JP2000128803A - Tissue factor pass way inhibitor-2 antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain tissue factor pass way inhibitor-2 antibody free from alternation response with tissue factor pass way inhibitor-(TFPI)-1, and useful for vasucular smooth muscle proliferation modifier or diagnostic agent for disease relating to proliferation of vasucular tract. SOLUTION: The antibody or its fraction specific to TFPI-2 free from alternation response with TFPI-1 is obtained. The TFPI-2 originating from human is preferable. The TFPI-2 is obtained by carrying out purification from the conditioned medium of cell manifesting natural TFPI-2 using mitogenicity as the indicator, or purifying expressed TFPI-2 after integrating DNA coding for TFPI-2 in an expression vector and expressing in a proper host. The TFPI-2 antibody is easily obtained by immunizing a proper animal using the product TFPI-2 as an antigen. Vasucular smooth muscle proliferation modifier or the like are provided using obtained antibody as active component.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an antibody. For more information, see Tissue Factor Pathway
The present invention relates to an inhibitor-2 antibody or a fragment thereof, and a vascular smooth muscle growth regulator and a diagnostic agent containing such an antibody or a fragment thereof.

[0002]

BACKGROUND OF THE INVENTION Proliferation of vascular smooth muscle cells is closely related to the pathogenesis of arteriosclerosis and restenosis after percutaneous transluminal coronary angioplasty (Ross, R. (1993) Nature 362, 801-). 809). Vascular endothelial cells have an antithrombotic function composed of various mechanisms and maintain blood fluidity, but also have a thrombotic function to quickly form a thrombus and protect the site of injury when injured. I have Endothelial cells having such contradictory functions maintain blood homeostasis, and if the balance is disrupted due to arteriosclerosis, various cardiovascular diseases such as myocardial infarction and cerebral infarction are considered to be caused. . Regarding the antithrombotic properties of endothelial cells, the anticoagulant action of antithrombin III and thrombomodulin present on the cell surface, the antiplatelet action by the production of prostaglandin I ₂ (PGI ₂ ), the tissue plasminogen activator (t- Production of PA) and fibrinolysis promoting activity by binding of plasminogen are known.

Recently, a new type of inhibitor having anticoagulant activity has been shown to be produced in vascular endothelial cells, and has attracted attention. This inhibitor,
That is, Tissue Factor Pathway Inhibitor (TFPI)
(TFPI is sometimes referred to as TFPI-1; hereinafter, abbreviated as TFPI-1) is synthesized by vascular endothelial cells,
Present on endothelium or in plasma (Jesty, J. et al. (1994)
Biochemistry 33, 12686-12694; Sprecher, CA et al.
(1994) Proc. Natl. Acad. Sci. USA 91, 3353-3357; P
eterson, LC et al. (1996) Biochemistry 35, 266-272.
). TFPI-1 inhibits early steps in the extrinsic coagulation pathway and regulates congestion. TFPI-1 is an inhibitor of Factor Xa or Factor VIIa in the presence of Factor Xa.
It is an inhibitor of the factor-tissue factor (TF) complex.
TFPI-1 is a Kuni with three tandem domains
It is a tz-type protease inhibitor. The first inhibitory domain interacts with the Factor VIIa / TF complex to form the complex: Factor Xa / TFPI-1 / Factor VIIa / TF, thereby forming the protein of the Factor VIIa / TF complex. Inhibits degradation activity. The Factor Vila / TF complex converts Factor X and Factor IX into active enzymes. Second
Interacts with factor Xa and directly inhibits factor Xa. The function of the third inhibitory domain is to bind to heparin. Factor Xa along with Factor Va cleaves prothrombin to thrombin, leading to the generation of fibrin clots.

On the other hand, TFPI-2 is structurally similar to TFPI-1 (Sprecher, CA et al. (1994) Proc.
Natl. Acad. Sci. USA 91, 3353-3357; Peterson, L.
C. et al., (1996) Biochemistry 35, 266-272). TFPI
-2 is a short acidic amino-terminal region, three tandem Kuni
It has a tz-type protease inhibitory domain and a carboxy-terminal tail rich in basic amino acids. TFPI-2 is deamidating activity of trypsin and human factor VIIa and T
Inhibits the activity of the F complex. TFPI-2 is TFP
Compared with I-1, it shows only a weak inhibitory activity on the amidolytic activity of factor Xa. TFPI-2 is a placental protein 5, the same protein as PP5 (Miyagi,
Y. et al., (1994) J. Biochem. 116, 939-942). TFPI
-2 (PP5) is the previously reported factor VIIa / T
F complex and the ability to inhibit trypsin,
It is a strong inhibitor of factor Ia, plasmin, plasma kallikrein and chymotrypsin.

