JPH09255700A - VEGF binding polypeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a low mol.wt. VEGF(Vascular Endothelial cell Growth Factor) inhibitor capable of being utilized for the therapy of solid carcinoma and other vascularization-accompanying diseases. SOLUTION: This polypeptide comprising the first to third immunoglobulin- like domains, the first to fourth immunoglobulin-like domains or the first to fifth immunoglobulin-like domains in the extracellular region of a FLT acting as a VEGF receptor has a VEGF-inhibiting activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide useful as an angiogenesis inhibitor and a method for producing the same.

[0002]

2. Description of the Related Art It is known that some diseases are accompanied by pathological angiogenesis closely related to their symptoms and etiology. A typical disease among them is solid cancer, and in order for cancer tissue to grow over a diameter of 1 to 2 mm, it is necessary for new blood vessels to extend from existing blood vessels to reach cancer tissue (J. Folkman, J. .Natl. Cancer Inst., 82: 4 (199
0)), and when the blood vessels reach the cancer tissue, the proliferation of the cancer tissue is explosively accelerated. Also, diabetic retinopathy is accompanied by pathological neovascularization of the retina, which often causes blindness. Furthermore, in diseases such as rheumatoid arthritis, psoriasis, hemangioma, scleroderma, and neovascular glaucoma, pathological neovascularization is involved, which is one of the main symptoms (J. Folkman, N. Engle. J. Med., 320: 1211 (198
9)). Therefore, substances that inhibit angiogenesis may be useful for treating cancer and the aforementioned diseases.

Vascular endothelial cells are the cells that form the innermost layer of blood vessels. Angiogenesis is performed by vascular endothelial cells proliferating by being stimulated by growth factors, physiologically active substances, mechanical damage, or the like. BF as a growth factor that directly or indirectly stimulates the proliferation of vascular endothelial cells
GF (basic fibroblast growth factor), aFGF
(Acidic Fibroblast Growth Factor), VEGF (Vas
cular Endothelial cellGrowth Factor), PD-EC
GF (Platelet-Derived Endothelial Cell Growth Fac
tor), TNF-α (Tumour Necrosis Factor-α), P
DGF (Platelet Derived Growth Factor), EGF (E
pidermal Growth Factor), TGF-α (Transforming
Growth Factor-α), TGF-β (Transforming Grow
th Factor-β), HGF (Hepatocyte Growth Factor)
Is known (L. Diaz-Floreset al., Histol.Histo
path., 9: 807 (1994)). In particular, VEGF (vascular endothelial growth factor) can be distinguished from other growth factors in that it acts very specifically on vascular endothelial cells. In other words, VEGF
Is expressed only on a limited number of cells other than vascular endothelial cells.

VEGF is a glycoprotein having a molecular weight of 40,000 to 45,000 and exists as a dimer (PW Leung et al., S
cience 246: 1306 (1989), PJ Keck et al., Scienc
e: 246: 1319 (1989)). VEGF acts by binding to VEGF receptors, promoting cell proliferation and promoting membrane permeability.

The following reports suggest the relationship between VEGF and cancer. Many cancer cells secrete VEGF (S. Kondo et al., Bichem. Biophys. Res. Commu.
n., 194: 1234 (1993)). When a cancer tissue section is stained with an anti-VEGF antibody, the cancer tissue and surrounding new blood vessels are strongly stained (HF Dvorak et al., J. Exp. Med. 174:
1275 (1991)., LF Brown et al., Cancer Res., 53: 4.
727 (1993)). The growth of transplanted cancer is suppressed in mice in which one of the VEGF receptors is genetically inactivated (B. Millauer et al., Nature, 367: 576 (1994)).
Anti-VEGF neutralizing antibody shows antitumor activity against cancer-bearing mice (KJ Kim et al., Nature, 362: 841 (1993),
S. Kondo et al., Bichem. Biophys. Res. Commun., 19
4: 1234 (1993)). From the above facts, it is considered that VEGF secreted by cancer cells plays a major role in tumor angiogenesis.

The VEGF receptor is FLT in humans.
(M. Shibuya et al., Oncogene, 5: 519 (1990)) and K
DR (BI Terman et al., Bichem. Biophys. Res. C
ommun., 187: 1579 (1992)) are known.
As shown in FIG. 1, the extracellular region of FLT has a structure composed of seven immunoglobulin-like domains (C. DeVries et al., Science, 255: 989 (1992)). F
For LT, the soluble receptor cDNA has been cloned (RL Kendal and KA Thomas,
Proc. Natl. Acad. Sci. USA, 90: 10705 (199
3)). The polypeptide encoded by this cDNA is F
Of the seven immunoglobulin-like domains in the extracellular region of LT, it corresponds to the first to sixth immunoglobulin-like domains and has the same affinity as the original FLT.
And inhibited VEGF activity. Also for KDR, it is known that the first to sixth domains of the extracellular region expressed by genetic engineering bind to VEGF (R.
L. Kendal et al., Bichem. Biophys. Res. Commun., 2
01: 326 (1994)).

[0007]

Since the mouse anti-VEGF neutralizing monoclonal antibody exhibits antitumor properties, it can be expected that it can be used as an anticancer agent. However, when a mouse antibody is administered to a human, a human antibody against the mouse antibody may be produced, neutralized, or cause anaphylactic shock. To avoid this, chimerization of mouse antibodies (SL
Morrison et al., Proc. Natl. Acad. Sci. USA, 8
1: 6851 (1989)) or humanization, it is necessary to bring the amino acid sequence of the mouse antibody close to that of the human antibody while maintaining the neutralizing ability. This requires a high level of skill, knowledge, experience, and effort, and the results are not always successful on a case-by-case basis. Also, 100% cannot be humanized by this method. Other methods include immunization with transgenic mice that produce human antibodies themselves (S. Wagner et al., Nu.
cleic Acid Res., 22: 1389 (1994)), which also requires highly specialized technology.

As described above, the extracellular region of the VEGF receptor binds specifically to VEGF with high affinity and
Since it can inhibit EGF activity, it can be used as an angiogenesis inhibitor. Moreover, since the polypeptide is originally of human origin, it can be expected that the antibody appearance rate will be low even when administered to humans. However, it has been reported that when a polypeptide, which originally does not exist in large amounts in the body, is administered, it is metabolized very quickly. For example, the soluble half-life of the HIV receptor CD4 in blood is 15 minutes (DJ Capon et al., Nature, 337: 525 (198).
In the case of interferon γ, the half-life in blood was 30 minutes (I. Rutenfranz and H. Kirchner, J. Interfe).
ron Res., 8: 573 (1988)).

