JP3452946B2 - Anti-TGF-β masking protein monoclonal antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a transforming growth factor β (transforming growth fact).
or-β; hereinafter abbreviated as TGF-β) An anti-TGF-β masking protein monoclonal antibody that specifically reacts with a masking protein is provided. The antibody of the present invention is useful for treating diseases caused by TGF-β, such as arteriosclerosis,
Cancer, inflammation, fibrosis diagnostics, therapeutics and latent TGFs
It is useful as a control agent for the growth, function expression, and differentiation of cultured cells caused by -β activation.
In addition, using the antibody of the present invention, latent TG in a tissue section is
The localization of F-β, latent TGF-β masking protein or latent TGF-β binding protein can be analyzed immunochemically.

[0002]

2. Description of the Related Art TGF-β was first discovered as a canceration-promoting factor for certain types of cells [Proc. Natl. Acad. Sci, USA) 80 , 62
64-6268 (1983)] is now found to be a protein that regulates the proliferation and differentiation of many normal cells [J. Cell Biol.].
105, 1039-1045 (1987)]. Recently, glomerulonephritis (Nature 346, 371-374 (1990)) and pulmonary fibrosis (Journal of Experimental Medicine (J. Exp.Med.) 170, 727-737 ( 1989)], liver fibrosis (J. Clin. Invest.) 85 , 1833-1843 (1990)]
Such diseases that are thought to be caused by TGF-β are becoming clear.

TGF-β is produced in vivo as an inactive latent TGF-β which is non-covalently associated with a masking protein (hereinafter abbreviated as MP) which is a specific binding protein. . MP is TGF
-Latency associated peptide, which is the pro-peptide part of the β-precursor
de; hereinafter abbreviated as LAP) and latent TGF-β
Binding protein (latent TGF-β binding
protein; hereinafter abbreviated as LTBP) has a structure in which it is cross-linked by a disulfide bond [Journal of Biochemistry (J. Biochem.) 106, 304-310 (198
9)]. In addition, only low-molecular-weight latent T in which only LAP to which LTBP is not bound is non-covalently associated with TGF-β.
The existence of GF-β is also known [Mol. Cell. Biol. 7, 34
18-3427 (1987)]. Latent TGF-β is activated TGF-β and the binding protein are dissociated by chemical treatment such as acid treatment, urea treatment, and heat treatment in a test tube, and activated TGF-β.
It is known to be converted to β [biochemical
And Biophysical Research Communications (Biochem. Biophys. Res. Commun.) 133, 1026-
1034 (1985)]. Although the activation mechanism in vivo is not always clear, certain types of cultured cells such as vascular cells do not contain latent T
It is known to activate GF-β [J. Cell Biol. 109, 3
09-315 (1989)].

It has been reported that TGF-β neutralizing antibody reduces inflammation in glomerulonephritis [Nature].
(Nature) 346, 371-374 (1990)]. Arteriosclerosis is a pathological condition showing abnormal proliferation of smooth muscle cells and deposition of lipids and extracellular matrix associated with a persistent inflammatory reaction of blood vessel wall. The lesion is regarded as these morphological changes, and no specific diagnostic agent has been reported. It is considered that multiple cytokines are involved in arteriosclerosis, and it is known that interleukin-1, platelet-derived growth factor, tumor necrosis factor, etc. cause the onset and progress of arteriosclerosis [Latest Medical Sciences] , 46 , 485-490 (1991)]. Regarding the involvement of TGF-β in arteriosclerosis, it is known that in the rat arteriosclerosis model, TGF-β gene expression is enhanced in the lesion [Journal of Clinical Investigation ( J. Clin Invest.) 88 ,
904-910 (1991)]. In addition, analysis using TGF-β deficient mice revealed that TGF-β is responsible for suppressing excessive inflammatory reaction in vivo [Nature].
(Nature) 359, 693-699 (1992)], but the physiological significance of TGF-β at the site of arteriosclerosis is not clear.

As an antibody capable of immunohistochemical staining of arteriosclerotic lesions, an antibody against extracellular matrix vitronectin has been reported [The Journal of Biological Chemistry (J. Biol. Chem.) 265 . , 21232-2
1236 (1990)]. As an antibody against latent TGF-β, polyclonal antibodies against LAP and LTBP have been obtained and reported to be usable for immunostaining [J. Biol. Chem (J. Biol. Chem. .) 263, 7646-7654 (1988)
, Embo Journal (EMBO. J.) 18 , 1091-1101 (19
91)].

However, so far, latent TGF-
No monoclonal antibody that recognizes β or MP or its subunits is known. Furthermore, latent TG
Antibodies that suppress F-β activation are not known. In addition, latent TGF-β capable of immunostaining atherosclerotic lesions
There is no known monoclonal antibody against A. cerevisiae, nor is there any known diagnosis or treatment of arteriosclerotic lesions with antibodies against cytokines or related proteins.

