JPH02268694A - Thrombolytic agent - Google Patents

Abstract

PURPOSE:To obtain a thrombolytic agent which can be used as a remedy for patients with thrombosis by using a protein having an activity of tissue plasminogen activating protein and a monoclonal antibody inhibiting the plasmin- inhibiting activity of alpha2PI. CONSTITUTION:The subject thrombolytic agent contains, as active ingredients, a monoclonal antibody which is a monoclonal antibody against human alpha2Pl and inhibits the action of plasmin to inhibit the fibrinolytic action of plasmin and the segment having at least the Fab zone and another protein activating tissue plasminogen.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention relates to human α2-plasmin inhibitor ((X
2-plaslin inhibitor:
α 2 -anjlplasmin: Hereafter α2P
monoclonal antibodies directed against the fibrinolytic site of α2PI (reactiVe
5ite) as an antigen, and as a result suppresses the inhibitory activity of α2PI against the fibrinolytic action of plasmin and promotes fibrinolysis.
Thrombosis (thrombosis) with two active ingredients
), diffuse angiocoagulopathy (DIC: D lss
eminatecl intravascularco
The present invention relates to a thrombolytic agent used as a new therapeutic agent for thrombotic diseases such as agulation), myocardial infarction, cerebral infarction, deep vein thrombosis, thrombotic preparatory state with a strong tendency to arteriosclerosis, and a method for using the same.

In particular, the monoclonal antibody is linked to plasminogen activating proteins, especially tissue plasminogen activator (tisslIe plasminogen act+).
Vator (sometimes abbreviated as tPA in the future) can be used in combination or simultaneously to improve the effectiveness of the drug compared to administering each alone.
The present invention relates to a thrombolytic agent that exhibits a fibrinolytic effect and a method for administering the same. Furthermore, the monoclonal antibody was combined with plasmogen activation protein, especially tPA and 01
By using or co-administering tPA alone, it is possible to achieve the same level of fibrinolytic effect with a higher level of tpA suspension than when tPA is administered alone, and it is possible to reduce the amount of tI)A used. This invention relates to a thrombolytic agent and its administration method.

b. Prior art H1-C2-plasmin inhibitor was first isolated and produced by a tree and valve opening, and instantaneously inhibited the conistertase activity of plasmin, which is dissolved in a linear t/ft. A powerful plasmin inhibitor that inhibits
It is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 and containing 11.7% sugar [M
oroi &Aoki;Ding'
he Journal of Biolog
ical hemistry, 251.595
See 6-5965 (197G).

It is known that human α2PI has three types of active sites. The first is a site that inhibits plasmin's cytolytic action (hereinafter sometimes referred to as the "reactive site") [B, Woman &Co11en]
; The Journal of 3io1ogi
caChemistry, 254.9291-92
9i' (1979) @ Teru] 'c is present, the second is the plasmin binding site on the carboxyl terminal side [3, Woman
& D, Co11en;European
Journal of 3 iochemistry
, 84573-578 (1978) Reference 1, and the third is the fibrin binding site at the amine 15 end [
Y, 3akata.

et al, Thrombosia Qesea
rch, 16. 279-282 (1979) 1 If it is possible to provide a monoclonal antibody that selectively recognizes reactive sites 1 to 1 as antigens among these three types of active sites in d3 of human α21]I, by using this monoclonal antibody, This is very interesting because it can directly suppress the fibrinolytic inhibitory effect of human α2PI and promote fibrinolysis.

On the other hand, thrombotic diseases are caused by changes in blood properties and depression of blood flow.
It is a disease in which blood coagulates within blood vessels due to changes in the fn tube wall, etc., and DIC is caused by the invasion of coagulation activating factors into blood vessels.
It is a disease in which blood clots form in small blood vessels throughout the body. In these treatments, an enzyme for activating plasminogen, such as tPA, is administered for the purpose of dissolving thrombus.