As a method for extending the blood half-life, a method is known in which a fusion polypeptide with a molecule having a long blood half-life such as an antibody molecule is genetically engineered and used.
In the case of CD4, the blood half-life was extended from 15 minutes to 48 hours when the polypeptide was fused to the Fc region of antibody IgG1 (DJ Capon et al., Nature, 337: 525 (19
89)). In addition, an effector function possessed by the antibody, that is, complement-dependent cytotoxicity (DB Amos et al., Trans
plantation, 7: 220 (1969)) and antibody-dependent cytotoxicity (AY Liu et al., Proc. Natl. Acad. Sci. U.
SA, 84: 3439 (1987)) can be expected to be effective. Furthermore, since it is dimerized via the Fc region, one molecule can bind to the ligand at two positions. Therefore, when binding to the ligand on the solid phase such as the membrane surface or extracellular matrix, the affinity is high. However, it can be expected that the effect will be significantly improved.

When a fusion polypeptide with an antibody is used, it is desirable that the original polypeptide has a small molecular weight, because the fusion increases the molecular weight. The reason for this is that when the molecular weight is large, the molecular weight of the DNA to be handled becomes large when preparing a recombinant host that produces the fusion polypeptide by genetic engineering. Generally, the larger the molecular weight of the DNA to be introduced, the lower the efficiency of introduction into the host, and the lower the frequency of obtaining recombinants. In general, the larger the molecular weight of the recombinant polypeptide to be produced, the lower the production amount. Furthermore, when used for the treatment of solid cancer, it has been reported that a polypeptide having a large molecular weight has poor invasiveness to the affected area (DM Lane et al., Br. J.
Cancer, 70: 521 (1994)).

[0011]

The present inventors have found that VEGF
The inventors have made diligent efforts for the purpose of finding a polypeptide that can inhibit angiogenesis by specifically inhibiting the protein, particularly a polypeptide having a small molecular weight among the polypeptides relating to the extracellular region of the VEGF receptor. As a result, F
It was found that a polypeptide containing the first to third immunoglobulin-like domains in the extracellular region of LT binds to VEGF with high specificity and can inhibit VEGF activity. In addition, in the present specification, the “polypeptide” generally means that amino acids are covalently bonded to each other by a peptide bond, and the length is not limited.

The polypeptide of the present invention includes a polypeptide comprising the first to third immunoglobulin-like domains of the extracellular region of FLT, a polypeptide comprising the first to fourth immunoglobulin-like domains, and the first to the first. Included are polypeptides consisting of 5 immunoglobulin-like domains. In addition, since the polypeptide consisting of the first to sixth immunoglobulin-like domains and the polypeptide consisting of the first to seventh immunoglobulin-like domains have too large a molecular weight, "the expression by the recombinant DNA technique is easy and It is considered that the effect of the present invention that "infiltration is rapid" cannot be sufficiently exerted, and is excluded from the polypeptides of the present invention. The boundaries of the FLT domains are not clearly distinguished, but in the present specification, each domain is defined as a domain including an amino acid sequence having the following residue number. [The residue number is
Same as SEQ ID NO: 1. That is, the residue number counted from the N-terminus of mature FLT (“Ser” at position 1 of SEQ ID NO: 1) is shown. ] 1st immunoglobulin domain 1-110 2nd immunoglobulin domain 111-208 3rd immunoglobulin domain 209-311 4th immunoglobulin domain 312-407 5th immunoglobulin domain 408-535 6th immunoglobulin domain 536-640th 7 Immunoglobulin domain 641-736

BEST MODE FOR CARRYING OUT THE INVENTION

These peptides can be produced by the following procedure. Human vascular endothelial cells, such as human umbilical cord-derived vascular endothelial cells (sold by Iwaki Glass, Morinaga Milk Industry, Kurabo Industries, etc.) were cultured and subjected to the acidic phenol method (P. Chomzyn
ski and N. Sacchi, Anal. Biochem., 162: 156 (198
7)) is used to extract total RNA, and poly (A) ⁺ RNA is prepared with oligo dT cellulose. Single-stranded cDNA or double-stranded cDNA is synthesized using this as a template and reverse transcriptase and oligo dT (12-16) primer. For the preparation method of poly (A) ⁺ RNA and cDNA, see “J. Sambrook et al.,” Molecular Clonin.
g ", Cold Spring Harbor Laboratory Press, 1989". In addition, commercially available poly (A) ⁺ RNA
It can also be performed using a preparation reagent (Oligotex-dT30, manufactured by Takara Shuzo) or a cDNA synthesis kit (manufactured by Pharmacia Biosystems). When there is fltcDNA already cloned from the cDNA library, the DNA of the region to be expressed may be directly cut out with an appropriate restriction enzyme and introduced into an expression vector.

Next, PCR is carried out using the obtained cDNA as a template.
Law (“PCR Protocols”, Academic Press Inc., 199
According to "0"), the DNA of the target portion can be amplified. For example, the following primers may be used. The primer DNA can be synthesized with a DNA synthesizer (manufactured by Applied Biosystems, manufactured by Nippon Millipo Limited, etc.), or custom DNA can be ordered (Sawady Technology). For example, to obtain a cDNA encoding the first immunoglobulin-like domain to the fourth immunoglobulin-like domain, the upstream primer: 5'-N (3-5) X (6) CGTCGCGCTCACCATGGT
CAG-3 '(SEQ ID NO: 2) Downstream primer: 5'-N (3-5) Y (6) TTATTCGTAAATCTGGGG
TTTCAC-3 '(SEQ ID NO: 3) may be used. In the array,
N is any of A, C, G, and T, X or Y is a restriction enzyme recognition sequence, and the numbers in parentheses represent the number of bases. Specifically, N
(3 to 5) indicates that there are 3 to 5 of any one of A, C, G and T, and X (6) or Y (6) indicates a recognition site of a restriction enzyme that recognizes 6 bases. . These restriction enzyme recognition sequences are
It is desirable to use a sequence that does not exist in the DNA fragment to be amplified and the vector into which it is to be inserted. DN encoding a desired C-terminal by appropriately designing a downstream primer with reference to the nucleotide sequence of SEQ ID NO: 1
The A fragment can be amplified. Also, when incorporated into an expression vector, the coding sequence for the polypeptide must be oriented in the forward direction relative to the promoter. In the primer sequence, the portion corresponding to the fltDNA sequence is not necessarily limited to 21 bases, and 17-2
It may be about 5 bases. The PCR conditions are as described in “PCR Prot
Although the standard conditions described in “ocols” are acceptable, the progress of the reaction differs depending on the amount of template and the primer sequence, so in order to perform efficiently, each parameter (eg Mg ⁺⁺ concentration, annealing temperature, extended reaction time, number of cycles) Etc.) can be appropriately changed and optimized. D used for PCR
NA polymerase has a proofreading (3 'exonuclease) activity than Taq polymerase.
fu polymerase (Stratagene) or Taq polymerase with Pfu polymerase added is preferred.
Increased reliability (Fiderity) during PCR amplification (WM Barn
es, Proc. Natl. Acad. Sci. USA, 91: 2216 (199
Four)).

