is suppressed by introducing the sense oligonucleotide of the gene of the present invention into cancer cells or cancer tissues and tissues surrounding the cancer, the sense oligonucleotide may be, for example, the full-length TSA2306 gene. It is not necessary that the modified form described above be used, as long as it retains a function substantially equivalent to the function of promoting the expression of the TSA2306 gene, or a gene consisting of a partial sequence retaining the function. It may be.

Vectors for introducing the desired gene for both recombination and extrachromosomal maintenance are already known in the art, and any of such known vectors can be used in the present invention. For example, a viral vector or a plasmid vector containing a copy of a sense oligonucleotide of the TSA2306 gene linked to an expression control element and capable of expressing the sense oligonucleotide product in a target cell can be mentioned. As such a vector, the above-mentioned expression vector can be usually used, but preferably, for example, US Pat. No. 5,252,479 and P.S.
The vectors disclosed in CT International Publication WO 93/07282 (specifically, pWP-7A, pwP-19,
pWU-1, pWP-8A, pWP-21 and / or pRSVL, etc.) or pRC / CMV (Invitrogen
(Manufactured by K.K.). More preferably, the following various viral vectors can be mentioned.

As a promoter used for a vector used in gene transfer therapy, a promoter specific to an affected tissue to be treated for various diseases can be suitably used. Specific examples thereof include, for example, albumin, α-fetoprotein, α1-
Examples include antitrypsin, transferrin, and transstyrene. For the colon, examples thereof include carboxylate anhydrase I and carcinoembrogen antigen. For uterus and placenta, estrogen, aromatase cytochrome P450, cholesterol side chain cleavage P450, 17 alpha-hydroxylase P4
50 and the like.

For the prostate, the prostate antigen, gp91
-Fox gene, prostate-specific kallikrein and the like. For the breast, erb-B2, erb-
Examples include B3, β-casein, β-lactoglobin, whey protein and the like. For the lung, the activator protein C uroglobulin can be exemplified. For skin, K-1
Examples thereof include 4-keratin, human keratin 1 or 6, and leucrin. For the brain, examples include glial fibrillary acidic protein, mature astrocyte-specific protein, myelin, tyrosine hydroxylase pancreatic villin, glucagon, Langerhans islet amyloid polypeptide and the like.
For the thyroid gland, thyroglobulin, calcitonin and the like can be exemplified. For bone, α1 collagen, osteocalcin, bone sialoglycoprotein and the like can be exemplified. For the kidney, renin, liver / bone / kidney alkaline phosphatase, erythropoietin and the like can be exemplified, and for the pancreas, amylase, PAP1 and the like can be exemplified.

In the production of the sense oligonucleotide introduction vector, the introduced sense oligonucleotide (having all or a part of the sequence corresponding to the sequence of the gene of the present invention) contains the nucleotide sequence of the gene of the present invention. Based on the information, as described above, it can be easily manufactured and obtained by a general genetic engineering technique.

The introduction of such a sense oligonucleotide introduction vector into cells is already known in the art for introducing DNA into cells, including, for example, electroporation, calcium phosphate coprecipitation, and virus transduction. Can be performed according to various methods. The cells transformed with the above sense oligonucleotides themselves have an anti-angiogenic activity in an isolated state, and as a result, a drug for suppressing cancer or suppressing cancer metastasis, It can also be used as a model system for

In gene therapy, the sense oligonucleotide-introducing vector can be locally or systemically injected into a patient at or around a tumor site. This administration allows introduction into the tumor cells of the patient. In that case, systemic administration can reach any tumor cells that can metastasize to other sites. If the transduced gene is not permanently integrated into the chromosome of each target tumor cell, the administration can be repeated periodically.

The method for gene therapy of the present invention is characterized in that
An in vivo method in which a material for oligonucleotide introduction (sense oligonucleotide introduction vector) is directly administered into the body, or a method in which a target cell is once taken out of the patient's body, and then the gene is introduced outside the body, and then And ex vivo methods for returning the cells into the body. Gene therapy by directly introducing a sense oligonucleotide into cells is also possible.

The gene transfer vector containing the sense oligonucleotide of the gene sequence of the present invention and the gene therapy agent containing as an active ingredient a cell into which the sense oligonucleotide has been introduced by the vector are particularly useful for cancers in which angiogenesis is promoted. It can be used for the treatment of proliferative tissue diseases other than cancer, for example, diseases such as uterine fibroids, and can also be used for treatment for gene labeling. it can.

The target cells into which the sense oligonucleotide of the gene of the present invention is introduced can be appropriately selected depending on the target of gene therapy (treatment). For example, examples of the target cells include cells such as lymphocytes, fibroblasts, hepatocytes, hematopoietic stem cells, and the like, in addition to cancer cells and tumor tissues.

The method for introducing a sense oligonucleotide in the above gene therapy includes a viral introduction method and a non-viral introduction method.

As a method for viral introduction, for example, a method using a retrovirus vector as a vector in consideration of the fact that the sense oligonucleotide to be introduced is a foreign substance that is expressed particularly in normal liver and kidney cells. Can be mentioned. Other viral vectors include adenovirus vectors, HIV (humanimm
unodeficiency virus) vector, adeno-associated virus vector (AAV), herpes virus vector, herpes simplex virus (HSV)
Vector, Epstein-Barr virus (EBV, Epst
ein-Barr virus) vector.

[0153] Non-viral gene transfer methods include:
Calcium phosphate coprecipitation method: Membrane-fused liposome in which DNA-encapsulated liposome is fused with inactivated Sendai virus whose gene has been disrupted in advance by ultraviolet rays to produce a membrane-fused liposome, and directly fused with a cell membrane to introduce DNA into cells Method (Kato, K., et al., J. Biol. Chem., 266 , 220).
71-22074 (1991)]; a method of coating plasmid DNA with gold and physically introducing the DNA into cells by high-pressure discharge [Yang, NS et al., Proc. Natl. Acad. Sci., 87 ,
9568-9572 (1990)]; naked DNA for injecting plasmid DNA directly into organs and tumors in vivo
Method (Wolff, JA, et al., Science, 247 , 1465-1467 (199
0)]; Cationic liposome method for introducing genes embedded in multilamellar positively charged liposomes into cells [Kunio Yagi, History of Medicine, Vol. 175, No. 9, 635-637 (1995)]; The ligand-DNA complex method of binding a ligand that binds to a receptor expressed in a target cell to DNA and administering the same (Frindeis, et al. , Trends Biote
chnol., 11 , 202 (1993); Miller, et al., FASEB J., 9 ,,
190 (1995)].

The ligand-DNA complex method includes, for example, a method using asialoglycoprotein as a ligand targeting asialoglycoprotein receptor expressed by hepatocytes [Wu, et al., J. Biol. Chem., 266 , 14338 (1991); F
erkol, et al., ASEB J., 7 , 1081-1091 (1993)],
A method using transferrin as a ligand targeting a transferrin receptor strongly expressed by tumor cells [Wagner et al., Proc. Natl. Acad. Ci., US
A., 87 , 3410 (1990)].

Further, the above-described gene transfer method may be a combination of the above-described various biological and physical gene transfer methods as appropriate. As a method based on the combination, for example, a plasmid DNA of a certain size
An example is a method in which the antibody is combined with a polylysine-conjugated antibody specific to a hexon protein. According to this method, the desired gene can be introduced by binding the obtained complex to the adenovirus vector and infecting the cell with the thus obtained trimolecular complex. In this method, before the DNA coupled to the adenovirus vector is damaged,
Efficient binding, internalization and endosomal degradation are possible. In addition, the liposome / DNA complex can directly mediate gene transfer in vivo.

Hereinafter, a specific method for preparing a viral vector for introducing a sense oligonucleotide and a method for introducing a sense oligonucleotide into a target cell or target tissue will be described.