If a monoclonal antibody that specifically recognizes latent TGF-β can be obtained, TGF- of arteriosclerosis and the like can be obtained.
It can be used for diagnosis and treatment of various diseases associated with β. Furthermore, if an antibody that suppresses activation of latent TGF-β is obtained, TG can be obtained in the same manner as the TGF-β neutralizing antibody.
It can be clinically applied as a therapeutic drug for a disease caused by F-β. The TGF-β neutralizing antibody targets the TGF-β that has been produced, but the antibody that suppresses the activation of latent TGF-β does not activate the latent TGF-β that has not been activated yet. By targeting, it may act more upstream in the etiology.

[0008]

The object of the present invention is to provide TG
It is intended to provide a latent TGF-β masking protein monoclonal antibody useful as a diagnostic and therapeutic agent for a disease caused by F-β.

[0009]

The present invention relates to an anti-TGF-β masking protein monoclonal antibody which specifically reacts with a TGF-β masking protein. In order to produce the anti-TGF-β masking protein monoclonal antibody of the present invention, first, a hybridoma is prepared by fusing antibody-producing cells of an animal immunized with a complex of MP or a subunit thereof and a carrier protein with mouse myeloma cells. And a hybridoma producing a monoclonal antibody that specifically reacts with latent TGF-β or MP or a subunit thereof is selected. Then, the hybridoma is cultured in a medium or administered to an animal to cause ascites tumor in the animal, and a monoclonal antibody is collected from the culture medium or ascites. A monoclonal antibody that reacts with latent TGF-β or MP or a subunit thereof and does not react with a carrier protein is selected from the obtained monoclonal antibodies. Furthermore, from the monoclonal antibody, a monoclonal antibody having an action of suppressing activation of latent TGF-β or immunohistological staining can be performed by using cultured cells that reproduce latent TGF-β activation in vitro. Select a monoclonal antibody.

The obtained monoclonal antibody or its active fragment F (ab ') ₂ , Fab' or Fab is
It can be used as a drug for suppressing activation of latent TGF-β or a drug for performing immunohistological staining.
Specific examples of hybridomas producing the monoclonal antibody of the present invention include hybridomas KM-704 and KM-699.
Can be given. Hybridoma KM-704, March 1992
FERM B to the Institute of Microbial Technology of the Institute of Industrial Science on 26th
Deposited as P-3809, hybridoma KM-699
Has been deposited as FERM BP-4181 with the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science as of February 9, 1993. The monoclonal antibodies produced by hybridoma KM-704 and hybridoma KM-699 are referred to as KM-704 and KM-699, respectively.

The method for producing the monoclonal antibody of the present invention will be described in detail below. (1) Preparation of Antigen Latent TGF-β can be prepared by known methods [Journal of
Biological Chemistry (J. Biol. Chem.) 263,
6407-6415 (1988)].
TGF-β is known to suppress antibody production by B lymphocytes [Journal of Immunology
(J. Immunol.) 137, 3855-3860 (1986)], it is considered that the purified latent TGF-β is activated in vivo when it is directly administered to animals as an antigen, and antibody production is suppressed. Therefore, the purified latent TGF-β is
It is necessary to dissociate TGF-β with an acid, urea, heat treatment, or the like, and use only MP, which is a binding protein, or its subunit as an antigen.

MP can be prepared, for example, by the following gel filtration method. Latent TGF-β was added to 8M urea and phosphate buffer (pH 7.4)
saline; hereinafter abbreviated as PBS) and dialyzed into MP and T
After dissociating GF-β, it is passed through a gel filtration column equilibrated with PBS containing 6M urea, and the MP fraction is collected.

(2) Immunization of animals and preparation of antibody-producing cells The MP or its subunit prepared in (1) is used as a hemocyanin keyhole limpet (hereinafter, abbreviated as KLH) in order to enhance antigenicity. A) and a carrier protein. The complex of the antigen and the carrier protein is obtained by crosslinking the antigen and the carrier protein with an appropriate crosslinking agent such as glutaraldehyde or m-maleimide-benzoyl-N-hydroxysuccil. It can be produced by

Mice aged 3 to 20 weeks are immunized with the above complex to prepare antibody-producing cells in the spleen, lymph nodes and peripheral blood of the animal. Immunization is performed by subcutaneously or intravenously or intraperitoneally administering an appropriate adjuvant [for example, Complete Freund's Adjuvant
vant), or aluminum hydroxide gel and B. pertussis vaccine, etc.]. Dosage 5-10 every 1-2 weeks after the first dose
Do it once. Blood is collected from the fundus venous plexus 3 to 7 days after each administration, and the following shows that the serum reacts with latent TGF-β or MP or a subunit thereof, enzyme-linked immunosorbentassay; Hereinafter abbreviated as ELISA) [Enzyme-linked immunosorbent assay: published by Ikusho Shoin
1976] and so on. Antibody-producing cells in the spleen, lymph nodes, or peripheral blood of an immunized animal whose serum shows a sufficient antibody titer against the antigen used for immunization are collected.