In addition to the above properties in the blood coagulation system, TF
The antiproliferative effects of PI-1 on intimal or smooth muscle cells in various arteries have been reported. In a model of arterial injury in atherosclerotic rabbits, treatment with recombinant TFPI-1 reduced angiographic restenosis and reduced neointimal hyperplasia (Jang, Y. et al. (1995) Circulation 92). , 304
1-3050). Inhibition of TF-mediated coagulation by administration of recombinant TFPI-1 during the first 24 hours following balloon-induced arterial injury in the carotid arteries of minipigs is effective in reducing subsequent neointimal formation and lumen stenosis (Oltrona, L. et al. (1997) Circulation 96, 646-65
2). Recombinant TFPI-1 shows growth inhibitory activity on cultured human neonatal aortic smooth muscle cells (Kamikubo, Y.
(1997) FEBS Lett. 407, 116-120). in this way,
Various effects of TFPI-1 have been elucidated, and application studies on pharmaceuticals have been actively conducted. On the other hand, TFPI-2 having a similar structure has little known physiological function.

[0006] Recently, a diagnostic agent containing an antibody recognizing TFPI-2 is commercially available.
At present, it is not possible to clarify the relevance to a clear pathological condition by diagnosis using the antibody because of cross-reaction with I-1.

[0007]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned prior art, and makes it possible to directly clarify the physiological role of TFPI-2.
To elucidate various pathological conditions involving TFPI-2 or to study therapeutic methods, TFPI does not cross-react with TFPI-1.
It is intended to provide an antibody or the like specific to PI-2.

[0008]

SUMMARY OF THE INVENTION To study the effect of endothelial cells on smooth muscle cell proliferation, we coated the cells with cultured human umbilical vein endothelial cells (HUVEC).
Examination using a culture insert with a 0 μm membrane and a microtest plate covered with bovine aortic smooth muscle cells showed that when the endothelial cells were present in the insert, proliferation of the smooth muscle cells was seen. . Thus, a substance having a smooth muscle cell growth activity (ie, mitogenic activity) is purified from the conditioned medium of cultured HUVEC,
When the terminal amino acid sequence was analyzed, it was unexpectedly found that the mitogen substance was TFPI-2 having a similar structure to TFPI-1 having a growth inhibitory activity on vascular smooth muscle cells.
It was discovered that. Furthermore, the present inventors have prepared antibodies specific to TFPI-2 using the purified recombinant TFPI-2 as an antigen, thereby completing the present invention.

That is, the gist of the present invention is to provide (1) a tissue factor pathway inhibitor (TF)
(2) an antibody or fragment thereof specific for TFPI-2, which does not cross-react with PI) -1, (2) the antibody or fragment thereof according to (1) above, wherein TFPI-2 is of human origin;
A vascular smooth muscle growth regulator comprising the antibody or fragment thereof according to (1) or (2) as an active ingredient;
(4) A diagnostic agent for a disease associated with blood vessel proliferation, comprising the antibody or the fragment thereof according to (1) or (2).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The antibody or a fragment thereof of the present invention comprises:
It is an antibody or a fragment thereof specific for tissue factor pathway inhibitor (TFPI) -2 and does not cross-react with TFPI-1.

[0011] The antibody of the present invention or a fragment thereof is TFPI
As long as it does not cross-react with -1, it may be any of a monoclonal antibody, a polyclonal antibody or a fragment thereof. The classes of such antibodies are IgA, IgD, IgE,
Examples include IgG and IgM, with IgG being preferred. Here, the antibody fragment includes Fab, F (ab) ₂ and Fc.