The retrovirus vector system comprises a virus vector and a helper cell (packaging cell).
Consists of Here, the helper cells previously express genes such as retroviral structural proteins gag (structural protein in virus particles), pol (reverse transcriptase), and env (envelope protein). Say not cells. On the other hand, viral vectors use packaging signals or LTRs (long terminal repeats).
s), but gag, po necessary for virus replication
It does not have structural genes such as l and env. The packaging signal is a sequence that serves as a tag during assembly of the virus particles. The selection gene (neo, hyg) and the desired sense oligonucleotide for introduction incorporated in the cloning site are inserted instead of the virus gene.
Here, in order to obtain a high titer virus particle, the insert should be as short as possible, the packaging signal should be wide including a part of the gag gene, and the ATG of the gag gene should be increased.
It is important not to leave any

By transferring the vector DNA incorporating the desired sense oligonucleotide into the helper cells, the vector genomic RNA is packaged by the viral structural proteins produced by the helper cells to form virus particles and secreted. . After a virus particle as a recombinant virus infects a target cell, DNA reversely transcribed from viral genomic RNA is incorporated into a cell nucleus, and a sense gene inserted into a vector is expressed.

As a method for increasing the efficiency of introducing a desired gene, a method using a fragment containing a cell adhesion domain of fibronectin, a heparin binding site and a junction segment [Hanenberg, H., et al., Exp. Hemat., 23 , 747 (1995))
Can also be adopted.

The vector used in the above retrovirus vector system includes, for example, a retrovirus derived from mouse leukemia virus [McLachlin,
JR, et al., Proc. Natl. Acad. Res. Molec. Bio
l., 38 , 91-135 (1990)].

The method using an adenovirus vector will be described in detail.
Berkner, KL, Curr.Topics Microbiol.Imm
unol., 158 , 39-66 (1992)), Yasuhiro Setoguchi et al. (Setoguch
i, Y., et al., Blood, 84 , 2946-2953 (1994)), Hiromi Kaneka et al. (Experimental Medicine, 12 , 28-34 (1994)) and Kenner et al. (Ketner, G., et al.) ., Proc. Natl. Acad. Sci., US
A., 91 , 6186-6190 (1994)].

For example, to construct a non-competent adenovirus vector, the E.
The 1 and / or E3 gene region is removed. Next, a desired expression unit of a desired foreign gene (constituted by a desired sense oligonucleotide for introduction, a promoter for transcribing the sense oligonucleotide, and a polyA for imparting stability to the transcribed gene) And a plasmid vector containing a part of the adenovirus genomic DNA and a plasmid containing the adenovirus genome are simultaneously transfected into, for example, 293 cells. By causing homologous recombination between the two and replacing the gene expression unit with E1, a non-proliferative adenovirus vector which is a vector containing the desired sense oligonucleotide can be prepared. . In addition, an adenovirus genomic DNA can be incorporated into a cosmid vector to prepare a 3 'adenovirus vector to which a terminal protein has been added. Furthermore, a YAC vector can also be used for producing a recombinant adenovirus vector.

Briefly describing the production of the adeno-associated virus (AAV) vector, AAV was discovered as a small virus contaminating the adenovirus culture system. This confirms the existence of the genus Parvovirus, which grows autonomously in host cells without requiring a helper virus for virus replication, and the genus Dependovirus, which requires a helper virus. The AAV is a common virus that has a wide host range and infects a variety of cells. The virus genome is composed of linear single-stranded DNA having a size of 4680 bases, and 145 bases at both ends are ITRs (inverted terminal).
exists) with a characteristic sequence called "repeat". This ITR portion serves as an origin of replication and serves as a primer. Furthermore, the ITR is also essential for packaging into virus particles and integration into chromosomal DNA of host cells. As for viral proteins, the left half of the genome encodes a nonstructural protein, that is, Rep, a regulatory protein that controls replication and transcription.

The production of recombinant AAV was performed by
It can be carried out using the property of being incorporated into NA, and thus a desired gene transfer vector can be prepared. More specifically, in this method, first, a plasmid (AAV vector plasmid) in which ITRs at both ends of 5 ′ and 3 ′ of wild-type AAV are left and a desired sense oligonucleotide for introduction is inserted therebetween is prepared. On the other hand, viral proteins required for virus replication and virus particle formation are supplied by another helper plasmid. It is necessary to prevent a common base sequence from being present between the two, and to prevent the appearance of wild-type virus due to genetic recombination. Thereafter, both plasmids are introduced, for example, by transfection into 293 cells, and further infected with an adenovirus (if 293 cells are used, a non-proliferating type) may be used as a helper virus to obtain a desired non-proliferating set. A recombinant AAV is produced. Subsequently, since the recombinant AAV is present in the nucleus, the cells are freeze-thawed and recovered, and the contaminating adenovirus is inactivated by heating at 56 ° C. Further, if necessary, the recombinant AAV is separated and concentrated by ultracentrifugation using cesium chloride. As described above, a desired recombinant AAV for gene introduction can be obtained.

The EBV vector can be produced, for example, according to the method of Shimizu et al. [Norio Shimizu, Jinzo Takada,
Cell Engineering, 14 (3), 280-287 (1995)].

The production of an EBV vector for introducing a sense oligonucleotide according to the present invention will be briefly described.
Epstein-Barr virus (EBV) was introduced in 1964 by Epstein et al. In Burkitt.
A virus belonging to the family Herpes isolated from cultured cells derived from lymphoma (Kieff, E. and Liebowitz, D .: Vi.
rology, 2nd ed. Raven Press, New York, 1990, pp.18
89-1920]. Since the EBV has the activity of transforming cells, a virus lacking this transforming activity must be prepared in order to use it as a vector for gene transfer. This can be done as follows.

That is, first, the EBV genome near the target DNA into which the desired foreign gene is to be incorporated is cloned. The DNA fragment of the foreign gene and the drug resistance gene are inserted therein, and used as a vector for producing a recombinant virus. Next, the EBV-positive Akata cells are transfected with the vector for producing a recombinant virus cut out by an appropriate restriction enzyme. Recombinant virus generated by homologous recombination can be recovered together with wild-type Akata EBV by stimulating virus production by anti-surface immunoglobulin treatment. This is EBV
By infecting negative Akata cells and selecting resistant strains in the presence of the drug, Akata cells infected with only the desired recombinant virus without wild-type EBV can be obtained. Further, by inducing the virus activity in the recombinant virus-infected Akata cells, a large amount of the desired recombinant virus vector can be produced.

The production of a non-viral vector in which a desired sense oligonucleotide is introduced into a target cell without using a recombinant viral vector can be carried out, for example, by a gene transfer method using membrane-fused liposomes.
This is a method in which membrane liposomes (vesicles composed of a lipid bilayer membrane) are given a fusion activity to a cell membrane, thereby directly introducing the contents of the liposome into cells.

The introduction of the sense oligonucleotide using the membrane-fused liposome can be carried out, for example, by the method of Nakanishi et al. [Nakanishi, M., et al., Exp.
s., 159 , 399-499 (1985); Nakanishi, M., et al., Gene i.
ntroduction into animal tissues.In Trends and Futu
re Perspectives in Peptide and Protein Drug Delive
ry (ed. by Lee, VH et al.)., Harwood Academic Pu
blishers Gmbh. Amsterdam, 1995, pp. 337-349].

Hereinafter, the sense fusion with the membrane-fused liposome
An outline of the method for introducing the oligonucleotide will be described. That is, a Sendai virus whose gene is inactivated by ultraviolet rays and a liposome encapsulating a polymer substance such as a desired sense oligonucleotide or expressed protein are fused at 37 ° C. This membrane fusion liposome has a liposome-derived cavity inside,
It has a structure called a pseudovirus with the same spikes as the virus envelope on the outside. After purification by sucrose density gradient centrifugation, the membrane-fused liposome is adsorbed at 4 ° C. to the target cultured cells or tissue cells.
Then, at 37 ° C., the contents of the liposome are introduced into the cells, and the desired sense oligonucleotide can be introduced into the target cells. Here, the lipid used as the liposome is 50% (molar ratio) cholesterol, lecithin, and a synthetic phospholipid having a negative charge.
It is preferable to prepare and use unilamellar liposomes.

[0171] Methods for introducing a sense oligonucleotide into target cells using another liposome include:
A sense oligonucleotide introduction method using cationic liposomes can be mentioned. The method can be carried out according to the method of Yagi et al. [Yagi, K., et al., BBR
C., 196, 1042-1048 (1993)]. This method focuses on the fact that both plasmids and cells are negatively charged, and gives a positive charge to both the inside and outside of the liposome membrane to increase plasmid uptake by static electricity and enhance interaction with cells. Is what you do. The liposome used here is a multilamellar large liposome having a positive charge.
Large vesicles (MLV) is useful but large unilamellar vesicles (LUV)
And small unilamellar liposomes (small unilamellar vesicl
(es: SUV) can be used to create a complex with the plasmid to introduce the desired sense oligonucleotide.