ELISA : A 96-well enzyme immunoassay plate [manufactured by Greiner] was diluted with PBS to 10 μg / ml of the antigen solution at 50 μg / ml.
Dispense μl / well and leave overnight at 4 ° C to coat the plate with antigen. After removing the antigen solution, 100 μl of PBS solution containing 1% BSA (hereinafter abbreviated as BSA-PBS)
The wells are dispensed and left at room temperature for 1 hour to block (block) the binding residues with the protein remaining on the plate. After that, BSA-PBS was removed, and the wells were thoroughly washed with PBS containing 0.05% Tween (Tween) -20 (hereinafter abbreviated as PBS-Tween), and the wells were washed with BSA-PBS as the primary antibody.
Approximately 1000-fold diluted sample (mouse or rat serum, hybridoma culture supernatant, purified monoclonal antibody) 10
Dispense at 0 μl / well and leave at 4 ° C overnight. Then, after removing the primary antibody and thoroughly washing with PBS-Tween,
Peroxidase-labeled anti-rat or anti-mouse immunoglobulin antibody as secondary antibody [DAKO]
50 μl / well, and leave at room temperature for 2 hours.

The plate subjected to the above operations was washed with PBS-Tween after removing the secondary antibody, and then the ABTS substrate solution [2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] was added. 550 mg of diammonium is dissolved in 1 liter of 0.1 M citrate buffer (pH 4.2), and 1 μl / ml of hydrogen peroxide is added to the solution immediately before use] to develop color and measure the absorbance at 415 nm. .

(3) Preparation of myeloma cells As myeloma cells, cell lines obtained from mice are used. For example, 8-azaguanine resistant mice (BALB / c
Origin) Myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current
Current Topics in Microbiology and Immun
ology) 81 , 1-7 (1987)], P3 / NS1 / 1-Ag4-1 (NS-1)
(European Journal of Immunology (Eur
opean J. Immunology) 6, 511-519 (1976)], SP2 / 0-Ag
14 (SP-2) (Nature ( 276 ) , 269-270 (1978)
], P3-X63-Ag8 653 (653) (Journal of Immunology (J. Immunol.) 123, 1548-1550 (1979)),
P3-X63-Ag8 (X63) (Nature 256, 495-497
(1975)] is used. These cell lines are 8-
Medium containing azaguanine [RPMI-1640 medium with glutamine
(1.5 mM), 2-mercaptoethanol (5 × 10 ^-5 M), gentamicin (10 μl / ml) and fetal calf serum (FC
S) (CSL; 10%) added to the normal medium,
-Azaguanine (15 μg / ml) -containing medium] to give an 8-azaguanine resistant strain.
Secure ⁷ or more cells to use as the parent strain for cell fusion.

(4) Preparation of hybridoma A hybridoma is prepared by fusing the antibody-producing cells prepared in (2) with the myeloma cells prepared in (3). Cell fusion can be achieved by polyethylene glycol (polyethylene glycol;
Hereinafter, abbreviated as PEG), a method using a fusion promoter such as Sendai virus, and a physical method using freeze-thawing, electric pulse and the like can be used.

For example, when PEG is used, the antibody-producing cells and the myeloma cells are mixed so that the cell number becomes 5 to 10: 1, and the mixture is stirred in a medium containing PEG having an average molecular weight of 1,000 to 6,000. To prepare a hybridoma. Selection of hybridoma and other unfused cells can be performed using a selection medium. As the selective medium, for example, HA
T medium (normal medium containing hypoxanthine (10 ^-4 M), thymidine
(1.5 × 10 ⁻⁵ M) and aminopterin (4 × 10 ⁻⁷ M) ^-containing medium] can be used. Unfused antibody-producing cells and myeloma cells die within 10 days in HAT medium and only hybridomas grow, so cells that grow for 2 weeks or more in HAT medium are collected as hybridomas.

The prepared hybridoma is measured for antibody titer against MP or its subunit. The antibody titer can be measured by, for example, dispensing an appropriately diluted hybridoma suspension into a 96-well culture plate, culturing for 7 to 14 days, collecting a part of the culture supernatant, and measuring MP or its MP by ELISA or the like. Select cells in wells that show a binding reaction with the subunit. Re-dilute the cells in the selected wells
Dispense into a 96-well culture plate, repeat cloning by the limiting dilution method, and select a stable stable antibody titer as an anti-TGF-β masking protein monoclonal antibody-producing hybridoma.