TFP which is recognized by the antibody or its fragment of the present invention and which is also used as an antigen for producing the antibody
Examples of I-2 include those derived from mammals such as humans, rabbits, and rats, but human-derived TFPI-2 described in SEQ ID NO: 1 is preferred. TFPI-2 may be either a natural protein or a recombinant protein, and may be a partial peptide of such a protein as long as it has antigenicity.

As a method for producing TFPI-2, for example, a method for purifying natural TFPI-2 from a conditioned medium of cells expressing natural TFPI-2 using mitogenic activity as an index, and a method for producing TFPI-2 And then purifying the expressed recombinant TFPI-2 after incorporating the DNA encoding 2 into an expression vector and expressing it in an appropriate host.

As a method for purifying natural TFPI-2, for example, as described in Reference Example 1, a conditioned medium of human umbilical vein endothelial cells (HUVEC) is concentrated by ultrafiltration, and a heparin affinity column is used. After elution of the mitogen-active fraction, LC-6 using a ProRPC HR 5/10 column (Amersham Pharmacia Biotech)
A A method in which a mitogen-active fraction is eluted using a high-performance liquid chromatography (HPLC) system (manufactured by Shimadzu).

A method for purifying recombinant TFPI-2 is described in, for example, Reference Examples 2 to 4.
DNA is prepared by PCR or the like, an expression vector cloned into a mammalian expression vector pK4K is constructed, and then baby hamster kidney (BHK) tk ^- ts13 cells or the like are transformed by a calcium phosphate precipitation method or the like, and are treated with methotrexate (MTX) or the like. After the selection, the conditioned medium is recovered and purified in the same manner as the natural TFPI-2.

As used herein, "not cross-reacting with TFPI-1" means that TFPI-1 cannot be measured by a conventional enzyme immunoassay (EIA) system using TFPI-2 antibody, or TFPI-1 cannot be measured. -1 and TFPI-2 were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by a conventional method and subjected to Western blotting.
It means that only 2 can be detected.

The anti-TFPI-2 antibody of the present invention is, for example,
Antibodies; A Laboratory Manual, edited by Lane.HD et al., Cold Spring Harber Laboratory Press N
ew York 1989. An antibody that recognizes TFPI-2 or an antibody that neutralizes the activity by immunizing a suitable animal with an appropriate method using the purified TFPI-2 as an antigen according to the method described in 1989 or the like. Can be easily prepared and purified. Furthermore, the produced antibody is TFP
By confirming that it does not cross-react with I-1,
An antibody specific for TFPI-2 is obtained.

The fragment of the anti-TFPI-2 antibody of the present invention can be obtained by allowing a proteinase such as papain or pepsin to act on the purified antibody.

Uses of the antibody or its fragment include reagents for research such as affinity chromatography and screening of cDNA library, immunological diagnostic agents, pharmaceuticals and the like.

The present invention also provides a vascular smooth muscle growth regulator comprising the antibody or a fragment thereof as an active ingredient.

When the antibody of the present invention or a fragment thereof is contained in a vascular smooth muscle growth regulator, the form may be a solution, suspension, emulsion, lyophilized product or the like. The lyophilized product is used by dissolving it in an appropriate solvent, saline, buffer or the like immediately before use.

The antibody of the present invention or a fragment thereof is TFPI
-2, which has an action of specifically binding to -2 to neutralize its activity, and thus the vascular smooth muscle growth regulator of the present invention containing such an antibody or a fragment thereof as an active ingredient has the following effects. . That is, it is possible to inhibit the growth promoting effect of vascular smooth muscle cells and the inhibition of blood coagulation effect of TFPI-2 in vivo or in vitro. Further, it is also useful as a therapeutic or preventive agent for diseases involving TFPI-2.

When the vascular smooth muscle proliferating agent of the present invention is used as a therapeutic or preventive agent for the above-mentioned diseases, it is preferably administered parenterally. That is, they can be injected intravenously, subcutaneously, intramuscularly, etc. as liquids, emulsions, suspensions, liposomes and the like, and can also be administered rectally as suppositories. Such dosage forms include the usual pharmaceutically acceptable carriers, excipients, binders, stabilizers, emulsifiers, buffers, solubilizers,
It can be produced by mixing an isotonic agent or the like with the antibody of the present invention or a fragment thereof.