An outline of the method for preparing plasmito-embedded cationic MLV is as follows.
(α-trimethylammonioacetyl) -didodecyl-D-glutamate
chloride), DLPC (dilauroyl phosphatidylcholin)
e) and DOPE (dioleoylphosphatidylethanolamin)
A chloroform solution (1 mM in lipid concentration) containing e) in a molar ratio of 1: 2: 2 is prepared. Next, a total amount of 1 μmol of lipid is put into a Spitz-type test tube, and chloroform is removed under reduced pressure using a rotary evaporator to prepare a lipid thin film. Further, chloroform is completely removed under reduced pressure and dried. Then, 0.5 ml of Dulbecco's phosphate buffered saline containing Mg and Ca containing 20 µg of the plasmid for gene transfer was added, and after replacement with nitrogen gas, the mixture was stirred with a vortex mixer for 2 minutes to give a desired sense oligo. A plasmid-embedded cationic MLV suspension containing nucleotides can be obtained.

One example of using the plasmid-embedded cationic MLV obtained above as a gene therapy agent is as follows:
For example, an expression plasmid incorporating the expression-purpose sense oligonucleotide is embedded in the above cationic MLV so as to have a DNA amount of 0.6 μg and a liposome lipid amount of 30 nmol, and this is suspended in 2 μl of a phosphate buffered saline. An example is a method of administering to target cells or a patient tissue extracted from a patient in a turbid state every other day.

Gene therapy is defined in the Ministry of Health and Welfare guidelines as "administration of a gene or a cell into which a gene is introduced into a human body for the purpose of treating a disease". However, the gene therapy according to the present invention means, in addition to the definition of the guideline, only the treatment of various diseases such as cancer due to the anti-angiogenic effect by introducing the DNA of the sense oligonucleotide into the target cells described above. In addition, the method also includes introducing a gene serving as a label or a cell into which a gene serving as a label has been introduced into a human body.

In the gene therapy of the present invention, the method for introducing a desired gene into a target cell or target tissue typically includes the following two methods.

In the first method, target cells are collected from a patient to be treated, and the cells are cultured outside the body, for example, under the addition of interleukin-2 (IL-2) or the like. Is a method (ex vivo method) of re-transplanting the obtained cells after introducing the target sense oligonucleotide contained in the above. The method is suitable for treating, for example, a genetic disease or cancer caused by a defective gene.

The second method is a direct gene transfer method (direct method) in which a sense oligonucleotide of interest is directly injected into a target site such as a patient's body or tumor tissue.

The first method of the gene therapy is performed in more detail, for example, as follows. That is, mononuclear cells collected from a patient are collected from monocytes using a blood separator, and the collected cells are cultured in an appropriate medium such as AIM-V medium for about 72 hours in the presence of IL-2, Add the vector containing the sense oligonucleotide to be introduced. To increase the introduction efficiency of sense oligonucleotide,
After centrifugation at 32 ° C. for 1 hour and 2500 rpm in the presence of protamine, the cells may be cultured at 37 ° C. for 24 hours under a 10% carbon dioxide gas condition. After repeating this operation several times, I
Cultured for 48 hours in an AIM-V medium or the like in the presence of L-2,
The cells are washed with saline, the number of viable cells is calculated, and the efficiency of sense oligonucleotide introduction is determined by the in situ PCR.
Alternatively, for example, if the desired target is an enzyme activity, the degree of the activity is measured to confirm the effect of introducing the desired sense oligonucleotide.

[0179] In addition, the safety is checked by culturing bacteria and fungi in the cultured cells, the presence or absence of mycoplasma infection, and the search for endotoxin. After confirming the safety, the expected effective dose of sense oligonucleotide is determined. The introduced cultured cells are returned to the patient by intravenous drip. Gene therapy is performed by repeating such a method at intervals of several weeks to several months, for example.

Here, the dose of the viral vector is appropriately selected depending on the target cells to be introduced. Generally, the virus titer is, for example, 1 × 1 for 1 × 10 ⁸ target cells.
It is preferable to adopt a dose in the range of 0 ³ cfu to 1 × 10 ⁸ cfu.

As an alternative to the first method, a virus-producing cell containing a retroviral vector containing a target sense oligonucleotide and, for example, a patient's cell are co-cultured, and the target cell is subjected to the sense oligonucleotide. Can be adopted.

In carrying out the second method (direct method) of gene therapy, it is determined whether or not the target sense oligonucleotide is actually introduced by the gene transfer method by a preliminary experiment especially in vitro.
It can be confirmed by PCR method or in situ PCR method, or it can be confirmed that the desired therapeutic effect based on the introduction of the target sense oligonucleotide, such as an increase in specific activity, an increase in growth of target cells, or inhibition of growth. desirable. When a viral vector is used, a search for a replication-competent retrovirus or the like is performed by the PCR method, reverse transcriptase activity is measured, or a membrane protein (env) gene is subjected to PCR.
Needless to say, it is important to confirm the safety of the introduction of sense oligonucleotides during gene therapy, for example, by monitoring with a method.

In the gene therapy method of the present invention, particularly when targeting cancer or malignant tumor, after collecting cancer cells or perituclear cells from a patient and performing enzyme treatment or the like to establish cultured cells, for example, retrovirus Introduced into cancer cells targeting the desired sense oligonucleotide at G418
After screening in cells, the expression level of IL-12 or the like is measured (in vivo), then radiation treatment is performed, and a cancer treatment method inoculated into a patient's tumor or a para-tumor can be mentioned as an example.

The present invention also relates to a vector for introducing the sense oligonucleotide of the present invention or a cell into which a sense oligonucleotide of interest has been introduced as an active ingredient, which is used together with a pharmaceutically effective amount and a suitable nontoxic pharmaceutical carrier or diluent. A pharmaceutical composition or a pharmaceutical preparation (gene therapeutic agent) containing the same is also provided.

Pharmaceutical carriers that can be used in the pharmaceutical composition (pharmaceutical preparation) of the present invention include fillers, extenders, binders, humectants, disintegrants, and the like, which are usually used according to the use form of the preparation.
Diluents or excipients such as surfactants and lubricants can be exemplified, and these can be appropriately selected and used depending on the dosage unit form of the resulting preparation.

The dosage unit form of the pharmaceutical preparation of the present invention includes
The same ones as described in detail for the above-mentioned polypeptide preparation of the present invention can be mentioned, and can be appropriately selected from various forms depending on the purpose of treatment.

For example, a pharmaceutical preparation containing the sense oligonucleotide-introducing vector of the present invention can be infected with a virus including a retrovirus vector containing the sense oligonucleotide in a form in which the vector is embedded in a liposome. It is prepared in the form of cultured cells.

These are phosphate buffered saline (pH
7.4), can be prepared in a form mixed in Ringer's solution, injection for intracellular composition liquid, etc., or can be prepared in a form to be administered together with a substance such as protamine which enhances gene transfer efficiency. .

The administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like.

The amount of the active ingredient to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and may be determined according to the desired therapeutic effect, administration method, treatment period, patient age, gender and other conditions. It is appropriately selected from a wide range.

In general, the dosage of a retroviral vector containing the desired sense oligonucleotide as a pharmaceutical preparation is from about 1 × 10 ³ pfu to 1 × 10 ³ pfu / kg body weight per day, for example, as a retrovirus titer.
^It is good to be about ¹⁵ pfu.

In the case of cells into which the desired sense oligonucleotide for introduction has been introduced, 1 × 10 ⁴ cells / body
It is suitably selected from the range of about 1 × 10 ¹⁵ cells / body.

The preparation may be administered once or several times a day, or may be administered intermittently at intervals of one to several weeks. Preferably, it can be co-administered with a substance such as protamine which enhances gene transfer efficiency or a preparation containing the substance.

When the gene therapy according to the present invention is applied to the treatment of cancer, the above-described various gene therapies can be appropriately combined (combined gene therapy). , Radiation therapy, immunotherapy and the like. Furthermore, the gene therapy of the present invention can be carried out with reference to NIH guidelines including its safety [Recombinant DNA Advi
sory Committee, Human Gene Therapy, 4 ,, 365-389 (19
93)].