However, it was not effective because plasmin was quickly inhibited by α2PI in the blood. Therefore, α2 in the blood
It is predicted that if a monoclonal antibody that directly suppresses the inhibitory effect of PI on plasmin activity is used in conjunction with enzyme therapy, thrombi can be rapidly eliminated.

C1 Structure of the Invention The present invention first provides a monoclonal antibody against human α2Pl, which suppresses the inhibitory effect of plasmin on the cytoplasmic lytic zone of human α2PI, or a fragment thereof containing at least a Fab portion. It is a thrombolytic agent whose active ingredients are plasminogen and tissue plasminogen activating protein.

The anti-hi-α2PI monoclonal antibody, which is one of the components of the thrombolytic agent of the present invention, suppresses the activity of human α2PI, and binds directly to the reactive site among the three types of active sites, or It binds to its vicinity and suppresses its activity.

These monoclonal antibodies are not limited to mouse-derived animals, but are also applicable to guinea pigs 1-. It may be derived from rats, rabbits, sheep, sheep, goats, pigs, dogs, cats, cows, horses, etc. Furthermore, Kudo et al.
et al, J, [mmunol, 135°64
2-645 (1985)), chimeric antibodies may be used, such as those in which the variable region of an antibody derived from a non-human animal and the constant region of an antibody derived from a human are combined.

Naturally, such an antibody does not have to be used in its entirety, but may be reduced in molecular weight by enzymatic treatment or produced directly as a low molecular weight product by genetic manipulation. Examples of such low molecular weight substances include (Fab')z and Fat). Also, Bird et al.
242. 423 (1988)) may be a single heavy chain type antibody. All of these will hereinafter be collectively referred to as ['fO, clonal antibodies].

The amount of such monoclonal antibodies may be extremely small, for example, 0.01 uM or more, preferably 0.1 μM or more. Furthermore, if it exceeds 5.0 μM, there is virtually no effect of increasing the amount, so it is not necessarily necessary to have such a multilevel presence. Usually, it may be used at a concentration of 0.8 to 1.2 μM.

An example of a protein having the activity of tissue plasminogen activating protein, which is the other component, is so-called tPA. Examples of tpΔ include two-chain m tPA and single-chain t P A /JX.

These drugs begin to exhibit effects even after administering only 1 nQ/d. It is preferably iong/'d or more.

Once you exceed 120 ngl, the meaning of using the above-mentioned antibodies in combination becomes even more important. Therefore, preferably 120ng
/d, more preferably 1100n/d or less, more preferably 6
0ugld or less.

Although these monoclonal antibodies and the protein may be mixed in their original form, they are preferably dissolved in an aqueous medium. As such an aqueous medium, a conventionally known medium such as physiological saline, which does not contain components that impair protein activity, is used.

Other additives may be mixed into the solution thus obtained.

As mentioned above, the thrombolytic agent of the present invention is effective for thrombosis.

It is used to treat diffuse intravascular coagulation, myocardial infarction, cerebral infarction, deep venous thrombosis, and a thrombotic semi-IFI state with a strong tendency to 8 artery sclerosis. It is performed by injecting it into a blood vessel when a dangerous situation occurs.

One of the basic technical ideas of the present invention is that when trying to dissolve a thrombus using a protein that controls P△ activity, the anti-inflammatory protein prevents [)△ from disappearing in the body in an extremely short period of time. α2PI
Although the aim is to prevent thrombolysis using monoclonal antibodies, such effects can be achieved without necessarily using the above-mentioned thrombolytic agents. That is, the anti-α2P [monoclonal antibodies] are injected before the protein with tPA activity is injected, and when the protein is injected later, they end up in the body and the two coexist, thereby achieving the objective. achieve.

Thus, as a second invention, the present application also includes the monoclonal antibodies as a pre-administration agent prior to administration of a protein having tPA activity. The time taken for the monoclonal antibodies to disappear in the body is considerably longer than that of the protein, and therefore, the effect is exerted even when administered 40 hours before administration of the protein having tPA activity. Of course, even shorter time intervals may be used, such as 24 hours ago, or several minutes for a 6-hour history.