The dose of the vascular smooth muscle growth regulator of the present invention can be appropriately adjusted depending on the disease to be treated or prevented, the age and weight of the patient, etc., but is usually 0.001 per dose.
The dose is from 01 mg to 1000 mg, preferably from 0.001 mg to 1000 mg, which is preferably administered about 1 to 3 times per day.

Further, the present invention provides a diagnostic agent containing the antibody of the present invention or a fragment thereof.

In order to include the antibody of the present invention or a fragment thereof in a diagnostic agent, the form can be a solution, suspension, emulsion, lyophilized product, or the like. Lyophilized products are
Immediately before use, it is used by dissolving it in an appropriate solvent, saline, buffer or the like. The content of the antibody or its fragment may be appropriately determined according to the diagnostic method used.

The diagnostic agent of the present invention is preferably used for an immunological diagnostic method. Specifically, an immunoblot method, a radioimmunoassay (RIA), an enzyme immunoassay (EL)
ISA), a fluorescence measurement method or a luminescence measurement method.

Further, the diagnostic agent may contain a reagent for performing an immunological reaction and a reagent for detecting such a reaction, depending on the intended diagnostic method. Reagents for performing an immunological reaction include buffers, salts and the like. As the detection reagent, a conventional immunological method such as a labeled secondary antibody recognizing the antibody of the present invention or a fragment thereof, or a complex of the antibody of the present invention or a fragment thereof and an antigen, a substrate corresponding to the label, etc. Reagents used in diagnostic methods are included. Here, examples of the label include biotin, alkaline phosphatase and the like.

The diagnostic agent of the present invention has an effect of specifically diagnosing a disease involving TFPI-2, unlike conventional diagnostic agents.

[0030]

The present invention will be described in more detail with reference to the following examples, which are not intended to limit the present invention.

Reference Example 1 Purification of TFPI-2 Human umbilical vein endothelial cells (HUVEC) were obtained from Warishi
iishi, S.) et al., Biochem. Biophys. Res. Commun. 216,
The cells were cultured according to the method described in 729-735 (1995), and TFPI-2 was purified from the conditioned medium. 800m
of the conditioned medium at 4 ° C. with an ultrafiltration membrane (YM1
0, manufactured by Amicon). This 20
ml of the solution in advance with 50 mM Tris-HCl (pH
The sample was applied to a HiTrap Heparin affinity column (5 ml, manufactured by Amersham Pharmacia Biotech) equilibrated in 7.4). After application of the sample, the column was washed with 50 ml of a 50 mM Tris-HCl (pH 7.4) solution. The mitogen-active fraction containing TFPI-2 was 1
50 mM Tris-HCl containing M NaCl (pH
Eluted in 20 ml of 7.4). Centriprep eluate
10 (manufactured by Amicon) and concentrated to 0.5 ml. The concentrated solution is subjected to LC-6A high performance liquid chromatography (HP
LC) system (manufactured by Shimadzu) using ProRPC HR
It was injected into a 5/10 column (Amersham Pharmacia Biotech). The flow rate was 1 ml / min and the peak was monitored at 280 nm. The column was eluted with a gradient formed from 0.1% trifluoroacetic acid (TFA) in 15% acetonitrile to 0.1% TFA in 100% acetonitrile (FIG. 1). The mitogen-active fraction eluted from the column was lyophilized. Mitogenic activity was measured using the method described in Reference Example 5 below.

Table 1 shows the activity of the mitogen fraction in each purification step for bovine aortic smooth muscle cells whose growth was stopped. Through the above series of purification steps, it was revealed that the substance having mitogenic activity was purified about 420-fold from the HUVEC concentrated condition medium.

[0033]

[Table 1]

The mitogen-active fraction (0.1 μg) after the final purification step was used in a Phast System (Amersham Pharmac.
ia Biotech) and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The protein after the electrophoresis was stained with Coomassie brilliant blue. The molecular weight markers used were obtained from Amersham Pharmacia Biotech. As a result, it was found that a protein having a molecular weight of 32 kDa was purified (FIG. 2, lane B).