Also according to the present invention, to detect the presence of the TSA2306 gene, a biological sample, such as blood or serum, is prepared, nucleic acids are extracted if desired, and analyzed for the presence of the TSA2306 gene. It is possible.

The detection method is, for example, the method of
An NA fragment is generated and designed to be used for screening and / or amplification of the TSA2306 gene. More specifically, for example, those having a property as a probe in plaque hybridization, colony hybridization, southern blotting, northern blotting, and the like, and those amplified by the polymerase chain reaction (PCR) in which a nucleic acid sequence is amplified with a polymerase. A probe having properties as a probe for obtaining a DNA fragment of the invention gene in whole or in part can be prepared. For this purpose, first, a primer having the same sequence as the TSA2306 gene is prepared, and this is used as a screening probe. The primer is reacted with a biological sample (nucleic acid sample) to confirm the presence of the TSA2306 gene sequence. it can. The nucleic acid sample may be prepared by various methods that facilitate detection of the target sequence, such as denaturation, restriction digestion, electrophoresis or dot blotting.

The screening method includes, in particular,
From the viewpoint of sensitivity, it is preferable to use the CR method.
The method is not particularly limited as long as the method uses a fragment of the gene of the present invention as a primer, and conventionally known methods (Science, 230 ,
1350-1354 (1985)) and a newly developed or future modified PCR method (Yoshiyuki Sakaki, et al., Ed., Yodosha, Experimental Medicine, extra edition, 8 (9) (1990); Enzymes, extra editions, Kyoritsu Shuppan Co., Ltd., 35 (17) (1990)) can be used.

The DNA fragment used as the primer is a chemically synthesized oligo DNA.
NA can be synthesized using an automatic DNA synthesizer or the like, for example, a DNA synthesizer (Pharmacia LKB Gene Assembler Plus: manufactured by Pharmacia). The length of the synthesized primer (sense primer or antisense primer) is preferably about 10 to 30 nucleotides. The probe used for the above screening is usually a labeled probe, but may be unlabeled or may be detected by specific binding to a directly or indirectly labeled ligand. Suitable labels, and methods for labeling probes and ligands, include:
Known in the prior art of the present invention, for example, Nick
These techniques include radiolabels, biotin, fluorescent groups, chemiluminescent groups, enzymes, antibodies, and the like, which can be incorporated by known methods such as translation, random priming, kinase treatment, and the like.

The PCR method used for detection is, for example, the RT-PCR method, but various modifications used in the art can be applied.

Further, the above-mentioned measuring method is based on the method of measuring TSA2306 in a sample.
By using a reagent kit for gene detection, it can be easily carried out.

Accordingly, the present invention also provides a reagent kit for detecting the TSA2306 gene containing the DNA fragment of the gene of the present invention.

The reagent kit comprises at least SEQ ID NO: 2
If a DNA fragment that hybridizes to a part or all of the base sequence shown in or a complementary base sequence thereof is contained as an essential component, a labeling agent, P
Indispensable reagents for CR method (for example, Taq DNA polymerase, deoxynucleotide triphosphate, primer, etc.)
May be included.

Examples of the labeling agent include a radioactive isotope or a chemically modified substance such as a fluorescent substance.
The fragment itself may be conjugated with the labeling agent in advance. Further, the reagent kit may contain a suitable reaction diluent, a standard antibody, a buffer, a detergent, a reaction stop solution, etc., for the benefit of performing the measurement.

Further, the present invention also provides a method for diagnosing cancer using the above-mentioned measuring method, a diagnostic agent used for the method, and a diagnostic kit.

Further, by using the above method, the sequence of the TSA2306 gene obtained from the test sample can be directly or indirectly sequenced to obtain the wild-type TSA2306 gene.
Related genes related to the new TSA2306 gene, which is a homologue highly homologous to the gene, can be found.

[0206] Therefore, the present invention provides a human T cell in a test sample by such measurement and sequencing of the TSA2306 gene in the test sample.
The present invention also provides a method for screening a related gene related to the SA2306 gene.

The protein encoded by the human TSA2306 gene of the present invention (polypeptide having the amino acid sequence represented by SEQ ID NO: 1) or one or several amino acids in SEQ ID NO: 1 are deleted, substituted or added. By using a polypeptide having the amino acid sequence or a fragment thereof, and using an antibody against each of these proteins, it becomes possible to measure wild-type TSA2306 and / or mutant TSA2306.

Therefore, the present invention provides a method for assaying an antibody or antigen for wild-type TSA2306 and / or mutant TSA2306. According to the measurement method, it is also possible to detect the degree of damage of the neoplastic state or the degree of malignancy of the malignant tumor based on the change of the wild-type TSA2306 (polypeptide). Such alterations can also be detected by analysis of the sequence of TSA2306 by conventional techniques in the art, and more preferably, using antibodies (polyclonal or monoclonal antibodies) to determine differences in TSA2306 polypeptides or TSA2306 polypeptides.
It can be detected by detecting the presence or absence of the 306 polypeptide.

A specific example of the measurement of the wild type and / or mutant TSA2306 is described below. That is, the TSA2306 antibody immunoprecipitates the TSA2306 polypeptide from a solution containing a biological material sample collected from a human such as blood or serum, and reacts with the TSA2306 polypeptide on a Western blot or immunoblot of a polyacrylamide gel. it can. The TSA2306 antibody can also detect TSA2306 polypeptide in paraffin or frozen tissue sections using immunohistochemical techniques. Since antibody production technology and purification technology are well known in the art, these technologies can be appropriately selected.

More preferred embodiments related to the method for detecting wild-type TSA2306 or a mutant thereof include enzyme-linked immunosorbent assay (ELISA), including a sandwich method using monoclonal and / or polyclonal antibodies, radioimmunoassay. Assays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA).

[0211] The present invention can also provide a TSA2306 receptor present on the cell membrane fraction or the cell surface having binding activity to the TSA2306 polypeptide. The TSA2306 receptor is obtained by conjugating a labeled TSA2306 polypeptide in a biomaterial sample containing a cell membrane fraction, extracting and isolating a binding reaction, purifying, and specifying the amino acid sequence of the isolate. Achieved. Obtaining and sequencing the TSA2306 receptor polypeptide will be apparent to those skilled in the art.

The present invention also provides a method for screening a compound (TSA2306 receptor) using a TSA2306 receptor binding reactant or a binding fragment thereof for any of a variety of drug screening techniques.
2306 receptor reactant: The compound can be screened for low molecular weight compounds, high molecular weight compounds, proteins, protein partial fragments, antigens, antibodies, etc.). Preferably, the TSA2306 receptor is utilized. The TSA2306 receptor polypeptide or fragment thereof used in such a screening test may be free in solution attached to a solid support or carried on the cell surface.

As an example of drug screening, for example, a prokaryotic or eukaryotic host cell stably transformed with a recombinant protein expressing a TSA2306 polypeptide or a fragment thereof is used, preferably in a competitive binding assay. can do. Such cells, either in free or fixed form, can also be used in standard binding assays. More specifically, the formation of a complex between the TSA2306 receptor polypeptide or fragment thereof and the substance to be tested is measured, and the complex between the TSA2306 receptor polypeptide or fragment thereof and the TSA2306 polypeptide or fragment thereof is measured. Compounds can be screened by detecting the extent to which the formation of is inhibited by the substance being tested.

Thus, the present invention provides for contacting such a substance with a TSA2306 receptor polypeptide or a fragment thereof by methods known in the art,
Agent screening methods can be provided that measure for the presence of a complex between an A2306 receptor polypeptide or a fragment thereof, or for the presence of a complex between a TSA2306 receptor polypeptide or a fragment thereof and a ligand. In addition, TSA2306 receptor activity is measured to determine whether such substances can inhibit the TSA2306 receptor and thus regulate the activity of TSA2306 as defined above, e.g., cell growth or the ability to modulate protein-protein interactions or complex. Can be adjusted. In such a competitive binding assay, more specifically, the TSA2306 receptor polypeptide or a fragment thereof is labeled. Free TSA2306 receptor polypeptide or a fragment thereof is separated from that present in the protein: protein complex, and the amount of free (uncomplexed) label is determined by the amount of binding or T
SA2306 receptor: A measure of inhibition of TSA2306 polypeptide binding. Small peptides of the TSA2306 polypeptide (peptide mimetics) can be analyzed in this manner to determine those that have TSA2306 receptor inhibitory activity.