When used as such a pre-administration agent, the administration format is as follows:
As mentioned above, the monoclonal antibodies are preferably dissolved in an aqueous medium and used as an injection solution.

The content of the monoclonal antibodies in this injection solution is not particularly limited, but is usually 0.01 uM to 5.0 μM.
0 μM, preferably in the range of 0.8 μM to 1.2 μM. As the aqueous medium, the aqueous medium mentioned above in the description of the thrombolytic agent can be used as is.

The pre-administration solution thus formed may contain various additives in addition to the above-mentioned components.

By administering the pre-administration agent thus obtained before tPA administration, the practical efficiency of tPA can be significantly increased, and thus the use of tPA can be significantly reduced.

Furthermore, the monoclonal antibodies used in the invention also have the effect of helping plasminogen bind to blood clots, thereby promoting thrombolysis. However,
Another invention encompassed by the present application is a drug comprising the monoclonal antibodies that promotes plasminogen binding to blood clots.

The thrombus/plasminogen binding promoter is composed of the monoclonal antibodies, and is preferably used in a form dissolved in an appropriate aqueous medium.

In this case, the aqueous medium described in the description of the thrombolytic agent can basically be used.

These may contain added auxiliary agents used in general injection solutions.

The content of the monoclonal antibodies in the aqueous medium is usually 0.01 to 5. OAlM, preferably 0.
It is 8-1.2 μM.

As mentioned above, the present inventors have demonstrated that by adding the monoclonal antibody, spontaneous lysis of fibrin clots (spontaneous lysis)
taneous 1ysis) and elucidated its mechanism. First, we changed the m degree of the monoclonal antibody in the blood and observed the degree of thrombolysis.
At 0.4 to 0.8 μM, fibrinolysis was significantly promoted, and at 1.2 μM, the thrombus was dissolved in a short time almost the same as α2PI-depleted plasma. In a thrombolytic experiment using TH plasma, it was found that in the presence of tPA, the monoclonal antibody was activated and the blood clot was dissolved in a short period of time.

It was found that the monoclonal antibody promotes thrombolysis when added before thrombus formation, but it also promotes thrombolysis in almost the same way when added after thrombus formation, demonstrating a synergistic effect in the treatment of thrombosis, etc. It was found that the thrombolysis promoting effect was dramatically increased. Furthermore, tPA, 2
It has also been found that it has the effect of reducing the time between uses of plasminogen activation proteins such as heavy chain tPA. In an example of an experiment conducted by the present inventors, by using the monoclonal antibody in combination with t,PA (2 bottles of 1 tPA), 1/12 of the amount of tPA (double-chain tPA) was used alone. We have obtained the fact that tPA (2 pieces of 11 [PA)] can exhibit the same or higher fibrinolytic activity. Currently, intravenous drip injection is used for fibrinolytic therapy by administering tPA due to the large dose, but it has been pointed out that it is difficult to use, there are concerns about side effects, and it is expensive. However, by instilling it simultaneously with the monoclonal antibody, it is possible to greatly reduce the time required for tPA use. In addition, by administering the monoclonal antibody once or in multiple doses in conjunction with the tPA drip administration period, αzPIfl cross-linked in the blood and/or thrombi can be temporarily reduced for treatment. It is possible.

Furthermore, it is also possible to use plasminogen-activated proteins such as tPA by frequent administration rather than continuous administration such as by infusion.

Furthermore, in the presence of the monoclonal antibody, between plasminogen bound to fibrin clots, GIL
It was also found that the amount of J plasminogen increased to about twice that in the absence of the monoclonal antibody. From this fact, it was confirmed that the euclonal antibody effectively promotes thrombolysis.