DN to be amplified by PCR in this case
Since the A fragment has a known sequence, its size is confirmed by agarose gel electrophoresis after amplification, and it is recovered from the gel, digested with an appropriate restriction enzyme, and its electrophoresis pattern is examined to obtain the target DNA fragment. You can judge whether or not. Agarose gel electrophoresis, recovery of DNA fragments from gel, and digestion with restriction enzymes are described in "Molecula" above.
r Cloning ”. For recovering DNA from the gel, a commercially available kit using glass beads (for example, Prep-A-Gene, Bio-Rad) can be used.

The recovered DNA fragments are X (6) and Y (6)
Both ends of the fragment are digested with a restriction enzyme capable of cleaving the enzyme, deproteinized by phenol treatment, and ethanol precipitated, then an appropriate buffer such as TE (10 mM Tris-HCl (pH 7.5) / 1
Dissolve in mM EDTA). Similarly, the cloning site of an appropriate expression vector is cleaved with a restriction enzyme capable of cleaving X (6) and Y (6), and agarose gel electrophoresis is performed to recover the vector DNA. In this way small fragments between the X (6) and Y (6) cleavage sites can be eliminated. The DNA fragment to be inserted and the cleaved vector DNA are added so that the ratio of vector DNA: inserted DNA fragment is 1: 5 to 1:10,
Ligation reaction is performed using T4 DNA ligase. The ligation product is added to E. coli competent cells, transformation is performed, and antibiotic-resistant transformants are first transformed in a medium containing an antibiotic corresponding to a vector-encoded selection marker (eg, ampicillin resistance, kanamycin resistance, etc.). select.

The recombinant in which the DNA fragment is inserted into the expression vector is selected by examining the restriction enzyme cleavage pattern of the plasmid possessed by each antibiotic-resistant transformant. Alternatively, each transformant is used as a template for each bacterial cell, and PCR is performed using a primer that amplifies the DNA fragment to be inserted, to determine whether it is a recombinant by whether the target DNA fragment is amplified or not. You can The series of procedures for obtaining these E. coli recombinants is described in "Molecular Clon" above.
ing ”.

A variety of hosts can be utilized to produce the polypeptides of the present invention. For example, E. coli (Esc
herichia coli), Pseudomonos bacteria, Bacillus subtilis, Bacillus brevis
(Bacillus brevis), Bacillus licheniformis (Baci
llus liqueniformis), Bacillus thuringiensis (Baci
llus thuringensis) and other Gram-negative or Gram-positive bacteria, Pichia pastoris, Schizosaccharomyces pombe,
Saccharomises cerevisia
e) such as yeast, fungi such as Aspergillus genus, Sf9 (from Spodoptera frugiperda),
Sf21, TN5 (from Trichoplusia ni), BN4 (Bombyx moli
Origin) etc., mammalian cells such as CHO (Chinese hamster ovary origin), COS cells (monkey kidney origin) etc. can be used. As the vector, a vector suitable for each species of host may be used. It may be easier to obtain recombinant DNA once in E. coli using a shuttle vector of the host to be produced the polypeptide of the present invention and E. coli before obtaining the final transformed cell. The transformation method for obtaining a recombinant host that produces the polypeptide of the present invention is a competent cell method for E. coli and a competent cell method for Bacillus (K. Bott and
GA Willson, J. Bacteriol., 94: 562 (1967)), protoplast method (M. Mandel and A. Higa, J. Mol. Bio
l., 53: 159 (1970)), the yeast protoplast method (M. B.
roker et al., BioTechniques, 5: 516 (1987)), for insect cells and mammalian cells, the lipofectin method (RW Ma
lone et al., Proc. Natl. Acad. Sci. USA, 86: 6
077 (1989)), calcium phosphate method (FL Graham and
AJ van der Eb, Virology, 52: 456 (1973)). In addition, the electroporation method (BI
The ORAD pamphlet, etc.) can be applied to all the cells described above.

Basically, a plasmid or viral DNA replicable in the host to be used may be used, and the DNA encoding the part to be expressed may be incorporated downstream of a strong promoter functioning in the host. If the gene to be expressed does not have a translation initiation codon, it must be added. When using a prokaryotic cell as a host, the ribosome binding sequence (JR MacLaughlin et al., J. B
iol. Chem., 256: 11283 (1981)). It is also possible to use a method in which a vector having a part of the host chromosomal DNA and not replicable in the host is used to cause homologous recombination with the host chromosome and the vector is integrated into the host chromosome (Japanese Patent Laid-Open No. 278092/1992). Gazette, DJ King et al.,
Biochem. J., 281: 317 (1992)). Also, a method using an animal or plant individual as a host instead of a cultured cell can be used. For example, by producing a recombinant virus of BmNPV, which is a silkworm virus, and inoculating the silkworm, the polypeptide will be obtained with higher productivity as compared to the case where the cultured cells from the silkworm body fluid are used as the host (Kawai Hideki,
Yoichiro Shimogan, Bioindustry, 8:39 (1991)). p
It is also possible to recover the recombinant polypeptide from ascites by transplanting mouse myeloma cells transformed with the recombinant of the SV type vector into the abdominal cavity of SCID mice. D of the present invention
Transgenic animals using NA (G. Wright at a
l., Bio / Technology, 9: 830 (1991)) or transgenic plants (M. Owen et al., Bio / Technology, 10: 7).
90 (1992)) could be used as a host.