[0035] The purified protein was purified from Applied Biosystems.
Amino acid sequence analysis was performed by automated Edman degradation using a 470A gas phase sequencer (Perkin-Elmer). As a result, the protein was added to the N-terminus by DAAQEP.
It was found to have the amino acid sequence of TGNNAEI (SEQ ID NO: 3). This N-terminal amino acid sequence is
TFPI-2 (PP
It was the same N-terminal amino acid sequence as in 5). TFPI-
SEQ ID NO: 1 shows the entire amino acid sequence of No. 2.

Reference Example 2 Construction of Expression Vector Containing TFPI- 2 cDNA Niidome, T. et al., Biochem. Biophys. Res. C
ommun. 203, 1821-1827 (1994),
An expression vector named pK4KT2 was constructed using the mammalian expression vector pK4K containing single restriction sites for BamHI and HindIII. That is, TFPI-2c
The DNA was ligated to a sense oligonucleotide 5'-ATGGACCCCGCTCCGCC- corresponding to nucleotides 39-54 and 763-782 of TFPI-2, respectively.
3 '(SEQ ID NO: 4) and antisense oligonucleotide 5'-GCCATAAAGACAAACAAGA
It was prepared by polymerase chain reaction (PCR) using T-3 ′ (SEQ ID NO: 5). The template was TFPI-2
pBluescript II SK (-) containing cDNA
(Stratagene). The obtained PCR product is
DNA Blunting Kit (Takara Biomedical
s) and ligated to the pK4K vector, which was similarly blunt-ended. The sequence of the PCR product was determined using an automatic DNA sequencer 373A (Applied Biosyst.
ems, Perkin-Elmer Corp.), it was confirmed to have a nucleotide sequence corresponding to nucleotides 39 to 782 of SEQ ID NO: 2.

Reference Example 3 Transformation and cell culture Baby hamster kidney (BHK) not transformed
k ^- ts13 cells were purified from Dulbecco's modified Eagl supplemented with 5% fetal calf serum (FCS), streptomycin (30 μg / ml) and penicillin (30 units / ml).
e Grow in medium (DMEM). Cell Phe
ct Transfection Kit (Amersham Pharmaci
a, BHKtk ^- ts13 cells (2 × 10
⁵ cells) with 5 μg of pK4KT2 prepared in Reference Example 2.
Transformed with the vector. Transformed cells, designated BHKT2, were grown in DMEM containing 5% FCS. After sorting with 250 nM methotrexate (MTX), cell culture supernatant (condition medium) was collected and used for isolation of recombinant TFPI-2 (rTFPI-2).

Reference Example 4 Purification of Recombinant TFPI-2 (rTFPI-2) Using the same method as described in Reference Example 1, the condition medium (1000 ml) of BHKT2 obtained in Reference Example 3 was used.
Was purified from rTFPI-2. Purified rTFPI
The SDS-PAGE of -2 is shown in FIG.

As a result, it was revealed that a protein having the same molecular weight (32 kDa) as natural TFPI-2 was purified.

Reference Example 5 Mitogenic Activity of rTFPI-2 Bovine aortic smooth muscle cells were isolated from the middle layer of the adult aorta by a modified method of Ross's explant technique.
(Shirotani, M. et al., J. Pharmacol.Exp.Ther. 259, 738
-744 (1990)). 10% FCS, penicillin (100U
/ Ml) and kanamycin (100 μg / ml) were grown at 37 ° C. in a humidified atmosphere of air containing 5% CO ₂ in DMEM. Propagated. Add 3 culture media (DMEM + 10% FCS)
It was changed every day, and after about 7 days, a confluent smooth muscle cell monolayer was obtained. Cells were used from passage 2 to passage 6. The cells were treated with 0.1% trypsin-0.02% EDT
Collected with solution A, 96-well plate (Nunk)
Were seeded at a density of 3,000 cells per well. 4
Eight hours later, the cells were grown in DM with 0.1% FCS.
Stopped with EM. After another 48 hours, fresh medium (D
(MEM + 0.1% FCS) and rTFPI-2 purified at various concentrations in Production Example 4 (final concentration: 0-500 nM)
Was added simultaneously to the growth arrested cells. 48 hours later,
Premix WST-1 cell proliferation assay system (Takara Biom
edical) was used to perform a cell proliferation assay. The system assayes formazone produced from tetrazolium at 420 nm with a reference wavelength of 600 nm. The obtained absorbance values were converted from the standard curve into cell numbers (FIG. 3).