(5) Preparation of Monoclonal Antibody As a method for obtaining a monoclonal antibody from the hybridoma obtained in (4), a method of culturing the hybridoma and recovering it from the culture supernatant, and the hybridoma is intraperitoneally administered to an animal The method of recovering from the ascites obtained by A method for obtaining ascites by intraperitoneally administering a hybridoma to an animal will be described below.

Obtained in (4) after administration of mineral oil such as pristane [2,6,10,14-tetramethylpentadecane (Pristane)] to animals or nude mice having major histocompatibility with hybridoma When the animals to which anti-TGF-β masking protein monoclonal antibody-producing hybridoma cells are administered are nude mice, 2 × 10 5
^{6 to} 5 × 10 ⁷ are intraperitoneally administered. The hybridoma becomes ascites tumor 10 to 21 days after administration.

Monoclonal antibodies are purified from the obtained hybridoma culture supernatant or ascites by commonly used chromatography such as ion exchange resins and gel filtration resins. When the monoclonal antibodies are of the IgG class, they have an affinity for IgG. Protein-A and Protein-
It is preferable to purify by affinity chromatography using a resin or the like on which G or the like is immobilized. The amount of purified IgG is calculated by the dye binding method or the absorbance at 280 nm.

The subclass of the obtained monoclonal antibody is determined by ELISA using an antibody specific to the subclass of the immunized animal.

(6) Antigen Specificity of Anti-TGF-β Masking Protein Monoclonal Antibody The antigen specificity of the anti-TGF-β masking protein monoclonal antibody obtained in (5) depends on the latent TGF-β or MP or its subunit. Responsiveness to ELIS
It can be confirmed using A or immunoblotting.

For example, Western blotting (we
stern blotting) method (Proc. Natl. Acad. Sci, USA) 76 , 4350-4354
(1979)] can be used to confirm the antigen specificity of the monoclonal antibody as follows. Treatment of latent TGF-β with a denaturing agent such as sodium dodecyl sulfate (hereinafter abbreviated as SDS)
And TGF-β dissociated from the sample, or treated with a reducing agent such as 2-mercaptoethanol to reduce MP to LTBP and L
The sample cleaved into AP was SD-treated according to the Laemmli method [Nature 227, 680-685 (1970)].
S-containing polyacrylamide gel electrophoresis (polyacry
lamide gel electrophoresis; hereinafter referred to as SDS-PAGE). In addition, non-denaturing latent TGF-β not treated with SDS and 2-mercaptoethanol was subjected to polyacrylamide gel electrophoresis without SDS (hereinafter referred to as NaDS).
abbreviated as tive-PAGE).

The latent TGF-β or MP or its subunits separated in the gel by Native-PAGE or SDS-PAGE is subjected to polyvinylidene difluoride (poly) according to the Western blotting method described above.
vinylidene difluolide (hereinafter abbreviated as PVDF)) and the like are transferred onto a membrane that binds a protein. Then, latent TG
A part of the membrane on which the F-β subunit is transferred is stained with a protein stain such as Coomassie brilliant blue (hereinafter abbreviated as CBB) -R250. The unstained transfer membrane is the anti-TGF-β selected in (5).
Masking protein monoclonal antibody (primary antibody)
And then reacted with a secondary antibody that is specific to the primary antibody and is labeled with an enzyme or the like. After the reaction, the substrate is reacted with the labeled secondary antibody on the membrane to develop the color of the transfer membrane.

In the above-described operation, the latent latent T which is not denatured
When GF-β was subjected to Native-PAGE, latent TGF-β was transcribed on the membrane and treated with a denaturing agent to give SDS-PA.
When subjected to GE, latent TGF-β showed MP and T
When dissociated into GF-β and transcribed, and treated with denaturing and reducing agents and subjected to SDS-PAGE, MP is LTBP.
And is cleaved into LAP and transcribed. Therefore, by comparing the immunostained band on the membrane with the protein-stained band, the protein to which the anti-TGF-β masking protein monoclonal antibody binds or its subunit can be determined.

(7) Selection of Monoclonal Antibody Inhibiting Latent TGF-β Activation Monoclonal antibodies inhibiting latent TGF-β activation are known latent TGF-β activating cells [protein nucleic acid enzyme, 36 , 1350-1356 (1991)].

For example, a coculture system of vascular endothelial cells and smooth muscle cells [Journal of Cell Biology (J. C
ell Biol.) 109, 309-315 (1989)] is as follows. Bovine aortic endothelial cells (bovine a) that have been confluent in a culture dish.
An ortic endothelial cell (hereinafter abbreviated as BAE) is injured with a razor blade to create a pseudo-wound state in which some BAE are detached. Bovine aortic smooth muscle cells (bMC) are added to this pseudo-wounded BAE and cocultured. The anti-TGF-β masking protein monoclonal antibody purified in (5) is added to this coculture system.