In order to secrete the polypeptide of the present invention extracellularly, when a eukaryotic cell is used as a host, the signal peptide coding portion of FLT may be used as it is. In bacteria, the DNA encoding the signal peptide of the secretory polypeptide of the host used may be available. For example, in E. coli, the outer membrane protein Om
DNAs encoding signal peptides such as pA, OmpF, phosphatase PhoA, maltose-binding protein MalB, and amylase whose base sequence is known in Bacillus, alkaline phosphatase, serine protease, etc.
Can be used. Further, in the case of intracellular expression, the signal peptide coding portion other than the initiation codon may be removed and used. Inclusion body formation often occurs when a foreign polypeptide is highly expressed in bacterial cells. In that case, several μg after solubilization with 8M urea
It will be possible to recover some of the activity by diluting to a polypeptide concentration of / ml and gradually removing the urea by dialysis.

The polypeptide of the present invention obtained by the above-mentioned method can be purified by a general biochemical method. For example, ammonium sulfate precipitation, ion exchange chromatography, gel filtration, hydrophobic chromatography and the like can be used. Since the polypeptide of the present invention has heparin affinity, affinity chromatography using a heparin resin can be used. In the case of a fusion polypeptide with another polypeptide,
It is possible to purify by utilizing the property of the partner polypeptide (M. Uhlen et al., Methods Enzymol.,
185: 129 (1990)). For example, if the fusion polypeptide partner is the Fc region of an antibody, Protein A Sepharose or Protein G Sepharose (E. Harlow and D.
Lane, "Antibodies", Cold Spring Harbor Laboratory
Press, 1988), glutathione transferase (G
ST) glutathione sepharose (D.
B. Smith and FS Johnson, Gene, 67:31 (1988)),
Chloramphenicol sepharose for chloramphenicol transferase, Ni ⁺⁺ -NTA (nitryltriacetic acid) for histidine oligomers
Affinity chromatography using agarose (FH Arnold, Bio / Tecnology, 9: 151 (1991)) can be used.

The fraction containing the polypeptide of the present invention can be detected by EIA or Western analysis using an antibody reactive with the polypeptide. The antibody that reacts with the polypeptide of the present invention synthesizes an oligopeptide corresponding to the 24th to 30th amino acid sequences from the N-terminus,
Bovine serum albumin or KLH (keyhole lymphet hemoc)
yanine) and other carrier proteins and immunize rabbits with standard methods (E. Harlow anr D. Lane, "Antibodies", Col.
d Spring Harbor Press, 1988). Moreover, even if a fusion polypeptide of the polypeptide of the present invention and another polypeptide is produced in Escherichia coli and purified using the characteristics of the fusion partner polypeptide as described above and used as an immunogen, the polypeptide of the present invention is also used. An antibody that reacts with is obtained.

Since the polypeptide of the present invention binds to VEGF, it can be purified using its activity as an index.
For example, in the same manner as when preparing an antibody-immobilized plate for EIA, a solution containing the polypeptide of the present invention before purification is appropriately diluted, and a 96-well polystyrene microtiter plate is coated to perform blocking treatment. Make a plate. Since this plate specifically binds VEGF, if ¹²⁵ I-labeled VEGF is used, the binding can be confirmed from the radioactivity remaining in the wells. Fractions of chromatography performed to purify the polypeptide of the invention and
Pre-incubate ¹²⁵ I-VEGF and transfer to the wells of the plate to measure residual radioactivity. The presence of the polypeptide of the present invention in the fraction can be confirmed by binding to VEGF during preincubation and competing with the polypeptide of the present invention on the plate to make it difficult to bind to the plate.

The polypeptides of the present invention bind VEGF with high affinity and inhibit the binding of VEGF to the VEGF receptor. Therefore, the polypeptide of the present invention can inhibit VEGF activity at low concentrations. Since the polypeptide of the present invention inhibits VEGF activity, it inhibits VEGF-stimulated vascular endothelial cell proliferation. Moreover, the polypeptide of the present invention inhibits the promotion of vascular permeability by VEGF. Furthermore, the polypeptides of the present invention may be used in vivo for VEGF.
Inhibits angiogenesis and inhibits tumor growth.

[0025]

【Example】

[Example 1] Preparation of recombinant baculovirus expressing FLT extracellular region 1) Preparation of recombinant virus expressing FLT first to third immunoglobulin-like domains Basically, J. Sambrook "Molecular
The vector was constructed according to the procedure described in "Cloning" according to the procedure shown in FIG. To obtain a recombinant virus expressing the 1st to 3rd immunoglobulin-like domains of FLT, plasmid pflt3-7 (M. Shibuya et al., On.
Cogene, 5: 519 (1990)) was digested with restriction enzymes EcoRI and TaqI, and an EcoRI-TaqI DNA fragment of about 1.3 kbp separated by agarose gel electrophoresis was prepared. On the other hand, the DNA of the plasmid pVL1393 was digested with restriction enzymes EcoRI and NotI, and separated by agarose gel to obtain about 9.8 kbp EcoRI-N.
An otI DNA fragment was prepared. TaqI end not
As an adapter for converting to I-terminal, "5'CGACAA
ACCAATATAATCTAAC3 '"(SEQ ID NO: 4) and"5'GGCCGTTA
The oligonucleotide of "GATTATATTGGTTTGT3 '" (SEQ ID NO: 5) was mixed at room temperature, and the "1.3 kbp E
coRI-TaqI DNA fragment "and" 9.8 kbp E
coRI-NotI DNA fragment "and mixed with T4DN
A ligation reaction was performed using A ligase, and it was introduced into E. coli. The resulting plasmid DNA was EcoRI and No.
The fragment of the obtained 1.3 kbp DNA was digested with tI and the fragment was recovered, and EcoRI / of pVL1393 (PharMingen) was recovered.
It was introduced at the NotI site. This plasmid DNA was purified to obtain a recombinant virus using BaculoGold (PharMingen), which is baculovirus DNA lacking the pohedrin coding region, according to the manual. This recombinant baculovirus was named "B3N". Recombinant baculovirus "B3N" was amplified using Sf9 cells according to the manual and used for the subsequent experiments. In addition, "B3
"N" contains the DNA encoding the amino acid sequence from the N terminus of FLT to residue 339.