FIG. 3 shows that rTFPI-2 increases cell proliferation in a dose-response manner (1-500 nM).

Reference Example 6 Induction of DNA Synthesis by rTFPI-2 (1) As in Reference Example 5, bovine aortic smooth muscle cells were seeded in a 96-well plate, and cell growth was stopped by DMEM containing 0.1% FCS. I let it. 48 hours later, fresh medium (D
(MEM + 0.1% FCS) and rTFPI-2 purified at various concentrations in Reference Example 4 (final concentration: 0 to 500 nM)
Was added simultaneously to the growth arrested cells. 24 hours later,
5-Bromo-2'-deoxyuridine (BrdU) was added and the Biotrak cell proliferation ELISA system (Amersham)
Pharmacia Biotech Co., Ltd.)
DNA synthesis was detected by measuring the incorporation of rdU into DNA 12 hours later (FIG. 4).

FIG. 4 shows that BrdU is incorporated into DNA in a dose-dependent manner by rTFPI-2 stimulation.

Reference Example 7 Induction of DNA Synthesis by rTFPI-2 (2) In Reference Example 6, 10 ng was used instead of 0.1% FCS.
/ Ml platelet-derived growth factor (PDGF, manufactured by Upstate) in the same manner as in Reference Example 6 except that rTFP was used.
DNA synthesis was induced by I-2 (FIG. 5).

As shown in FIG. 5, 10% FCS was used instead of 0.1% FCS.
Even when using ng / ml PDGF, BrdU
Was found to be incorporated into DNA in a dose-dependent manner with rTFPI-2.

Production Example 1 Production and Purification of Anti-TFPI-2 Polyclonal Antibody Purified in Reference Example 4 dissolved in 1 ml of PBS
μg of rTFPI-2 was mixed with an equal amount of Freund's complete adjuvant (manufactured by WAKO) to form an emulsion, and then a primary immunization was performed by subcutaneous injection into Japanese white rabbits. Fifty days later, 100 μg of rTFPI-2 in 1 ml of PBS was mixed with an equal amount of Freund's incomplete adjuvant (manufactured by WAKO) to form an emulsion, which was used for boosting. 20, after boost
On the 40th day, blood was collected from rabbit artery and serum was separated.
Stored frozen at -80 ° C.

The IgG fraction was purified from the obtained antiserum using a protein G column according to a conventional method.

Example 1 TFPI-2 with anti-TFPI-2 polyclonal antibody
Detection (1) of rTFPI-2 purified in detecting Reference Example 4 of rTFPI-2 purified in Example 4 by Western Blotting SDS-PAGE
And Western blotting was performed by a conventional method. The blotted membrane was incubated with the anti-TFPI-2 polyclonal antibody purified in Production Example 1 and then with a commercially available alkaline phosphatase-conjugated anti-rabbit antibody, and rTFPI-2 was detected by color development.

As a result, it was found that the antibody of the present invention detects rTFPI-2 having a molecular weight of 32 kDa.

(2) Detection of TFPI-2 in Human Blood by Western Blotting A 10 ml sample of human heparin collected during cardiac catheterization was subjected to affinity concentration using a heparin column, and then the above-mentioned (1) was performed.
TFPI-2 was detected as described above.

As a result, it was found that the antibody of the present invention detects TFPI-2 in human blood. By using the antibody of the present invention for ELISA, TFPI in blood
It is also possible to determine -2.

Example 2 Cross-reactivity of anti-TFPI-2 polyclonal antibody TFPI-1 (manufactured by Sanko Junyaku) and rTFPI-2 purified in Reference Example 4 were subjected to Western blotting in the same manner as in Example 1 (1). As a result of detection by a color reaction using alkaline phosphatase, the antibody of the present invention is:
It was found that it did not recognize TFPI-1.

Example 3 TFPI-2 with anti-TFPI-2 polyclonal antibody
In Reference Example 6, rTFPI-2 (final concentration: 0-5)
00 nM), and purified with the anti-TFPI-
After further addition of serial dilutions of the two polyclonal antibodies, detection of DNA synthesis in bovine aortic smooth muscle cells by BrdU incorporation was performed.

As a result, it was found that the addition of the antibody suppressed the DNA synthesis, and the antibody of the present invention inhibited the vascular smooth muscle proliferation effect of TFPI-2.