In the present invention, another method for drug screening is a screening method for a compound having an appropriate binding affinity for the TSA2306 receptor polypeptide. Is synthesized on a solid support, such as the surface of a plastic pin or other material, and then the peptide test compound is reacted with the TSA2306 receptor polypeptide,
An example of a method of washing and then detecting a reaction-bound TSA2306 receptor polypeptide using a known method (P
(CT Patent Publication No .: WO84-03564). Purified TSA2306 receptor can be coated directly onto plates used in the drug screening techniques described above. The antibody can be supplemented with a non-neutralizing antibody against the polypeptide to immobilize the TSA2306 receptor polypeptide on a solid phase.

The present invention is further directed to the use of competitive drug screening assays. According to this, TSA230
6. A test compound competes with a neutralizing antibody capable of specifically binding to the TSA2306 receptor polypeptide for binding to the receptor polypeptide or a fragment thereof. The competition by the antibody makes it possible to detect the presence of any peptide having one or more antigenic determinants of the TSA2306 receptor polypeptide.

Further, with respect to drug screening, additional methods include the use of host eukaryotic cell lines or cells that contain a non-functional TSA2306 gene. After growing a host cell line or cell in the presence of a drug compound for a period of time, the rate of growth of the host cell is measured to determine whether the compound is capable of regulating cell growth or protein-
To confirm whether it is possible to regulate protein mutual binding or complex forming ability. As one means of measuring the growth rate,
It is also possible to measure the biological activity of the TSA2306 receptor.

Also according to the present invention, to develop more active or stable forms of TSA2306 polypeptide derivatives or agents, for example, to enhance or interfere with the function of TSA2306 polypeptide in vivo. Interact with a biologically active polypeptide of interest or a structural analog thereof, such as a TSA2306 agonist, TSA
2306 antagonists, TSA2306 inhibitors and the like can be made. The structural analog is, for example,
The three-dimensional structure of a complex of a TSA2306 polypeptide and another protein can be determined by X-ray crystallography, computer modeling, or a combination thereof.
Information on the structure of the structural analog can also be obtained by modeling a polypeptide based on the structure of a homologous protein.

More active or stable forms of TSA2
As a method for obtaining the 306 polypeptide derivative, analysis can be performed by, for example, alanine scanning.
The method involves substituting Ala for an amino acid residue and measuring each of the amino acid residues of the peptide in a manner that measures its effect on the activity of the peptide, thus determining regions important for the activity and stability of the peptide. Is the way. By this method, more active or stable TSA2306 polypeptide derivatives can be designed.

It is also possible to isolate the target-specific antibody selected by the functional assay and then analyze its crystal structure. In principle, this approach allows the pharmacoma (pharmac
ore). By generating anti-idiotypic antibodies to functional pharmacologically active antibodies, it is possible to identify and isolate peptides from a bank of chemically or biologically generated peptides. Therefore, it is expected that the selected peptide can also act as a pharmacore.

Thus, a drug having improved TSA2306 activity or stability or action as an inhibitor, agonist, antagonist or the like of TSA2306 activity can be designed and developed.

Further, according to the present invention, by producing a TSA2306 gene-containing knockout mouse (mutant mouse), which site of the TSA2306 gene sequence affects various TSA2306 activities in vivo as described above, TSA
The function of the 2306 gene product and the modified TSA2306 gene product in vivo can be confirmed.

This method is a technique for intentionally modifying the genetic information of an organism using a homologous recombinant gene, and can be exemplified by a method using mouse embryonic stem cells (ES cells) (Capeccchi). , MR, Science, 244 , 1288-1292 (198
9)).

The method for producing the above mutant mouse is already a common technique for those skilled in the art,
Mutant mice can be easily prepared by adapting human wild type TSA2306 gene and mutant TSA2306 gene to (Tetsuo Noda, Experimental Medicine, extra edition, 14 (20) (1996), Youtosha). Therefore, by adaptation of the above technique, a drug having improved TSA2306 activity or stability or an action as an inhibitor, agonist, antagonist or the like of TSA2306 activity can be designed and developed.

[0225]

According to the present invention, a novel TSA2306 gene having an angiogenesis inhibitory action is provided. The gene of the present invention has similarity to angiopoietin 2 and is specifically expressed in liver and kidney, and its expression is not observed in cancer tissues of liver and kidney. It is considered to have an effect of suppressing the growth of these tissues and an effect of suppressing the metastasis of these tissues to cancer. Further, the gene of the present invention is useful for gene therapy and as a gene therapy agent.

According to the provision of the gene of the present invention, a large amount of the polypeptide encoded by the gene can be produced by genetic engineering. According to the provision of the polypeptide, TSA230 can be produced.
Functions such as 6-activity and TSA2306 polypeptide binding activity can also be examined.

The TSA2306 polypeptide is useful for elucidation, diagnosis, treatment, and the like of diseases associated with the gene of the present invention and its expression product (eg, diseases and cancers related to angiogenesis in the liver and kidney, particularly cancer). .

According to the present invention, there are further provided a sense oligonucleotide (polynucleotide) of the TSA2306 gene, a gene transfer vector containing the same, for gene therapy,
A cell into which the sense oligonucleotide has been introduced and a gene therapy agent comprising the vector or the cell as an active ingredient,
In addition, a gene therapy method using the same is provided.

Further, according to the present invention, the active ingredient has an inhibitory effect on the progression of liver and kidney cancers and metastasis, and is used as an active ingredient in a sense oligonucleotide used for treating these cancer diseases and conditions. Can also be provided.

[0230]

The present invention will now be described in further detail with reference to examples.

[0231]

EXAMPLE 1 (1-1) [.gamma. ³³ P] exposed labeled with [.gamma. ³³ P] ATP to confirm the human gene expressed in expression method tissue-specific technique labeled with ATP The method was used. The procedure of the method is essentially as follows:
(Liang P., et al., Science, 257 , 967-971 (1992)).

That is, 13 human tissues (adult brain, fetal brain,
Lung, liver, stomach, pancreas, spleen, mammary gland, prostate, placenta, testis, kidney and heart: 0.2 μg of polyARNA isolated from each of Mix with 25 pmol of 3'-Uncard oligo dT primer G (T) 15MA (M is a mixture of G, A and C)
Heated for minutes. 4 μl of 5 × first strand buffer (BRL), 2 μl of 0.1 MDT
T (BRL), 1 μl of 250 mM dNTPs (B
RL), 1 μl ribonuclease inhibitor (40 units; TOYOBO) and 1 μl Superscript II reverse transcriptase (200 units; BRL)
Was added. The final volume of each reaction was 20 μl. After culturing each solution at 37 ° C. for 1 hour, the solution was diluted by a factor of 2.5 by adding 30 μl of distilled water, and -20 until use.
Stored at ° C.

The cDNA was amplified by PCR in the presence of [γ- ³³ P] ATP-labeled (Amersham) 3′-uncarded primer. P of this cDNA
CR amplification was performed as follows.

That is, each 20 μl of the PCR mixture was
1 RT reaction mixture, 2 μl of 10 × PCR buffer (Takara), 4 μl of 2.5 mM dNTPs, 0.25
μl of Ex Taq DNA polymerase (5 units / ml:
Takara Co., Ltd.), were labeled with [γ- ³³ P] ATP 25pmol
3'-Uncard oligo-dT primer and 2
It contained 5 pmol of 5'-primer (10-mer deoxyoligonucleotide primer of sequence shown in SEQ ID NO: 4). The PCR was performed under the following conditions.
That is, one cycle of 95 ° C. for 3 minutes, 40 ° C. for 5 minutes and 72 ° C. for 5 minutes was performed, and then 95 ° C. for 0.1 minute.
40 cycles of 5 minutes, 2 minutes at 40 ° C. and 1 minute at 72 ° C. were performed, and finally, the reaction was performed at 72 ° C. for 5 minutes.