2) Preparation of Recombinant Virus Expressing 1st to 4th Immunoglobulin-like Domains of FLT Basically, "Molecul of J. Sambrook et al.
The vector was constructed by the procedure shown in FIG. 3 according to the method described in “ar Cloning”. To obtain a recombinant virus expressing the 1st to 4th immunoglobulin-like domains of FLT, the plasmid pflt3-7 (M. Shibuya et al.,
Oncogene, 5: 519 (1990)) with the restriction enzyme Eco
About 1.6 kbp EcoRI-NdeI digested with RI and NdeI and separated by agarose gel electrophoresis.
A DNA fragment was prepared. On the other hand, the plasmid pME18SN
eo DNA was digested with restriction enzymes EcoRI and XhoI, and separated by agarose gel electrophoresis to about 5.5.
A kbp EcoRI-XhoI DNA fragment was prepared.
"5'TATTAATGATCTAGATGAC 3 '" (SEQ ID NO: as an adapter for converting the NdeI end into an XhoI end)
6) and "5'TCGAGTCATTCTAGATCATTAA 3 '" (SEQ ID NO:
The oligonucleotides of 7) are mixed at room temperature, and the "1.6 kbp EcoRI-NdeI DNA fragment" and the "5.5 kbp EcoRI-XhoI DNA fragment" are further mixed.
Were mixed and ligated using T4 DNA ligase, and introduced into E. coli. Obtained recombinant plasmid DN
Digestion of A with EcoRI and NotI resulted in 1.
The 6 kbp DNA fragment was recovered and pVL1393 (Phar
Mingen) EcoRI / NotI site. BaculoGold (Ph, which is a baculovirus DNA lacking the pohedrin coding region, was purified from this plasmid DNA.
Recombinant virus was obtained according to the manual using arMingen). This recombinant baculovirus was named "B4N". Recombinant baculovirus "B4N" was added to Sf9
The cells were used for amplification according to the manual and used for the subsequent experiments. “B4N” is 45 from the N-terminal of FLT.
It contains DNA encoding an amino acid sequence of up to 7 residues.

3) Preparation of Recombinant Virus Expressing 1st to 5th Immunoglobulin-like Domains of FLT Basically, "Molecul of J. Sambrook et al.
According to the method described in “ar Cloning”, the vector was constructed by the procedure shown in FIG. Plasmid pflt3-7D
The NA was cut with EcoRI and HindIII and 1.9.
A kbp EcoRI-HindIII DNA fragment was prepared. The oligonucleotide "5'AGCTTTTAATGATC" was used as an adapter for converting the HindIII end into XhoI.
TAGAATGAC3 '"(SEQ ID NO: 8) and"5'TCGAGTCATTCTAGA
TCATTAAA 3 '"(SEQ ID NO: 9) was mixed and added, and 5.5 kbp E of the above-mentioned plasmid pME18SNeo was added.
It was ligated with the coRI-XhoI DNA fragment, and E. coli was transformed with this, to obtain a recombinant plasmid. The obtained recombinant plasmid DNA was transformed with EcoRI and Not.
Digested with I, the obtained 1.9 kbp DNA fragment was recovered, and EcoRI / N of pVL1393 (PharMingen) was recovered.
It was introduced at the otI site. This plasmid DNA was purified and a recombinant virus was obtained according to the manual using BaculoGold (PharMingen), which is baculovirus DNA lacking the pohedrin coding region. This recombinant baculovirus was named "B5N". Recombinant baculovirus "B5N" was amplified using Sf9 cells according to the manual and used for the subsequent experiments. In addition, "B5
"N" includes DNA encoding the amino acid sequence from the N terminus of FLT to the 560th residue.

4) Preparation of Recombinant Virus Expressing 1st to 7th Immunoglobulin-Like Domains of FLT Basically, "Molecular of J. Sambrook et al.
According to the method described in "Cloning", the vector was constructed by the procedure shown in FIG. To obtain a recombinant virus expressing the 1st to 7th immunoglobulin-like domains of FLT, plasmid pflt3-7 (M. Shibuya et al., On
cogene, 5: 519 (1990)) was digested with restriction enzymes EcoRI and HindIII, and separated by agarose gel electrophoresis to obtain about 1.9 kbp EcoRI-HindII.
An I DNA fragment was prepared. On the other hand, plasmid pVL13
The DNA of 93 was digested with restriction enzymes EcoRI and NotI, and an EcoRI-NotI DNA fragment of about 9.8 kbp separated by agarose gel was prepared. Hind
Primer A "5'ACATAAGCTTTTATATCACA3 '" as an adapter for converting the III end into a NotI end
(SEQ ID NO: 10) and primer B “5′TTATGCGGCCGCTT”
ATCCTTGAACAGTGAGGTA3 '"(SEQ ID NO: 11) by PCR for residues 1840-2400 of pflt3-7.
Was amplified. The DNA fragment of about 600 bp was mixed with the "1.9 kbp EcoRI-HindIII DNA fragment" and the "9.8 kbp EcoRI-NotI DNA fragment" and ligated using T4 DNA ligase to introduce into E. coli. Obtained plasmid DNA
Was digested with EcoRI and NotI and the resulting 2.5
The kbp DNA fragment was recovered and pVL1393 (PharMin
gen) and introduced at EcoRI / NotI site. This plasmid DNA was purified to obtain BaculoGold (PharMin, which is baculovirus DNA lacking the pohedrin coding region).
Recombinant virus was obtained according to the manual. This recombinant baculovirus was named "B7N". Recombinant baculovirus "B7N" was amplified using Sf9 cells according to the manual and used for the subsequent experiments. “B7N” is 750 from the N-terminal of FLT.
It contains DNA encoding the amino acid sequence up to the residue.