In the above co-culture system, latent TGF
Whether BAE activation was suppressed is described below.
It is judged from the change in the number of migrating cells. BAE in pseudo wound condition
In single culture, cells migrate to the detached dish, but coculture with SMC activates latent TGF-β produced by BAE and SMC to suppress BAE migration. Therefore, if the added anti-TGF-β masking protein monoclonal antibody has an activity of suppressing the activation of latent TGF-β, BAE migration is restored.

The number of BAEs that have migrated is measured as follows. After 20 hours of co-culture, the medium is removed and the cells are fixed with methanol. The immobilized cells were stained by the Giemsa method (TISSUE CULTURE Methods and Applications, published by Academic Press (1973)), and the number of BAE migrating from the exfoliated end was optically determined. Measure with a microscope. BAE is derived from the morphology of the whole cell and nucleus.
It can be distinguished from SMC.

Based on the above, the number of BAEs that have migrated is increased as compared with the case where no monoclonal antibody is added or the case where a control monoclonal antibody which does not have an action of suppressing activation of latent TGF-β is added. Can be selected as an antibody having an action of suppressing activation of latent TGF-β.

(8) Selection of monoclonal antibody capable of immunohistostaining A monoclonal antibody that specifically recognizes an arteriosclerotic lesion can be obtained by a known immunohistostaining method [Antibodies-A Laborator].
y Manual, Cold Spring Harbor Laboratory, 1988)
(Actor / Hist Chemistry / Site Chemistry (A
Cytachemtochem. Cytochem.) 23 , 647-661 (1990)], using the monoclonal antibody obtained in (5) as the first antibody against the tissue section, and selecting one that stains the tissue section positively. be able to.

Examples of the present invention will be shown below.

[0036]

【Example】

(1) Preparation of antigen MP, which is an antigen, is purified from latent TGF-β to TG
It was prepared by dissociating F-β. Latent TGF
-Β is a known method [The Journal of Biological Chemistry (J. Biol. Chem.) 263, 6407-64].
15 (1988)] and purified from human platelets. MP was prepared from latent TGF-β by the gel filtration method shown below.

The latent TGF-β was dialyzed against PBS containing 8M urea and passed through a Superose 6 column (Pharmacia) equilibrated with PBS containing 6M urea to collect the MP fraction.
The recovered MP was dialyzed against PBS and used for the next operation.

(2) Preparation of antibody-producing cells MP which is an antigen and KLH which is a carrier protein are mixed at a protein ratio of 1: 5, and 0.02M glutaraldehyde is used in the presence of 0.1M ammonium acetate. Known methods (Proceeding of Society of Experimental Biological Medicine (Proc.
Soc. Exp. Biol. Med.) 148, 784-789 (1975)], and MP-KLH complex was prepared and dialyzed against PBS to obtain an immunogen. 100 for each rat and mouse
μg of MP-KLH complex was administered with 2 mg of aluminum gel and 1 × 10 ⁹ cells of pertussis vaccine (Chiba Serum Research Institute), and 2 weeks later, 100 μg of MP-KLH complex was administered.
The complex was administered once a week for a total of 6 times.

Rats and mice immunized as described above were respectively bled from the fundus venous plexus and the serum antibody titer was determined by ELIS.
Spleens were excised from the rats and mice showing sufficient antibody titer at 3 days after the final immunization. The extracted spleen is
Shred in Eagle essential basic medium (minimum essential medium; Nissui Pharmaceutical Co., Ltd .; hereinafter abbreviated as MEM medium),
Centrifuge at 1,200 rpm for 5 minutes, remove the supernatant, treat with Tris-ammonium chloride buffer (pH 7.65) for 1-2 minutes to remove erythrocytes, and wash the cells with MEM medium 3 times. It was subjected to fusion.

(3) Preparation of mouse myeloma cells P3-U1 which is a mouse myeloma cell line subcultured in 8-azaguanine medium was subcultured in normal medium 4 days before cell fusion, and 2 × 10 5 cells were cultured on the day of cell fusion. A cell number of ⁷ or more was obtained and used as a parent strain for cell fusion.