The thrombolytic promoting effect of the monoclonal antibody is
This is because the monoclonal antibody binds to the reactive site of α2PI or its vicinity and inactivates the plasmin activity inhibitory effect of α2PI. Therefore, not only the monoclonal antibody that the present inventors actually used in the experiment, but also any antibody that abolishes the plasmin activity inhibitory effect of α2PI can be found to have the thrombolysis-promoting activity described here by the present inventors. It can also be used as a thrombolytic promoter. Furthermore, as mentioned above, the species of monoclonal antibody is not limited to the mouse mentioned here, but also guinea pigs.

It is possible to use rats, rabbits, humans, sheep, goats, pigs, dogs, cats, cows, horses, etc.
o A, et at,, J, lll1nunol
, 135°642-645 (1985))
Chimeric antibodies made by inheriting the variable region of a foreign animal and the constant region of a human, as reported by
It is also possible to use an antibody.

The present invention will be described in detail below with reference to Examples.

Example 1 (1) Preparation of human plasma 2.360 ml according to the blue water and open valve method described above.
7.7 mg of human α2PI was obtained from d.

(■ Mouse immunized male [3alb/c mice with human (22PI100
Complete Freund's adjuvant
uvant)? The mice were immunized intraperitoneally twice with emulsion at an interval of 21 days. 7 more
Days later and 88 days later, 130 μg of human αzP was additionally administered intravenously together with physiological saline. Four days after the final immunization, the spleen cells were used for cell fusion.

(3) Preparation of spleen cell suspension The spleen was aseptically removed and passed through a stainless steel mesh to obtain a single cell suspension. Cells L-
Glutamine 0.399/ρ.

is Kanamycin 0.2g/fl and Na)-1cO
The cells were transferred to RPM I-1640 medium (manufactured by GIBCO) supplemented with 32,07/wheat. Proliferated cells are RPMI
-1640 and resuspended in RPM1-1640 medium.

Preparation of Myeloma Cells Mouse myeloma cells P3-Ul have 0.3-glutamine
9g/U, kanamycin sulfate 0.2y/'1. Na
HCO32,0g/U and 'CF10% (7) Bovine fl
The cells were cultured in RPM 1-1640 medium (abbreviated as 10% FC8-RPM 1-1640) supplemented with f'j baby serum. Myeloma cells were in the log phase of cell division at the time of cell fusion.

(5) Serum-free RP with cell fusion spleen cells and myeloma cells at a ratio of 10:1
Suspended in M1-1640 medium and centrifuged at approximately 200 g for 5 minutes. After removing the supernatant medium, the precipitate was incubated with 1 d of 50% polypolylene glycol solution (pH 8,2) with an average molecular weight of 1,540 for 2 minutes at 37°C. Then serum-free RPM 1-1640
tB 9d was added and the cells were carefully resuspended for 5 minutes. The suspension was then centrifuged at approximately 200 g for 5 min, then resuspended in 1o% FC8-RPM l-1640 medium to obtain a concentration of 8 X 106 cells/d, and then plated onto a 96 microwell plate. Approximately 100 microns per well). This fused cell was heated at 37°C.
The cells were cultured using 5% CO2.

One day after cell fusion, add HAT medium at 10 ml per well.
0μρ was added. Thereafter, at intervals of 2 days, add half the amount of the medium to fresh H.
The medium was replaced with AT medium and cultured.

881, Hi l~α in the culture supernatant of hybridoma cells
Screening for antibodies against 2PI was performed by enzyme immunoassay. The antigen used in the screening was human 2PI, and the second antibody was a rabbit anti-mouse antibody labeled with alkaline phosphatase.

All of the 349 wells were positive by enzyme immunoassay, indicating that they produced antibodies against α2P1.

When active growth of II cells was observed, HT medium was added. Change the medium using HT medium 4 times at 1-day intervals, and then use regular 10% FC8-RPM.
The cells were cultured using l-1640 medium.

Among the hybridoma cells producing antibodies against human α2PI described above, hybridoma cells producing antibodies having the function of suppressing the lytic lysis inhibitory activity of human α2PI were screened by the following method.