[Example 2] Immunochemical analysis of expression product Incubation of insect cells HiFive (manufactured by Invitrogen Corp.) with ExC
The cells were cultured in ell400 medium (manufactured by Iwaki Glass Co., Ltd.), infected with recombinant baculovirus "B3N", "B4N", "B5N" or "B7N", and the culture supernatant was collected. This sample was subjected to SDS-polyacrylamide gel electrophoresis and subjected to Western blotting. Rabbit anti-FLT extracellular domain polyclonal antibody was used as the primary antibody, alkaline phosphatase-labeled anti-rabbit IgG was used as the secondary antibody, and NTB (Nitroblue tetazolium chrolide) / B was used.
CIP (5-Bromo-4-chrolo-3-indolylphosphate p-tolui
The color was developed with a dine salt) (manufactured by Gibco BRL). As a result, specific reactivity with this antibody was confirmed (Fig. 6). Lane 1 is B3N infected cell culture supernatant, lane 2 is B3N infected cell extract, lane 3 is B4N infected cell culture supernatant 2μ.
1, lane 4 was 10 μl of B4N infected cell extract, lane 5 was 2 μl of B5N infected cell culture supernatant, lane 6 was B5N.
10 μl of infected cell extract, lane 7 is the B7N infected cell culture supernatant, and lane 8 is the B7N infected cell extract. The mobility of immunochemically reacted bands was consistent with the expected molecular weight. Thereafter, these products were labeled with "3N-
It is referred to as "FLT", "4N-FLT", "5N-FLT", and "7N-FLT" (FIG. 1). In addition, it was determined that the culture supernatant sample contained about 4 μg / ml of “4N-FLT” or “5N-FLT”.

[Example 3] Affinity of expression product with VEGF Radioiodinated VEGF ₁₆₅ (= ¹²⁵ I-VE)
The binding assay between GF) and FLT extracellular domain was basically according to the method of Duan et al. (Duan et al., 1991). The purified FLT extracellular domain protein was treated with 10 mM phosphate buffer (pH 6.2) to give 10
The mixture was diluted to a concentration of about 0 ng / ml, 100 μl was dispensed into a 96-well plate, and then adsorbed at 4 ° C. for 12-16 hours. After adsorption, the 96-well plate was washed with PBS (-), and the binding buffer (DNEM / 25 mM HEPES-NaOH [pH
(7.6] /0.3% BSA) was dispensed into each well in an amount of 200 μl, left at room temperature for 2-3 hours to block non-specific adsorption sites, and then washed twice with binding buffer for use in the experiment. ¹²⁵ I-VEGF (Amersham: specific activity: 60-65
Tbq / mmol; 80,000-100,000 cpm / ng) using binding buffer from 16 ng / ml to 0.125 ng / ml
Dilute stepwise up to 2 times and add 100 μl of each dilution to F
It was dispensed into a 96-well plate to which LT protein was attached and reacted at room temperature for 3 hours. After completion of the reaction, the wells were washed 3 times with binding buffer, each well was divided, the radiation dose of each well was measured with a γ-ray counter (FIG. 7), and scatchard analysis was performed (FIGS. 8 and 9). 8A,
B shows the results using plates coated with “B3N”, “B4N”, and expression culture supernatant, respectively.
B is the result of using plates coated with “B5N” and “B7N” expression culture supernatants, respectively. At this time, since ¹²⁵ I-VEGF ₁₆₅ was hardly bound to the plate coated with the culture supernatant of the control virus-infected Sf9 cells, the bound radioactivity was changed from the inserted gene to the expression product.
It is considered to be the binding of ¹²⁵ I-VEGF ₁₆₅ . "B3
N, “B4N”, “B5N” and [B7N] -infected cells, the affinities of the expression products in the culture supernatant for VEGF ₁₆₅ have Kd values (dissociation constants) of 6.5 pM and 4.
It was 8 pM, 5.0 pM and 17.0 pM.

[Example 4] Inhibition of biological activity of VEGF by expression product 1) Inhibition of permeability enhancing activity of VEGF The inhibitory effect of "4N-FLT" or "5N-FLT" on the permeability enhancing activity of VEGF was examined. It was 0.5 ml of 1% Evans blue dye solution was injected into the heart of a guinea pig, and 30 minutes later, 0.2 ml of "4N-" was added together with VEGF.
The supernatant of the FLT "or" 5N-FLT "expressing culture was injected subcutaneously into the shaved skin, and after 30 minutes, leakage of the dye was observed (Table 1). From this result, "4N-FLT" and "5N-
-FLT "was found to inhibit the permeability enhancing activity of VEGF.

[0032]

[Table 1] Inhibitory effect of 4N-FLT and 5N-FLT on VEGF permeability enhancing activity Sample Dye leakage (relative value) ──────────────────────── ────────── PBS 0% 10 ng VEGF / 0.2 ml 100% 10 ng VEGF + Ca.400 ng 4N-FLT / 0.2 ml 5% 10 ng VEGF + Ca.400 ng 5N-FLT / 0.2 ml <5% ───────────────────────────────────

2) Inhibition of VEGF-dependent proliferation of rat sinusoidal endothelial cells by FLT extracellular domain The inhibitory effect of each FLT extracellular domain protein on VEGF-dependent proliferation was investigated. 1x1 non-parenchymal liver cells
Dispense 0 ⁵ each into a 24-well collagen plate and
After the time incubation, the plate was washed once with E-BM (Kurabo, Osaka). Then, "3N-FLT", "4N-F"
EG containing "LT", "5N-FLT" or "7N-FLT" at concentrations of 0, 10, 50 and 250 ng / ml.
Exchanged to MUV (Kurabo, Osaka). After changing the medium,
5 ng of purified human VEGF (165 amino acid type obtained by baculovirus protein expression system)
/ Ml was added. After 48 hours of culturing, the medium under the same conditions was exchanged and culturing was continued for another 48 hours. (E-GM
UV: vascular endothelial cell growth medium, 2% FCS, 10nG / ml EGF,
Contains 1 μg / ml hydrocortisone, 0.4% bovine brain extract. However, the medium alone cannot maintain the growth of sinusoidal endothelial cells. After the culture was completed, formalin was fixed and stained with crystal violet, and the number of sinusoidal wall endothelial cells was counted under a microscope (Fig. 10). From this result, "3N-
It can be seen that "FLT", "4N-FLT", "5N-FLT" and "7N-FLT" inhibit the endothelial cell proliferating activity of VEGF in a dose-dependent manner.