(4) Preparation of hybridoma Spleen cells obtained in (2) and myeloma cells obtained in (3)
After thorough washing with MEM medium, the spleen cell count was
Mixed to be 1: 1. Centrifuge at 1,200 rpm for 5 minutes
After that, the supernatant was removed. The precipitated cells were loosened well
Then, with stirring, at 37 ° C, 2 g of PEG-1,000 and 2 ml of MEM.
And a mixture of 0.7 ml of dimethyl sulfoxide to 10⁸ Individual
0.2-1 ml was added per cell. Every 1-2 minutes in this mixture
After adding 1-2 ml of MEM medium several times, add MEM medium and
The volume was 50 ml. After centrifugation at 900 rpm for 5 minutes, the supernatant
Discard the cells, loosen the cells gently, and then
The cells are gently sucked in and blown out with HAT medium (normal
Add hypoxanthine (10 ^-FourM), thymidine (1.5 × 10
^-FiveM) and aminopterin (4 × 10^-7M)
Ground] Suspended in 100 ml.

This cell suspension was dispensed into a 96-well culture plate at 100 μl / well and cultured at 37 ° C. for 14 days in a 5% CO ₂ incubator. After culturing, a part of the culture supernatant was collected, and those showing a binding reaction with MP were selected using ELISA. Then, the cloning was repeated twice by the limiting dilution method (the first time was HT medium (medium in which aminopterin was removed from HAT medium), the second time was normal medium), and a stable strong antibody titer was observed. Selected strains.

On a 96-well enzyme immunoassay plate [manufactured by Greiner], MP prepared in (1) was added to PBS.
The antigen solution diluted to 10 μg / ml with 50 μl / well was dispensed and left overnight at 4 ° C. to coat the plate with MP. After removing the antigen solution, 100 μl / well of BSA-PBS was dispensed and left at room temperature for 1 hour to block the binding residue with the protein remaining on the plate. After that, BSA-PBS
After removing the cells and thoroughly washing with PBS-Tween, 100 μl of the culture supernatant of the hybridoma strain selected above was used as the primary antibody.
Aliquots were dispensed at 1 / well and left overnight at 4 ° C.

After removing the primary antibody and washing thoroughly with PBS-Tween, a peroxidase-labeled anti-rat or anti-mouse immunoglobulin antibody [DAKO (DAK
O)) was dispensed at 50 μl / well and left at room temperature for 2 hours. After the secondary antibody was removed from the plate thus obtained and the plate was thoroughly washed with PBS-Tween, color was developed using an ABTS substrate solution, and the absorbance at 415 nm was measured. As a result, a strain showing an increase in absorbance was used as a hybridoma producing an anti-TGF-β masking putein monoclonal antibody, and a hybridoma KM-69 producing a rat anti-TGF-β masking protein monoclonal antibody.
8, KM-699, KM-700, KM-701, KM-702, KM-703, KM-704, KM-705
And KM-706, a hybridoma KM-7 producing a mouse anti-TGF-β masking protein monoclonal antibody
I got 07 respectively.

(5) Preparation of Monoclonal Antibody Pristane-treated 8-week-old female nude mice treated with 0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane intraperitoneally and fed for 2 weeks) were The hybridoma obtained in 4) was intraperitoneally injected with 2 × 10 ^{6 to} 5 × 10 ⁷ cells per mouse. The hybridoma developed ascites tumor 10 to 21 days after the injection.

1 to 1 per mouse from ascites-collected mice
8 ml of ascites was collected. The ascites was centrifuged at 3,000 rpm for 5 minutes to remove the solid content, and then passed through a Protein-G Sepharose (Pharmacia) column equilibrated in PBS to specifically adsorb IgG to the column. Column to PB
After washing with S, it was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.7). The IgG fraction of the eluate was collected and dialyzed against PBS to give a purified monoclonal antibody.

Hybridoma KM-698, KM-699, KM-700,
Purified monoclonal antibody KM-698, respectively from KM-701, KM-702, KM-703, KM-704, KM-705, KM-706 and KM-707
KM-699, KM-700, KM-701, KM-702, KM-703, KM-704, KM
-705, KM-706 and KM-707 were obtained. The protein concentration of each antibody is the molecular extinction coefficient of IgG [1.0 A ₂₈₀ (1 cm) = 1.4 mg / m
l] was calculated. The subclass of each monoclonal antibody was determined by ELISA using a rat monoclonal antibody typing kit (Nordic Immunology) or a mouse monoclonal antibody typing kit (Zymet).

(6) Antigen Specificity The antigen specificity of the anti-TGF-β masking protein monoclonal antibody obtained in (5) was confirmed using the Western blotting method shown below.