[0055]

According to the present invention, there is provided an antibody or a fragment thereof specific to TFPI-2, which does not cross-react with TFPI-1, and the physiological role of TFPI-2 is directly clarified by such an antibody. Elucidation of various pathological conditions involving TFPI-2 or research on therapeutic methods is progressing. In addition, a vascular smooth muscle growth regulator containing such an antibody or a fragment thereof is effective in treating or preventing various disease states involving TFPI-2. Further, a diagnostic agent containing such an antibody or a fragment thereof is useful for diagnosis of a disease involving TFPI-2.

[0056]

[Sequence List] SEQUENCE LISTING <110> SHIONOGI & CO., LTD. <120> Tissue Factor Pathway Inhibitor -2 Antibody <130> SG-10-002 <160> 5

<210> 1 <211> 235 <212> PRT <213> Homo sapiens <220> <221> SIGNAL <222> (1) ... (22) <400> 1 Met Asp Pro Ala Arg Pro Leu Gly Leu Ser Ile Leu Leu Leu Phe Leu 5 10 15 Thr Glu Ala Ala Leu Gly Asp Ala Ala Gln Glu Pro Thr Gly Asn Asn 20 25 30 Ala Glu Ile Cys Leu Leu Pro Leu Asp Tyr Gly Pro Cys Arg Ala Leu 35 40 45 Leu Leu Arg Tyr Tyr Tyr Asp Arg Tyr Thr Gln Ser Cys Arg Gln Phe 50 55 60 Leu Tyr Gly Gly Cys Glu Gly Asn Ala Asn Asn Phe Tyr Thr Trp Glu 65 70 75 80 Ala Cys Asp Asp Ala Cys Trp Arg Ile Glu Lys Val Pro Lys Val Cys 85 90 95 Arg Leu Gln Val Ser Val Asp Asp Gln Cys Glu Gly Ser Thr Glu Lys 100 105 110 Tyr Phe Phe Asn Leu Ser Ser Met Thr Cys Glu Lys Phe Phe Ser Gly 115 120 125 Gly Cys His Arg Asn Arg Ile Glu Asn Arg Phe Pro Asp Glu Ala Thr 130 135 140 Cys Met Gly Phe Cys Ala Pro Lys Lys Ile Pro Ser Phe Cys Tyr Ser 145 150 155 160 Pro Lys Asp Glu Gly Leu Cys Ser Ala Asn Val Thr Arg Tyr Tyr Phe 165 170 175 Asn Pro Arg Tyr Arg Thr Cys Asp Ala Phe Thr Tyr Thr Gly Cys Gly 18 0 185 190 Gly Asn Asp Asn Asn Phe Val Ser Arg Glu Asp Cys Lys Arg Ala Cys 195 200 205 Ala Lys Ala Leu Lys Lys Lys Lys Lys Met Pro Lys Leu Arg Phe Ala 210 215 220 Ser Arg Ile Arg Lys Ile Arg Lys Lys Gln Phe 225 230 235

<210> 2 <211> 979 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (39) ... (743) <400> 2 ggacgccttg cccagcgggc cgcccgaccc cctgcaccat ggaccccgct cgccccctgg 60 ggctgtcgat tctgctgctt ttcctgacgg aggctgcact gggcgatgct gctcaggagc 120 caacaggaaa taacgcggag atctgtctcc tgcccctaga ctacggaccc tgccgggccc 180 tacttctccg ttactactac gacaggtaca cgcagagctg ccgccagttc ctgtacgggg 240 gctgcgaggg caacgccaac aatttctaca cctgggaggc ttgcgacgat gcttgctgga 300 ggatagaaaa agttcccaaa gtttgccggc tgcaagtgag tgtggacgac cagtgtgagg 360 ggtccacaga aaagtatttc tttaatctaa gttccatgac atgtgaaaaa ttcttttccg 420 gtgggtgtca ccggaaccgg attgagaaca ggtttccaga tgaagctact tgtatgggct 480 tctgcgcacc aaagaaaatt ccatcatttt gctacagtcc aaaagatgag ggactgtgct 540 ctgccaatgt gactcgctat tattttaatc caagatacag aacctgtgat gctttcacct 600 atactggctg tggagggaat gacaataact ttgttagcag ggaggattgc aaacgtgcat 660 gtgcaaaagc tttgaaaaag aaaaagaaga tgccaaagct tcgctttgcc agtagaatcc 720 ggaaaattcg gaagaagcaa ttttaaacat tcttaatatg tcatcttg tt tgtctttatg 780 gcttatttgc ctttatggtt gtatctgaag aataatatga cagcatgagg aaacaaatca 840 ttggtgattt attcaccagt ttttattaat acaagtcact ttttcaaaaa tttggatttt 900 tttatatata actagctgct attcaatgt tt tt att gt att gt att gt tt tt tt g