[0034]

INDUSTRIAL APPLICABILITY Since the polypeptide of the present invention can inhibit VEGF-stimulated angiogenesis, it can be used for the treatment of solid cancer and other diseases accompanied by pathological angiogenesis.
In addition, since it consists of human-derived amino acids, it is difficult for antibodies to form even after long-term administration to humans. In addition, conventional polypeptides (RL Kendal and KA Thomas, Proc. Natl., Aca
d.Sci., USA, 90: 10705 (1993)), the expression by recombinant DNA is easy and the infiltration into the affected area is quick.

[0035]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1899 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: cDNA Origin organism name: Human cell type: Placental tissue Sequence features Characteristic symbols : CDS existing position: 250. . 1899 Characteristic determination method: E Characteristic symbol: sig peptide Location: 250. . 315 Method for determining characteristic: S Characteristic symbol: mat peptide Location: 316. . 1899 SEQ GCGGACACTCCTCTCGGCTCCTCCCCGGCAGCGGCGGCGGCTCGGAGCGGGCTCCGGGGC 60 TCGGGTGCAGCGGCCAGCGGGCCTGGCGGCGAGGATTACCCGGGGAAGTGGTTGTCTCCT 120 GGCTGGAGCCGCGAGACGGGCGCTCAGGGCGCGGGGCCGGCGGCGGCGAACGAGAGGACG 180 GACTCTGGCGGCCGGGTCGTTGGCCGGGGGAGCGCGGGCACCGGGCGAGCAGGCCGCGTC 240 GCGCTCACCATG GTC AGC TAC TGG GAC ACC GGG GTC CTG CTG TGC GCG 288 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala -20 -15 -10 CTG CTC AGC TGT CTG CTT CTC ACA GGA TCT AGT TCA GGT TCA AAA TTA 336 Leu Leu Ser Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu -5 1 5 AAA GAT CCT GAA CTG AGT TTA AAA GGC ACC CAG CAC ATC ATG CAA GCA 384 Lys Asp Pro Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala 10 15 20 GGC CAG ACA CTG CAT CTC CAA TGC AGG GGG GAA GCA GCC CAT AAA TGG 432 Gly Gln Thr Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp 25 30 35 TCT TTG CCT GAA ATG GTG AGT AAG GAA AGC GAA AGG CTG AGC ATA ACT 480 Ser Leu Pro Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr 40 45 50 55 AAA TCT GCC TGT GGA AGA AAT GGC AAA CAA TTC TGC AGT ACT TTA ACC 528 Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr 60 65 70 TTG AAC ACA GCT CAA GCA AAC CAC ACT GGC TTC TAC AGC TGC AAA TAT 576 Leu Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr 75 80 85 CTA GCT GTA CCT ACT TCA AAG AAG AAG GAA ACA GAA TCT GCA ATC TAT 624 Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr 90 95 100 ATA TTT ATT AGT GAT ACA GGT AGA CCT TTC GTA GAG ATG TAC AGT GAA 672 Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu 105 110 115 ATC CCC GAA ATT ATA CAC ATG ACT GAA GGA AGG GAG CTC GTC ATT CCC 720 Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro 120 125 130 135 TGC CGG GTT ACG TCA CCT AAC ATC ACT GTT ACT TTA AAA AAG TTT CCA 768 Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro 140 145 150 CTT GAC ACT TTG ATC CCT GAT GGA AAA CGC ATA ATC TGG GAC AGT AGA 816 Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg 155 160 165 AAG GGC TTC ATC ATA TCA AAT GCA ACG TAC AAA GAA ATA GGG CTT CTG 864 Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu 170 175 180 ACC TGT GAA GCA ACA GTC AAT GGG CAT TTG TAT AAG ACA AAC TAT CTC 912 Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu 185 190 195 ACA CAT CGA CAA ACC AAT ACA ATC ATA GAT GTC CAA ATA AGC ACA CCA 960 Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro 200 205 210 215 CGC CCA GTC AAA TTA CTT AGA GGC CAT ACT CTT GTC CTC AAT TGT ACT 1008 Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr 220 225 230 GCT ACC ACT CCC TTG AAC ACG AGA GTT CAA ATG ACC TGG AGT TAC CCT 1056 Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro 235 240 245 GAT GAA AAA AAT AAG AGA GCT TCC GTA AGG CGA CGA ATT GAC CAA AGC 1104 Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser 250 255 260 AAT TCC CAT GCC AAC ATA TTC TAC AGT GTT CTT ACT ATT GAC AAA ATG 1152 Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met 265 270 275 CAG AAC AAA GAC AAA GGA CTT TAT ACT TGT CGT GTA AGG AGT GGA CCA 1200 Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro 280 285 290 295 TCA TTC AAA TCT GTT AAC ACC TCA GTG CAT ATA TAT GAT AAA GCA TTC 1248 Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe 300 305 310 ATC ACT GTG AAA CAT CGA AAA CAG CAG GTG CTT GAA ACC GTA GCT GGC 1296 Ile Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly 315 320 325 AAG CGG TCT TAC CGG CTC TCT ATG AAA GTG AAG GCA TTT CCC TCG CCG 1344 Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro 330 335 340 GAA GTT GTA TGG TTA AAA GAT GGG TTA CCT GCG ACT GAG AAA TCT GCT 1392 Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala 345 350 355 CGC TAT TTG ACT CGT GGC TAC TCG TTA ATT ATC AAG GAC GTA ACT GAA 1440 Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu 360 365 370 375 GAG GAT GCA GGG AAT TAT ACA ATC TTG CTG AGC ATA AAA CAG TCA AAT 1488 Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn 380 385 390 GTG TTT AAA AAC CTC ACT GCC ACT CTA ATT GTC AAT GTG AAA CCC CAG 1536 Val Phe Lys Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln 395 400 405 ATT TAC GAA AAG GCC GTG TCA TCG TTT CCA GAC CCG GCT CTC TAC CCA 1584 Ile Tyr Glu Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro 410 415 420 CTG GGC AGC AGA CAA ATC CTG ACT TGT ACC GCA TAT GGT ATC CCT CAA 1632 Leu Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln 425 430 435 CCT ACA ATC AAG TGG TTC TGG CAC CCC TGT AAC CAT AAT CAT TCC GAA 1680 Pro Thr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu 440 445 450 455 GCA AGG TGT GAC TTT TGT TCC AAT AAT GAA GAG TCC TTT ATC CTG GAT 1728 Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp 460 465 470 GCT GAC AGC AAC ATG GGA AAC AGA ATT GAG AGC ATC ACT CAG CGC ATG 1776 Ala Asp Ser Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met 475 480 485 GCA ATA ATA GAA GGA AAG AAT AAG ATG GCT AGC ACC TTG GTT GTG GCT 1824 Ala Ile Ile Glu Gly Lys Asn Lys Met Ala S er Thr Leu Val Val Ala 490 495 500 GAC TCT AGA ATT TCT GGA ATC TAC ATT TGC ATA GCT TCC AAT AAA GTT 1872 Asp Ser Arg Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val 505 510 515 GGG ACT GTG GGA AGA AAC ATA AGC TTT TAT ATC ACA GAT GTG CCA AAT 1920 Gly Thr Val Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn 520 525 530 535 GGG TTT CAT GTT AAC TTG GAA AAA ATG CCG ACG GAA GGA GAG GAC CTG 1968 Gly Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu 540 545 550 AAA CTG TCT TGC ACA GTT AAC AAG TTC TTA TAC AGA GAC GTT ACT TGG 2016 Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp 555 560 565 ATT TTA CTG CGG ACA GTT AAT AAC AGA ACA ATG CAC TAC AGT ATT AGC 2064 Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser 570 575 580 AAG CAA AAA ATG GCC ATC ACT AAG GAG CAC TCC ATC ACT CTT AAT CTT 2112 Lys Gln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu 585 590 595 ACC ATC ATG AAT GTT TCC CTG CAA GAT TCA GGC ACC TAT GCC TGC AGA 2160 Thr Ile Met Asn Val Se r Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg 600 605 610 615 GCC AGG AAT GTA TAC ACA GGG GAA GAA ATC CTC CAG AAG AAA GAA ATT 2208 Ala Arg Asn Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile 620 625 630 ACA ATC AGA GAT CAG GAA GCA CCA TAC CTC CTG CGA AAC CTC AGT GAT 2256 Thr Ile Arg Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp 635 640 645 CAC ACA GTG GCC ATC AGC AGT TCC ACC ACT TTA GAC TGT CAT GCT AAT 2304 His Thr Val Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn 650 655 660 GGT GTC CCC GAG CCT CAG ATC ACT TGG TTT AAA AAC AAC CAC AAA ATA 2352 Gly Val Pro Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile 665 670 675 CAA CAA GAG CCT GGA ATT ATT TTA GGA CCA GGA AGC AGC ACG CTG TTT 2400 Gln Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe 680 685 690 695 ATT GAA AGA GTC ACA GAA GAG GAT GAA GGT GTC TAT CAC TGC AAA GCC 2448 Ile Glu Arg Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala 700 705 710 ACC AAC CAG AAG GGC TCT GTG GAA AGT TCA GCA TAC CTC ACT GTT CAA 2496 Thr Asn Gln Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln 715 720 725 GGA ACC TCG GAC AAG TCT AAT CTG GAG 2523 Gly Thr Ser Asp Lys Ser Asn Leu Glu 730 SEQ ID NO: 2 Sequence length: 21 Sequence Type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence CGTCGCGCTC ACCATGGTCA G 21 SEQ ID NO: 3 Sequence length: 24 Sequence type: Nucleic acid Number of strands : Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TTATTCGTAA ATCTGGGGTT TCAC 24 SEQ ID NO: 4 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Direct Chain type of sequence: Other nucleic acid (synthetic DNA) sequence CGACAAACCA ATATAATCTA AC 22 SEQ ID NO: 5 Sequence length: 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other Nucleic acid (synthetic DNA) sequence of GGCCGTTAGA TTATATTGGT TTGT 24 SEQ ID NO: 6 sequence Length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence TATTAATGAT CTAGAATGAC 19 SEQ ID NO: 7 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: Other nucleic acid (synthetic DNA) Sequence TCGAGTCATT CTAGATCATT AA 22 SEQ ID NO: 8 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single Strand topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence AGCTTTTAAT GATCTAGAAT GAC 23 SEQ ID NO: 9 Sequence length: 23 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence Type: other nucleic acid (synthetic DNA) sequence TCGAGTCATT CTAGATCATT AAA 23 SEQ ID NO: 10 sequence length: 20 sequence type: nucleic acid number of strands: single strand topology: linear sequence type: other nucleic acid ( Synthetic DNA) Sequence ACATAAGCTT TTATATCACA 20 SEQ ID NO. 11 Length of sequence: 33 SEQ types: the number of nucleic acid strands: single strand Topology: linear sequence type: other nucleic acid (synthetic DNA) sequence TTATGCGGCC GCTTATCCTT GAACAGTGAG GTA 33