The PCR reaction samples were extracted with ethanol, resuspended in formamide sequencing dye, and reacted on a 6% acrylamide 7.5M urea sequencing gel. The gel was dried without fixing and autoradiography was performed overnight. (1-2) Sub-cloning of amplified cDNA fragment A radioactive ink is previously marked on 3MM filter paper on which a dried gel has been placed, and this is combined with an autoradiogram to obtain a band containing the target cDNA. Is cut out together with the 3MM filter paper,
The mixture was stirred for 1 hour with 1 dH ₂ O. After removing the polyacrylamide gel and the filter paper,
μl of 10 mg / ml glycogen and 0.3 M NaOA
c in the presence of c.
Redissolved in μl dH ₂ O. 5 μl for reamplification
Of this solution was used. The PCR conditions and primers were the same as for the first PCR. The reamplified product of an appropriate size was recovered again as the first PCR product, and the PCR product was then transferred to the pUC118 vector (Takara).
Cloned at the Hinc II site. The nucleic acid sequence is ABI
It was determined by a 377 automatic sequencer (manufactured by Applied Biosystems).

As a result of comparing different expression patterns using mRNAs isolated from 16 human tissues by the above method, one PCR product specifically expressed in the lung was confirmed. This was named TSA2306DD.

This product consisted of 1461 nucleotides. FASTA Program (Person WR, et a
l., Proc. Natl. Acad. Sci., USA, 85 , 2444-2448 (19
Comparison of the DNA sequence with the DNA sequence in the GenBank / EMBL database using 88)) revealed that the PCR product had no homology to any other known DNA sequence. (1-3) Screening of cDNA A human normal liver cDNA library was constructed using oligo (dT) + random hexamer-primed human normal liver cDNA and Uni-ZAPTMXR (manufactured by Stratagene). The whole of 1 × 10 ⁶ clones were isolated by the above method, the screening was carried out using cDNA fragments labeled with [.gamma. ³³ P] -dCTP. Positive clones were selected and their inserted cDNA was
cript I cut it out in vivo in IISK (-).

As a result, the above TSA2306DD was approximately 1
00 plaques were identified. From these results, the amount of transcription between the total RNAs was calculated to be about 0.01%. TS
Assembled cDNA sequence homologous to A2306DD (TSA2306)
Contained 1461 nucleotides, including an open reading frame of 1380 nucleotides, encoding a 460 amino acid protein with a calculated molecular weight of 53637 Da.

From the primary sequence, the product of this gene (TSA2306
Protein) has a signal peptide-like sequence at the N-terminus, and was found to be a secreted protein.

The location on the chromosome is determined by the
1.3-p32.1

The gene of the present invention, TSA2306, has about 33% homology with angiopoietin 2 which is known to suppress angiogenesis and angiopoietin 1 which is known to promote angiogenesis, respectively. Had. (2) Expression in Tissues To examine the expression profile of TSA2306 in tissues, Northern blot analysis was performed using various human tissues.

For the Northern blot analysis, human MTN was used.
(Multiple-Tissue Northern) Blots I and II (Clontech) were used. The cDNA fragment consists of T3 and T
PC using a primer set of 7 promoter sequences
Labeled with [γ- ³³ P] -dCTP by R. The membrane containing the amplification product was pre-hybridized (the conditions were in accordance with the protocol of the product), and then hybridization was performed in accordance with the protocol of the product.

After hybridization, the washed membrane was exposed to autoradiography at -80 ° C for 24 hours. The result is as shown in FIG.

In the figure, the human tissues used were a heart, a brain, a placenta, and a lung.
g), liver (Liver), skeletal muscle (S. muscle), kidney (Kid
ney), pancreas (Pancreas), spleen (Spleen), thymus (Thy
mus), prostate (Prostate), testis (Testis), ovary (O
vary), small intestine, colon and peripheral blood leukocyte; PB
L.).

[0245] From the figure, a transcript homologous to TSA2306 was specifically observed in liver (liver) and kidney (kidney). (3) FISH FISH for chromosome alignment can be performed by a known method (Takahas
hi E., et al., Hum. Genet., 86 , 14-16 (1990)) using 0.5 μg of each cosmid DNA as a probe. FISH was captured by Provia 100 film (Fuji, ISO 100) or by a CCD camera system (Applied Imaging, Site Vision).

As a result, 100 typical R-bands
The signal examined in (pre) metaphase cells was localized to the chromosome 1 band p31.3-p32.1. Therefore, the chromosomal localization site of TSA2306 is 1p31.3-p
32.1 could be identified. (4) Expression of Transcripts in Cancer Cell Lines and Cancer Tissues by RT-PCR Analysis HuH 6 Clone5 (Life Sciences, 43 , 123)
3-1240 (1988)), HuH 7 (Cancer Res., 42 , 3858-3863)
(1982)), HLE (Gann, 66 , 385-392 (1975)) and P
LC (Sa Medical Journal, 50 , 509-515 (1976)) and renal cell carcinoma lines; Caki 1 (J. Natl. Cancer Int., 58 , 209-214).
(1977), VMRC-RCW (Catalog number JCRB0813, Human Science Foundation, Osaka), G402 (ATCC deposit number; CR
L-1440).

That is, 10 μl of total RNA isolated from cell lines and cancer tissues using ISOGEN (manufactured by Wako) was converted into 10 units of RNase-free DNase I.
(Boehringer Mainheim) for 15 minutes, extracted twice with phenol-chloroform, and precipitated with ethanol. The single-stranded cDNA was converted to Superscript ITMRN using oligo d (T) and random primers.
It was synthesized by aseH-reverse transcriptase (manufactured by Life Technology). 2 μl of each product was used for PCR amplification.

The primers P1 and P2 having the nucleotide sequences shown as SEQ ID NO: 5 and SEQ ID NO: 6 were used for 25 cycles of PCR amplification.

The PCR reaction was performed for 25 ng cDNA, 10
μM each primer, 2.5 mM dNTP and 0.25
The test was performed in a 20 μl solution containing U Extaq DNA polymerase (Takara). PCR products were dissolved in a 1.5% agarose gel stained with ethidium bromite.

As described above, normal liver and normal kidney tissues and seven cell lines (cell lines; lane 1 = normal liver tissue, lane 2 = HuH 6 Clone 5, lane 3 = HuH 7, lane 4 = HLE, lane 5 = PLC, lane 6 = normal kidney tissue,
The results of RT-PCR analysis of lane 7 = Caki 1, lane 8 = VMRC-RCW, lane 9 = G402) are as shown in FIG.

As can be seen from the figure, TSA2306 expression is known to be high in malignancy in HLE and PLC in hepatocyte cell lines.
It was confirmed that the expression was lost in all the cases of renal cell carcinoma investigated (lane 4 and lane 5 and lane 7 to lane 9). (5) Expression of TSA2306 gene in various cancers (Northern blot analysis) The expression of TSA2306 gene was determined in four cases each of RNA extracted from the same individual liver cancer tissue (T) and normal liver tissue (N) shown below (Invitrogen FIG. 3 shows the results of Northern blot analysis using the method of the present invention. From this figure, it was confirmed that the expression of the TSA2306 gene was lost in two cases of liver cancer tissues.

[0252] From these results, it is expected that the TSA2306 gene may act in a suppressive manner on the progress of cancer and function as a suppressor of angiogenesis.