The latent TGF-β prepared in (1) was added with 1% SD
After degrading by S and 2% 2-mercaptoethanol treatment (hereinafter referred to as reduction treatment) or 1% SDS treatment not containing 2-mercaptoethanol (hereinafter referred to as non-reduction treatment) to cleave into subunits , SDS-PAGE using polyacrylamide gel (SPG-520L, manufactured by Atto Co.) according to the Laemmli method. The native latent TGF-β not treated with SDS and 2-mercaptoethanol was subjected to Native-PAGE using polyacrylamide gel (Daiichi Pure Chemicals Co., Ltd., PAG plate 4/15 type). In addition, in each PAGE, as a protein for molecular weight assay, a standard protein (in SDS-PAGE, protein molecular weight marker manufactured by Daiichi Kagaku Co., Ltd. “Daiichi” II), in Native-PAGE, protein molecular weight marker manufactured by the same company "Daiichi" and I) were simultaneously run.

The latent TGF-β subunit separated in the gel by SDS-PAGE or Native-PAGE was electrophoretically converted into PVDF by Western blotting.
It was transferred onto a membrane (Millipore). Part of the PVDF membrane on which the latent TGF-β subunit was transferred was stained with CBB-R250, and the unstained PVDF membrane was blocked with 1% BSA solution, washed well with PBS-Tween, and then used as the primary antibody. As BS
Anti-TGF-obtained in (5) diluted to 10 µg / ml with A-PBS.
2 with β-masking protein monoclonal antibody at room temperature
Reacted for hours. Then, after thoroughly washing the PVDF membrane with PBS-Tween, anti-rat or anti-mouse immunoglobulin antiserum labeled with peroxidase (manufactured by Dako) was used as a secondary antibody and reacted at room temperature for 2 hours. After thoroughly washing the PVDF membrane that has undergone the above reactions with PBS-Tween,
The labeled secondary antibody on the membrane was developed using Konica Immuno Stain Kit (manufactured by Konica).

By comparing the band on the PVDF membrane immunostained as described above with the band subjected to CBB-R250 staining, the latent TGF-β subunit to which the anti-TGF-β masking protein monoclonal antibody binds was identified. You can decide. Table 1 shows the antigen specificity of the monoclonal antibody obtained in (5).

[0052]

[Table 1]

FIG. 1 shows the result of analyzing the antigen specificity of the monoclonal antibody KM-704. KM-704 is Native
-Western blotting by PAGE to latent type T
Reacting with GF-β and non-reducing SDS-PAGE
It was found that Western blotting according to the above-mentioned reaction to MP, and further Western blotting according to the reduction treatment by SDS-PAGE reacted to LAP of MP subunit. (7) Selection of monoclonal antibody that suppresses latent TGF-β activation The cells were cultured in α-MEM (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with 0.1% gelatin and 10% FCS to prepare an animal cell culture dish (diameter 35 m).
BAE that had become confluent in m) was injured with a razor blade to create a pseudo-wound condition in which some BAE were detached. Immediately apply 1 to this pseudo-wounded BAE.
Co-culture was performed by adding 5,000 cells / dish of SMC. In this co-culture system, anti-TGF-β masking protein monoclonal antibody KM-698, KM-699, KM-700, KM- prepared in (5)
701, KM-702, KM-703, KM-704, KM-705, KM-706 and KM-707
Was added to each so that the final concentration was 10 μg / ml.

After 20 hours of co-cultivation, the medium was removed, the cells were fixed with methanol, the cells were stained by the Giemsa method, and the number of BAE migrated from the exfoliation end was measured using an optical microscope. The result is shown in FIG. As shown in Figure 2, (5)
Among the TGF-β masking protein monoclonal antibodies prepared in step 2, BAE that migrated when KM-704 was added
The number of cells migrated in the BAE monoculture without SMC.
It recovered to the same level as the number of BAE. Therefore, KM-704 was selected as a monoclonal antibody that suppresses activation of latent TGF-β. In the above operation, the concentrations of the monoclonal antibodies KM-700 and KM-704 were adjusted to 0.3-
The antibody concentration dependence of the number of BAEs that migrated was changed to 30 μg / ml. The result is shown in FIG. As shown in FIG. 3, KM-704 completely restored the migration of BAE at 10 μg / ml, whereas monoclonal antibody KM-700 was also able to recover at 30 μg / ml.
Did not restore BAE migration.

(8) Selection of monoclonal antibody capable of immunohistostaining A monoclonal antibody capable of immunohistostaining uses a human arteriosclerotic lesion as a tissue section, and a known immunocolloid gold-silver method [Acta Hist Chemistry] is used.・ Site chemistry (Acta Histochem. Cytochem.) 23 , 647-661 (199
0)]. Specifically, it carried out as follows. The area of atherosclerotic lesion of human coronary artery obtained by autopsy was fixed with phosphate buffered paraformaldehyde, dehydrated with ethanol, treated with benzene, and embedded in paraffin. A 6 μm-thick section was prepared from this embedded tissue using a microtome, adhered to a slide glass coated with ovalbumin, deparaffinized with xylene, and hydrophilized with ethanol. The tissue sample prepared by the above procedure was washed with PBS, blocked with a 5% ovalbumin solution, and diluted to 10 µg / ml with the monoclonal antibody obtained in (5) (PBS containing 1% BSA). Was used as a primary antibody for 1 hour at room temperature. This was washed with PBS, blocked with a 5% ovalbumin solution, and then immunocolloidal gold solution [particle size 5 µm, diluted 80 times with PBS containing Janssen, 1% BSA] at room temperature. The reaction was carried out for 1 hour. After washing this with PBS, a physical developer containing bromohydroquinone (Acta Histochem. Cytoche
m.) 23 , 647-661 (1990), using Developer 2]. After washing this with running water and fixing with a photographic fixing solution (diluted 5 times with distilled water),
It was washed again with running water.