Hybridomas in each well were added to 10% FC8-RPM I.
-1640 medium to reduce the number of cells to approximately 2 x 10
There were 7 pieces. The cells were centrifuged at approximately 200 g for 5 minutes, the culture supernatant was removed, and the cells were then incubated with serum-free RPM I.
-1640 medium 10-. Approximately 2 more minutes
Centrifuge at 0Off, remove the supernatant, and dissolve the cells in 2-mercaftethanol 5. Od/u, insulin
7.5d/U, transferrin 5.0rd/U, ethanolamine 5. Od/Jl! , Sui ~ Thulium Rhenite 5.0d/f1. L-glutamine 0.39!
J/Jl! , @acid kanamycin 0.2g / text,
Hepes 2,38'j/n and Na HCO
RPMl-1640 supplemented with 31,5z/U:
Dulbecco's MEM: Hall'
The cells were suspended in 5F-12 (2:1:1) warmed serum-free medium (hereinafter abbreviated as MITES medium) and cultured for 3 days.

The culture supernatant was collected and suspended 25 times.

Add 04μq of human α2PIO to 25μ of this concentrated solution,
Incubation was at 37°C for 30 minutes. Then 0.025 units of plasminogen and 0.02 units of urokinase.
031 unit was added to make the liquid volume 40μ. 10 μm of this was placed on a fibrin plate. The fibrin plate was allowed to stand for 18 hours at 37° C. and a humidity of 95% or higher, and the dissolved area was measured.

As a result, it was found that human α2PI, added to the antibody produced by 1D10 hybridoma cells, has the ability to completely suppress the fibrinolytic inhibitory activity.

Example 3 Cloning of hybridoma cells: Hybridoma cells (IDIO) that showed positive results in the antibody activity test against human α2PI were cloned by the following method, and several antibody-producing cell clones were obtained. One type is 1D10B11 (FERM BP'
-1781).

1D10 cells were diluted to 0.9111 cells per well of a 96-well microtiter plate, and Ba
Add 1b/c mouse thymus w8 cells as feeder cells and distribute into plates with 10% FC8-RPM I-164.
The cells were cultured in 0 medium. Observation under a microscope confirmed that it was definitely a single cell colony. Antibodies against human hα2PI in the hybridoma cell supernatant were screened by enzyme immunoassay.

A total of 26 wells were positive by enzyme immunoassay and produced monoclonal antibodies against Hi1-αzPI.

Purification of monoclonal antibodies: To produce a large amount of human αzPI-bound monoclonal antibodies, approximately 10 7 hybridomas mu were injected intraperitoneally into 3alb/C mice pretreated with Blistane. Ey et al.'s method [P
,L,EV,S,J.

P. rowse and C. R., J. enkin.
Immunochem+5try.

15, 429-436 (1978), protein Δ Zepharose 4 B (protein Δ-
Antibodies were purified using a 8epharose 4B) column. 20 mg of a monoclonal antibody against human α2PI was obtained from 2.5 volumes of ascites fluid.

Characteristics of the purified monoclonal antibodies: The specific class of the purified monoclonal antibodies was determined by Ophthalonigel diffusion test using class-specific anti-mouse antiserum. The results are shown in Table 1 below. human α
Most of the antibodies against 2PI were directed against H and L chains.

Table Example 4 Suppression of human α2PI activity by antibody against human α2PI 11 μJ of human αzP, each monoclonal antibody, and 5 μ9 of each monoclonal antibody were dissolved in 50 u of 0.05 M phosphate buffered saline (hereinafter abbreviated as PBS) and incubated at 31°C.
and incubated for 30 minutes. followed by 0.025 units of plasminogen and 0.031 units of urokinase.
The unit was added to make the liquid volume 60ufl.

10 μm of this was placed on a fibrin plate.

The fibrin plate was allowed to stand for 18 hours at 37° C. and a humidity of 95% or higher, and the dissolved area was measured. The results are shown in Figure 2 below.

The area dissolved by 0.025 units of plasminogen and 0.031 units of urokinase was defined as 100%.