[0253]

[Sequence List] SEQUENCE LISTING <110> Otsuka Pharmaceutical Co., Ltd. <120> TSA2306 gene <130> 1069JP <140> <141> <160> 6 <170> PatentIn Ver. 2.0 <210> 1 <211> 460 <212> PRT <213> Human normal lung cDNA library <400> 1 Met Phe Thr Ile Lys Leu Leu Leu Phe Ile Val Pro Leu Val Ile Ser 1 5 10 15 Ser Arg Ile Asp Gln Asp Asn Ser Ser Phe Asp Ser Leu Ser Pro Glu 20 25 30 Pro Lys Ser Arg Phe Ala Met Leu Asp Asp Val Lys Ile Leu Ala Asn 35 40 45 Gly Leu Leu Gln Leu Gly His Gly Leu Lys Asp Phe Val His Lys Thr 50 55 60 Lys Gly Gln Ile Asn Asp Ile Phe Gln Lys Leu Asn Ile Phe Asp Gln 65 70 75 80 Ser Phe Tyr Asp Leu Ser Leu Gln Thr Ser Glu Ile Lys Glu Glu Glu 85 90 95 Lys Glu Leu Arg Arg Thr Thr Tyr Lys Leu Gln Val Lys Asn Glu Glu 100 105 110 Val Lys Asn Met Ser Leu Glu Leu Asn Ser Lys Leu Glu Ser Leu Leu 115 120 125 Glu Glu Lys Ile Leu Leu Gln Gln Lys Val Lys Tyr Leu Glu Glu Gln 130 135 140 Leu Thr Asn Leu Ile Gln Asn Gln Pro Glu Thr Pro Glu His Pro Glu 145 150 155 160 Val Thr Ser Leu Lys Thr Phe V al Glu Lys Gln Asp Asn Ser Ile Lys 165 170 175 Asp Leu Leu Gln Thr Val Glu Asp Gln Tyr Lys Gln Leu Asn Gln Gln 180 185 190 His Ser Gln Ile Lys Glu Ile Glu Asn Gln Leu Arg Arg Thr Ser Ile 195 200 205 Gln Glu Pro Thr Glu Ile Ser Leu Ser Ser Lys Pro Arg Ala Pro Arg 210 215 220 Thr Thr Pro Phe Leu Gln Leu Asn Glu Ile Arg Asn Val Lys His Asp 225 230 235 240 Gly Ile Pro Ala Glu Cys Thr Thr Ile Tyr Asn Arg Gly Glu His Thr 245 250 255 Ser Gly Met Tyr Ala Ile Arg Pro Ser Asn Ser Gln Val Phe His Val 260 265 270 Tyr Cys Asp Val Ile Ser Gly Ser Pro Trp Thr Leu Ile Gln His Arg 275 280 285 285 Ile Asp Gly Ser Gln Asn Phe Asn Glu Thr Trp Glu Asn Tyr Lys Tyr 290 295 300 Gly Phe Gly Arg Leu Asp Gly Glu Phe Trp Leu Gly Leu Glu Lys Ile 305 310 315 320 Tyr Ser Ile Val Lys Gln Ser Asn Tyr Val Leu Arg Ile Glu Leu Glu 325 330 335 Asp Trp Lys Asp Asn Lys His Tyr Ile Glu Tyr Ser Phe Tyr Leu Gly 340 345 350 Asn His Glu Thr Asn Tyr Thr Leu His Leu Val Ala Ile Thr Gly Asn 355 360 365 Val Pro Asn Ala Ile Pro Glu Asn L ys Asp Leu Val Phe Ser Thr Trp 370 375 380 380 Asp His Lys Ala Lys Gly His Phe Asn Cys Pro Glu Gly Tyr Ser Gly 385 390 395 400 400 Gly Trp Trp Trp His Asp Glu Cys Gly Glu Asn Asn Leu Asn Gly Lys 405 410 415 Tyr Asn Lys Pro Arg Ala Lys Ser Lys Pro Glu Arg Arg Arg Gly Leu 420 425 430 Ser Trp Lys Ser Gln Asn Gly Arg Leu Tyr Ser Ile Lys Ser Thr Lys 435 440 445 Met Leu Ile His Pro Thr Asp Ser Glu Ser Phe Glu 450 455 460 <210> 2 <211> 1383 <212> DNA <213> Human normal lung cDNA library <400> 2 aattaatgac atatttcaaa aactcaacat atttgatcag 240 tctttttatg atctatcgct gcaaaccagt gaaatcaaag aagaagaaaa ggaactgaga 300 agaactacat ataaactaca agtcaaaaat gaagagg tta agaag attagac attagaact gagacta 360 aattcaa aatcaacctg aaactccaga acacccagaa 480 gtaacttcac ttaaaacttt tgtagaaaaa caagataata gcatcaaaga ccttctccag 540 accgtggaag accaatataa acaattaaac caacagcata gtcaaataaa agaaatagaa 600 aatcagctca gaaggactag tattcaagaa cccacagaaa tttctctatc ttccaagcca 660 agagcaccaa gaactactcc ctttcttcag ttgaatgaaa taagaaatgt aaaacatgat 720 ggcattcctg ctgaatgtac caccatttat aacagaggtg aacatacaag tggcatgtat 780 gccatcagac ccagcaactc tcaagttttt catgtctact gtgatgttat atcaggtagt 840 ccatggacat taattcaaca tcgaatagat ggatcacaaa acttcaatga aacgtgggag 900 aactacaaat atggttttgg gaggcttgat ggagaatttt ggttgggcct agagaagata 960 tactccatag tgaagcaatc taattatgtt ttacgaattg agttggaaga ctggaaagac 1020 aacaaacatt atattgaata ttctttttac ttgggaaatc acgaaaccaa ctatacgcta 1080 catctagttg cgattactgg caatgtcccc aatgcaatcc cggaaaacaa agatttggtg 1140 ttttctactt gggatcacaa agcaaaagga cacttcaact gtccagaggg ttattcagga 1200 ggctggtggt ggcatgatga gtgtggagaa aacaacctaa atggtaaata taacaaacca 1260 agagcaaaat ctaagccaga gaggagaaga ggattat ctt ggaagtctca aaatggaagg 1320 ttatactcta taaaatcaac caaaatgttg atccatccaa cagattcaga aagctttgaa 1380 tga 1383 <210> 3 <211> 1461 <212> DNA <213> Human normal lung cDNA library <220> <221> CDS <222> (28) .. (1407) ) <400> 3 tgcttgaaat tgaaaatcaa gataaaa atg ttc aca att aag ctc ctt ctt ttt 54 Met Phe Thr Ile Lys Leu Leu Leu Phe 15 5 att gtt cct cta gtt att tcc tcc aga att gat caa gac aat tca Leca 102 Val Val Ile Ser Ser Arg Ile Asp Gln Asp Asn Ser Ser 10 15 20 25 ttt gat tct cta tct cca gag cca aaa tca aga ttt gct atg tta gac 150 Phe Asp Ser Leu Ser Pro Glu Pro Lys Ser Arg Phe Ala Met Leu Asp 30 35 40 gat gta aaa att tta gcc aat ggc ctc ctt cag ttg gga cat ggt ctt 198 Asp Val Lys Ile Leu Ala Asn Gly Leu Leu Gln Leu Gly His Gly Leu 45 50 55 aaa gac ttt gtc cat aag acg aag ggc caa att aat gac ata ttt caa 246 Lys Asp Phe Val His Lys Thr Lys Gly Gln Ile Asn Asp Ile Phe Gln 60 65 70 aaa ctc aac ata ttt gat cag tct ttt tat gat cta tcg ctg caa acc 294 Lys Leu Asn Ile Phe Asp Gln Ser Phe Tyr Asp Leu Ser Leu Gln Thr 75 80 85 agt gaa atc aaa gaa gaa gaa aag gaa ctg aga aga act aca tat aaa 342 Ser Glu Ile Lys Glu Glu Glu Lys Glu Leu Arg Arg Thr Thr Tyr Lys 90 95 100 105 cta caa gtc aaa aat gaa gag gta aag aat atg tca ctt gaa ctc aac 390 Leu Gln Val Lys Asn Glu Glu Val Lys Asn Met Ser Leu Glu Leu Asn 110 115 120 tca aaa ctt gaa agc ctc cta gaa gaa aaa att cta ctt caa caa Ser Lys Leu Glu Ser Leu Leu Glu Glu Lys Ile Leu Leu Gln Gln Lys 125 130 135 gtg aaa tat tta gaa gag caa cta act aac tta att caa aat caa cct 486 Val Lys Tyr Leu Glu Glu Gln Leu Thr Asn Leu Ile Gln Asn Gln Pro 140 145 150 gaa act cca gaa cac cca gaa gta act tca ctt aaa act ttt gta gaa 534 Glu Thr Pro Glu His Pro Glu Val Thr Ser Leu Lys Thr Phe Val Glu 155 160 165 aaa aaa caa gat aat agc atc aaa gac ctt ctc cag acc gtg gaa gac caa 582 Lys Gln Asp Asn Ser Ile Lys Asp Leu Leu Gln Thr Val Glu Asp Gln 170 175 180 185 tat aaa aaa caa tta aac caa cag cat agt caa ata aaa gaa ata gaa aat 630 Tyr Lys Gln Leu Asn Gln Gln His Ser Gln Ile Lys Glu Ile Glu Asn 190 195 200 cag ctc aga agg act agt att caa gaa ccc aca gaa att tct cta tct 678 Gln Leu Arg Arg Thr Ser Ile Gln Glu Pro Thr Glu Ile Ser Leu Ser 205 210 215 tcc aag cca aga gca cca aga act act ccc ttt ctt cag ttg aat gaa 726 Ser Lys Pro Arg Ala Pro Arg Thr Thr Pro Phe Leu Gln Leu Asn Glu 220 225 230 ata aga aat gta aaa cat gat ggc att cct gct gaa tgt acc acc att 774 Ile Arg Asn Val Lys His Asp Gly Ile Pro Ala Glu Cys Thr Thr Ile 235 240 245 tat aac aga ggt gaa cat aca agt ggc atg tat gcc atc aga ccc agc 822 Tyr Asn Arg Gly Glu His Thr Ser Gly Met Tyr Ala Ile Arg Pro Ser 250 255 260 265 aac tct caa gtt ttt cat gtc tac tgt gat gtt ata tca ggt agt cca 870 Asn Ser Gln Val Phe His Val Tyr Cys Asp Val Ile Ser Gly Ser Pro 270 275 280 tgg aca tta att caa cat cga ata gat gga tca caa aac ttc aat gaa 918 Trp Thr Leu Ile Gln His Arg Ile Asp Gly Ser Gln Asn Phe Asn Glu 285 290 295 acg tgg gag aac tac aaa tat ggt ttt ggg agg ctt gat gga gaa ttt Gp Trp 966 Thr T966 Asn Tyr Lys Tyr Gly Phe Gly Arg Leu Asp Gly Glu Phe 300 305 310 tgg ttg ggc cta gag aag ata tac tcc ata gtg aag caa tct aat tat 1014 Trp Leu Gly Leu Glu Lys Ile Tyr Ser Ile Val Lys Gln Ser Asn Tyr 315 320 325 gtt tta cga att gag ttg gaa gac tgg aaa gac aac aaa cat tat att 1062 Val Leu Arg Ile Glu Leu Glu Asp Trp Lys Asp Asn Lys His Tyr Ile 330 335 340 345 345 gaa tat tct ttt tac ttg gga aat cacga aac tat acg cta cat 1110 Glu Tyr Ser Phe Tyr Leu Gly Asn His Glu Thr Asn Tyr Thr Leu His 350 355 360 cta gtt gcg att act ggc aat gtc ccc aat gca atc ccg gaa aac aaa 1158 Leu Val Ala Ile Thr Gly Asn Val Pro Asn Ala Ile Pro Glu Asn Lys 365 370 375 gat ttg gtg ttt tct act tgg gat cac aaa gca aaa gga cac ttc aac 1206 Asp Leu Val Phe Ser Thr Trp Asp His Lys Ala Lys Gly His Phe Asn 380 385 390 tgt cca gag ggt tat tca gga ggc tgg tgg tgg cat gat gag tgt gga 1254 Cys Pro Glu Gly Tyr Ser Gly Gly Trp Trp Trp His Asp Glu Cys Gly 395 400 405 gaa aac aac cta aat ggt aaa tat aac aaa cca aga gca aaa tct a ag 1302 Glu Asn Asn Leu Asn Gly Lys Tyr Asn Lys Pro Arg Ala Lys Ser Lys 410 415 420 425 cca gag agg aga aga gga tta tct tgg aag tct caa aat gga agg tta 1350 Pro Glu Arg Arg Arg Gly Leu Ser Trp Lys Ser Gln Asn Gly Arg Leu 430 435 440 tac tct ata aaa tca acc aaa atg ttg atc cat cca aca gat tca gaa 1398 Tyr Ser Ile Lys Ser Thr Lys Met Leu Ile His Pro Thr Asp Ser Glu 445 450 455 agc ttt gaa tgaactgagg caaatt ttaaacatta 1447 Ser Phe Glu 460 acctcattcc aagt 1461 <210> 4 <211> <212> DNA <213> Primer sequence for PCR <400> 4 gatcaatcgc 10 <210> 5 <211> <212> DNA <213> P1 primer sequence for PCR <400> 5 gtgaagcaat ctaattatgt ttacgaa 27 <210> 6 <211> <212> DNA <213> P2 primer sequence for PCR <400> 6 agagtataac cttccatttt gagactt 27