The tissue section subjected to the above immunostaining was then subjected to nuclear staining with Cologne-Echroth's solution, dehydrated, sealed and examined under a microscope. As a result of the above operation, the anti-latent TGF-β monoclonal antibody (KM-699 to 707) obtained in (5)
Among them, KM-699 and KM-700 showed a strong immunostaining positive staining image in the intimal thickened part of the atherosclerotic lesion. KM-699
When stained with a monoclonal antibody other than KM-700 and KM-700, no immunostaining positive area was observed. Figure 4
Shows the results of immunostaining using KM-699.

[0057]

INDUSTRIAL APPLICABILITY According to the present invention, anti-TGF-useful as a diagnostic agent and therapeutic agent for diseases caused by TGF-β
A β-masking protein monoclonal antibody is provided.

[Brief description of drawings]

FIG. 1 is a diagram in which the antigen specificity of monoclonal antibody KM-704 is analyzed by Western blotting. A-
1 is Native of latent latent TGF-β that does not contain SDS
-CBB-R250 staining after PAGE, A-2 immunostaining after PAGE, B-1 non-reduced latent TGF-β SDS-PA.
CBB-R250 staining after GE, B-2 immunostaining after the same SDS-PAGE, C-1 staining CBB-R250 after SDS-PAGE of latent TGF-β subjected to reduction treatment, C-2 Shows immunostaining after the same SDS-PAGE. The leftmost lane (Marker) of each panel
Indicates CBB-R250 staining of standard protein, and the numerical value indicates molecular weight.

FIG. 2 shows migration BA in co-culture of BAE and SMC.
Monoclonal antibody to E number KM-698, KM-699, KM-70
0, KM-701, KM-702, KM-703, KM-704, KM-705, KM-706 and K
It is a figure showing the influence of addition of M-707. The left edge shows the number of migrated BAE when only BAE without SMC was cultured alone.

FIG. 3 is a diagram showing the numbers of migrated BAEs when the antibody concentrations of monoclonal antibodies KM-704 and KM-700 were changed to 0.3 to 30 μg / ml, respectively. SMC is not added to the left end
The number of migrated BAE when only BAE was cultured alone is shown.

FIG. 4 shows a micrograph of human arteriosclerotic lesions stained by immunocolloidal gold-silver method using monoclonal antibody KM-699. (A) is a photograph with a magnification of 4 times under a microscope, and (B) is a photograph with a magnification of 200 times. Although immunostaining spots of gold-silver staining positive are seen throughout the specimen section, strong immuno- colloidal gold-silver staining positive image is observed especially in the intimal thickening region of the arteriosclerotic lesion.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12R 1:91) (56) References Proc. Natl. Acad. Sc i. USA, 1990, Vol. 87, No. 22, pages 8835-9 Biochem. Biophys. Res. Commun. , 1986, Vol. 141, No. 1, pages 176-84 J. Biochem. , 1989, Vol. 106, No. 2, pages 304-10 FEBS Lett. , 1989, Vol. 242, No. 2, pages 240-4 (58) Fields investigated (Int.Cl. ⁷ , DB name) C07K 16/18 BIOSIS / WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A specific reaction to a latent transforming growth factor β (transforming growth factor-β; hereinafter abbreviated as TGF-β) masking protein and inhibition of activation of latent TGF-β. Group consisting of the following (a) and (b)
A monoclonal antibody or a binding active fragment thereof selected from (A) Monoclona produced by FERM BP-3809
Antibody (b) to which the monoclonal antibody described in (a) above binds
Monochrome binding to the TGF-β masking protein region
Internal antibody 2. The monoclonal antibody according to claim 1, which specifically reacts with the latency associated peptide in the TGF-β masking protein, or a monoclonal antibody thereof.
Binding active fragment. 3. A hybridoma which produces the monoclonal antibody according to claim 1 or 2. 4. The hybridoma according to claim 3, wherein the hybridoma is the hybridoma cell line FERM BP-3809. 5. A monoclonal antibody produced by the hybridoma according to claim 4.