Table 2 Example 5 L of various proteins! e! by 57! recognition α2 Pl, fibrinogen, Glu-plasminogen P l-3447-1, P I-3447-2,
and non-immune mouse antibody Na 1251 (18,2C
i/#) using the method of Mimuro J, etal, J, CI
In, Invest, 77, 1006-1013.
(1986)) (lactoperoxidase-glucose oxidase method;
e-glucoseoxidase l1lethOd
) was followed.

The specific activity of each protein obtained was 2,6x 10' cp
m/my, 3.2xtO' cI) IIl/#,
9.7X108cpIIl/m9゜7.5X 10ACDff1 /*, 7,8x10' cpm /r
ttg, and 6.5x 10” QpIR/Q tea
That's it.

Example 6 1125-labeled human α2PI 0.01 μM and α, PI
0.05μM of monoclonal antibody against 2% bovine serum albumin-0.05M Tris buffer (pH 7.4)
-0.15M Na Cl was added, and after incubation at 37°C for 30 minutes, it was left at 4°C overnight.

Add 2.5 mM Ca C to the Konoantigen-antibody reaction mixture.
1z.

A 7 μM fibrinogen fraction and 2 units/d thrombin were added to give a total volume of 100 μρ, and the mixture was incubated at 37° C. for 30 minutes to form a fibrin clot. After 30 minutes, 100 LIJ2 of 200 mM EDTA was added to remove calcium ions, and the coagulated product was wound up with a bamboo skewer. Roll the coagulate onto a bamboo skewer for 5 minutes.

3 washes [2% 8SA, 0.05M Tris buffer (pH 7,4), 0.15M NaCl,
211M EDTA]. Finally, the coagulated material was collected from the bamboo skewer into a test tube, and the radioactivity (cpm) of the coagulated material was measured.

Table 3 shows the ratio of the radioactivity of the coagulated product to the radioactivity in the original reaction mixture.

Table 3 also shows the results of using a commercially available Mouse I (IG) as a comparison antibody.

From this table, each human α2P? It can be seen that the monoclonal antibody against this antibody does not recognize the fibrin-binding site of α2PI.

This example investigated the effect of a monoclonal antibody against α2PI on the inactivation of plasmin by human α2PI.

α2PI 0.15 μM and monoclonal antibody against C2-plasmin inhibitor 0.15 μM were added to 2% raw serum albumin solution in 0.05 M Tris buffer ([lH7
, 4), 37 in 0.15 M Na Cf]
C. for 30 minutes and left at 4.degree. C. overnight.

Add 60μ of this reaction mixture and plasmin solution (0.47μM
> Mix 20up, 0.05 M to 1,1 s! 1 concentration iW(pi-17,4), 0.15 M Na
Two bottles were prepared for each sample by adding C1 and polishing the total volume to 500μ, and incubated at 37°C for 2 or 20 minutes. Next, 3.5 IBM synthetic substrate S-2251 (HD-valyl-L-isoyl-L-lysyl-〇-bihuman 1]anilide di[i salt) was added to 200μρ
In addition, spectrophotometric H4 (Becktan, DU-
8>, the change in absorbance at a wavelength of 405% m per unit time was measured. As a control, changes in absorbance were similarly examined for a sample in which only plasmin was reacted and a sample in which human α2PI and plasmin were reacted without adding a monoclonal antibody. The results are shown in Table 4 below.

From the results of Examples 5 and 6 shown in the table above, it was found that the monoclonal antibody of the present invention specifically recognized the human α2Pl reactant, and did not recognize either the plasmin-binding site or the fibrin-binding site.

Example 8 Monoclonal antibody 1D IOC11 described in the above example that specifically recognizes human α2PI glue active site
+11! Dissolve j in solution [2 ml EDTA.

12.5 mM cysteine, 50 mM Tris M buffer (pH 7.4), 0.15 M NaCff1]
100 u4Q of papain solution dissolved at 300 μM and 1 μ/da degree was added, and the mixture was reacted at 37° C. for 18 hours.