[Brief description of the drawings]

FIG. 1 is a drawing substitute photograph showing the results of Northern blot analysis using various human tissues performed to examine the expression profile of TSA2306 in the tissues according to Example 1 (2).

FIG. 2 is a photograph as a substitute for a drawing, showing the results of RT-PCR analysis performed to examine the expression of transcripts in cancer cell lines and cancer tissues by RT-PCR analysis according to (1) of Example 1.

FIG. 3 shows TSA2306 in various cancers according to Example 1 (5).
3 is a photograph as a substitute of a drawing, showing the results of Northern blot analysis of gene expression.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/475 C07K 16/22 16/22 C12N 1/15 C12N 1/15 1/19 1/19 1/1 / 21 1/21 C12Q 1/68 A5 / 10 A61K 37/02 C12Q 1/68 C12N 5/00 A F term (reference) 4B024 AA01 AA11 AA12 BA21 CA04 CA09 CA12 CA20 DA01 DA02 DA05 DA11 DA12 EA04 GA11 HA13 HA14 HA17 4B063 QA01 QA19 QQ02 QQ43 QQ52 QQ53 QR08 QR32.

Claims (19)

[Claims] 1. A gene containing the following polynucleotide (a) or (b): (a) a polynucleotide encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or a complementary chain thereof, (b) A polynucleotide having at least 95% homology to a polynucleotide encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or a complementary strand thereof. 2. A gene encoding the following polypeptide (a) or (b): (a) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, and (b) a polypeptide having the amino acid sequence represented by (a). Alternatively, a polypeptide comprising an amino acid sequence in which a plurality of amino acids are deleted, substituted or added, and having angiogenesis inhibitory activity or antitumor activity. 3. A gene comprising the following polynucleotide (a) or (b): (a) a nucleotide sequence represented by SEQ ID NO: 2; (b) a polynucleotide having the above nucleotide sequence (a) and a stringent A polynucleotide that hybridizes under mild conditions. 4. The gene according to claim 1, which is a polynucleotide encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. 5. The gene according to claim 3, which is a polynucleotide having the nucleotide sequence of SEQ ID NO: 2. 6. A recombinant expression vector having the gene according to claim 1. 7. A host cell having the recombinant expression vector according to claim 6. 8. A polynucleotide comprising at least 10 consecutive nucleotide sequences in the nucleotide sequence represented by SEQ ID NO: 2. 9. The method according to claim 8, comprising at least 30 consecutive nucleotide sequences in the nucleotide sequence represented by SEQ ID NO: 2.
Polynucleotide according to 1. 10. A gene therapy agent comprising the polynucleotide according to claim 8 or 9 or a vector carrying the polynucleotide as an active ingredient. 11. A polynucleotide probe comprising the nucleotide sequence represented by SEQ ID NO: 2. 12. A polynucleotide probe comprising at least 10 continuous nucleotide sequences in the nucleotide sequence shown by SEQ ID NO: 2. 13. The polynucleotide probe according to claim 12, comprising at least 30 consecutive nucleotide sequences in the nucleotide sequence represented by SEQ ID NO: 2. 14. A polypeptide of the following (a) or (b): (a) a polypeptide of the amino acid sequence represented by SEQ ID NO: 1, (b) one or more amino acids in the amino acid sequence of the above (a) A polypeptide comprising an amino acid sequence in which is deleted, substituted or added, and having angiogenesis inhibitory activity or antitumor activity. 15. A gene expression product obtained by expressing the gene according to claim 2. 16. A pharmaceutical composition comprising the polypeptide according to claim 14 as an active ingredient. 17. The pharmaceutical composition according to claim 16, which is an angiogenesis inhibitor. 18. The pharmaceutical composition according to claim 16, which is an antitumor agent. (19) an antibody or a fragment thereof having specific binding to the polypeptide of (14);