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the constitution of a domain of FLT extracellular region.

FIG. 2 is a diagram showing an outline of a construction process of a recombinant baculovirus expressing 3N-FLT.

FIG. 3 is a diagram showing an outline of a construction process of a recombinant baculovirus expressing 4N-FLT.

FIG. 4 is a diagram showing an outline of a construction process of a recombinant baculovirus expressing 5N-FLT.

FIG. 5 is a diagram showing an outline of a construction process of a recombinant baculovirus expressing 7N-FLT.

FIG. 6 shows Western blots of culture supernatants and extracts of cells infected with B3N, B4N, B5N and B7N recombinant baculovirus.

FIG. 7 is a diagram showing a VEGF saturation binding curve of each FLT fragment.

FIG. 8 shows Scatchard analysis of the interaction between 3N-FLT, 4N-FLT and VEGF fragment.

FIG. 9 shows Scatchard analysis on the interaction between 5N-FLT, 7N-FLT and VEGF fragment.

FIG. 10: 3N-FLT, 4N-FLT, 5N-FL
It is a figure which shows VEGF-dependent cell growth inhibition of T and 7N-FLT.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location C12N 15/09 G01N 33/68 C12P 21/02 A61K 37/02 ADU G01N 33/53 C12N 5/00 B 33 / 68 9282-4B 15/00 A // (C12P 21/02 C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (6)

[Claims] 1. A polypeptide comprising the first to third immunoglobulin-like domains, the first to fourth immunoglobulin-like domains, or the first to fifth immunoglobulin-like domains of the extracellular region of FLT which is a VEGF receptor. . 2. A DNA encoding the polypeptide according to claim 1. 3. A vector containing the DNA according to claim 2. A transformant carrying the vector according to claim 3. 5. A medicine comprising the polypeptide according to claim 1. 6. An analytical reagent comprising the polypeptide according to claim 1.