This reaction solution was subjected to liquid chromatography to separate Fab components. The molecular weight of this antibody was measured by 5DS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions.
It was confirmed that it was a Fab component consisting of a fragment with a molecular size of 23,000 from the 7-mino group end of n) and the entire L chain with a molecular size of about 23,000.

Example 9 Inhibition of α2PI activity Human α2P11μ3 and 3μ9 of each monoclonal antibody were dissolved in 50μρ of 0.05M PBS and incubated at 37℃ for 30 minutes.
Incubated for minutes. Next, 0.025 units of plasminogen and 0.034 units of urokinase were added to bring the solution ω to 60μ. 10 μm of this was placed on a fibrin plate. fibrin plate is 31
The solution was allowed to stand for 18 hours at a temperature of 95% humidity or higher, and the dissolved area was measured. The results are shown in Table 5 below. The values in the table below are based on the area dissolved by 0.025 units of plasminogen and 0.031 units of urokinase.
It is a relative value when expressed as %.

Table 5 In addition, the suppression of human α2Pl activity by the Fab component of this monoclonal antibody was investigated in the same manner as in Example 6.

Monoclonal antibody 40p101 against α2PI such as α2PIO06μy (9pLIol) was added to 2% bovine serum albumin solution [0.05M I-Lis buffer (1) H7,4
).

0.15M NaCf] dissolved in 60 μN, 37°C,
Incubate for 30 minutes and store at 4°C overnight.

This reaction mixture and 20μ of plasmin solution (0.47μM)
Mix ρ, 0. O5M Tris buffer (pH 7,4) 0
．． 15 M Na Cl was added to bring the total volume to 500 μρ.

Next, add 3.511 μM synthetic substrate S-2251 aqueous solution to 20
0 μm was added, and the change in absorbance per unit time at a wavelength of 405 μm was measured using a spectrophotometer (Hitachi 100-50). As a control, changes in absorbance were similarly examined for a sample in which only plasmin was reacted and a sample in which human α2PI and plasmin were reacted without adding a monoclonal antibody. The results are shown in Table 6 below.

From the results of Example 8 shown in the table above, it was found that the monoclonal antibody having the Fab of the present invention specifically recognizes the reactive site 1 of human α2PI, and suppresses the fibrinolysis-inhibiting effect of human α2PI. Ta.

Example 10 Thrombolysis test using human plasma Thrombin lysis M (200 units/d, 60μ) was added to 150μ of normal human plasma, and the mixture was heated at 37°C for 2 minutes to coagulate the plasma to obtain a clot. On the other hand, normal human plasma 290μ
In the statement, monoclonal antibody solution against α2Pl < 1
27 μm of DloC1; 3.39 Rg/d) was added, and the mixture was heated at 37° C. for 30 minutes. To this, 100 μp of plasmin solution (i, ooo units)/me) was added, and the coagulated mass was immersed at the same time and heated at 37°C. As a comparison, the monoclonal antibody solution was replaced with phosphate buffered saline and the clot dissolution time was compared. As a result, when a monoclonal antibody against α2PI was used, the clot was completely dissolved in about 2 hours, whereas when no monoclonal antibody was used, it took more than 10 hours.

Example 11 Thrombolysis test using human blood 60 μρ of thrombin solution (200 units/Id) was added to 150 μρ of normal human blood, and the mixture was heated at 37° C. for 2 minutes to coagulate the blood to obtain a clot. On the other hand, normal human blood 300
A monoclonal antibody solution against α2P [I
DloC1: 3,39 II! g/d) to 27μ
The mixture was then heated at 37°C for 30 minutes. To this was added 100 μp of plasmin solution (1,000 units/d), and at the same time, the coagulated mass was immersed and heated at 37°C. As a comparison, the monoclonal antibody solution was replaced with phosphate buffered saline and the clot dissolution time was compared.

As a result, when a monoclonal antibody against α2PI was used, the clot was completely dissolved in about 2 hours;
When a monoclonal antibody was not used, more than 10 hours were required.