<210> 3 <211> 13 <212> PRT <213> Homo sapiens <400> 3 Asp Ala Ala Gla Gln Glu Pro Thr Gly Asn Asn Ala Glu Ile 5 10

<210> 4 <211> 16 <212> DNA <213> Artifical Sequence <400> 4 atggaccccg ctcgcc 16

<210> 5 <211> 20 <212> DNA <213> Artifical Sequence <400> 5 gccataaaga caaacaagat 20

[Brief description of the drawings]

FIG. 1 shows the results of reversed-phase high-performance liquid chromatography using a ProRPC HR 5/10 column. In the figure, the dotted line is from 0.1% trifluoroacetic acid (TFA) in 15% acetonitrile to 0.1% TF in 100% acetonitrile.
The gradient up to A is shown, and the black peak indicates the cell proliferative activity on bovine aortic smooth muscle cells whose growth has been stopped.

FIG. 2 shows SDS-PA of purified native TFPI-2 (lane B) and rTFPI-2 (lane C).
It is a photograph of the electrophoresis which shows the result of GE. Lane A is
A molecular weight marker is shown, and each band is, from the top, phosphorylase b: 94 kDa; bovine serum albumin: 67 kD
a; ovalbumin: 43 kDa; carbonic anhydrase b: 30 kDa; soybean trypsin inhibitor: 20.1 kDa; α-lactalbumin: 14.4
kDa.

FIG. 3 is a graph showing the mitogenic activity of rTFPI-2 at various concentrations (0-500 nM) on bovine aortic smooth muscle cells. Each point in the graph indicates the average of 6 times, and the vertical line indicates the standard deviation.

FIG. 4 shows various concentrations (0-500) of bovine aortic smooth muscle cells arrested with low concentrations of serum.
nM) rTFPI-2 mitogenic activity was BrDU
4 is a graph measured by taking in a sample. Each point in the graph indicates the average of 6 times, and the vertical line indicates the standard deviation.

FIG. 5 is a graph showing the mitogenic activity of rTFPI-2 at various concentrations (0 to 500 nM) on bovine aortic smooth muscle cells whose growth was stopped by DMEM supplemented with PDGF, as measured by BrDU incorporation. Each point in the graph indicates the average of 6 times, and the vertical line indicates the standard deviation.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) C12N 5/10 C12P 21/02 C 15/09 ZNA G01N 33/50 T C12P 21/02 33/53 D G01N 33/50 C12N 5/00 B 33/53 15/00 ZNAA // (C12P 21/02 C12R 1:91) F term (reference) 2G045 AA13 AA25 CA25 DA36 DA77 FB03 4B024 AA01 AA11 BA61 BA80 CA04 EA04 GA11 HA11 4B064 AG01 AG21 AG26 BA14 CA10 CA19 CC24 DA01 DA13 4C085 AA13 AA19 CC03 EE01 GG01 4H045 AA11 BA10 CA40 DA75 DA86 EA23 EA24 EA50 FA72 FA74 HA05

Claims (4)

[Claims] 1. Tissue factor pathway
Does not cross-react with inhibitor (TFPI) -1, T
An antibody or a fragment thereof specific for FPI-2. 2. The method of claim 1, wherein the TFPI-2 is of human origin.
The antibody or the fragment thereof according to the above. 3. A vascular smooth muscle growth regulator comprising the antibody or fragment thereof according to claim 1 or 2 as an active ingredient. 4. A diagnostic agent for a disease associated with blood vessel proliferation, comprising the antibody or the fragment thereof according to claim 1 or 2.