Example 12 Fibrin clot dissolution test 2 using purified system! l! g of 3+-labeled fibrinogen (XI[l
Factor) 100,000 cpm and Glu-plasminogen 0.2! f! g, t-pA and r of 0.4 units
J O, 06rtyi (Dα2PI 11R1! 03
01% Bovine Serum Albumin Solution (0.05Ml-Lis Slow I
M ([lH7,4), o, 1MNact,
It was dissolved in 0.01% bovine serum albumin 1, and to this was added 1 unit of α-thrombin and CaCu5 to a final concentration of 2.51M. A fibrin clot was formed by incubating at 37°C for 10 minutes. The resulting fibrin clot was rolled up with a bamboo skewer and returned to a fresh 0.01% raw serum albumin solution.

Next, p l -3447- was added at concentrations of 0.4f1M, 0.8μM, 1.2μM and 5.0μM.
1 or 5.0 μM of J T
P I-2 was added. While incubating at 37°C, 50 μm of liquid was collected after 8 and 10 hours at 2, 4, 6°C, and the inoculations contained therein were transferred to Curran 1. 12
Dissolved IyIIffi for the amount of layer label of 5I
Figure 2 shows the ratio of As a result, it was found that fibrinolysis increased in proportion to the amount of P I-34471 up to 1.2 μMil. Also Incubatecon 10
The values after hours showed that when the concentration of PI-3447-11 was 2 μM, the dissolution of the fibrin clot was almost the same as in the case of α2PI-removed plasma.

Example 13 Thrombolysis test using human plasma (II) Venous thrombosis plasma, or healthy person plasma, or tPA-depleted plasma 11d,
1251 to give a final concentration of 100,000 cpm/ate.
Labeled fibrinogen was added, followed by 1 unit of α-thrombin and CaCN2 to a final concentration of 18 mM.
and incubated at 37° C. for 15 minutes to form a thrombus. Wind up the thrombus with a bamboo skewer and add 1°2μM1
PI-34471 monoclonal antibody-containing double-chain turokinase-depleted serum containing PI-34471 monoclonal antibody, or healthy subject plasma as a control. Thrombolysis was measured by C according to the method shown in Example 12. The result is the third
As shown in the figure. The fibrinolytic promoting effect of the P I-3447-1'U nickclonal antibody was comparable between Kinase-depleted plasma and healthy human plasma, but it showed no effect at all on tPA-depleted plasma. Ta.

Example 14 Binding of anti-α2PI monoclonal antibody to plasma thrombus - α-1~thrombin was added to 1 ml of healthy human plasma or α2PI-deficient patient plasma at a final concentration of 1 unit/d to a final concentration of 18 mM. Add CaCN2 to 0 and add 3
By incubating at 7° C. for 15 minutes, a thrombus in which α2PI in plasma was cross-linked to fibrin (hereinafter sometimes referred to as a cross-linked thrombus) was created.

Well, healthy human plasma 1Rf with citric acid added! to final concentration 2
．． 5 J4 added EDT△. α-thrombin is added to this at a final concentration of 1 unit l-/d to form a thrombus in which α2PI in the plasma is not cross-linked to fibrin (hereinafter this may be referred to as a non-bridged hematoma).
It was created. These thrombi were treated with the 1251-labeled Pl-3447-1 monoclonal antibody in Example 5, or with P
I-3447-2 monoclonal antibody or non-immune mouse antibody was added to plasma and incubated at 37°C for 1 hour. The thrombus was then treated with 1% bovine serum albumin and 100 units/'d of apro cotinine.
The cells were thoroughly washed in a Tris buffer solution containing <tinin>. The amount of I251 contained in the thrombus after washing was measured using a gamma counter.

As a result, as shown in Figure 4, P I-3447-1
It was found that the monoclonal antibody and the PI-3447-2 monoclonal antibody bind well to α2PI on cross-linked thrombi.

Example 15 Thrombolysis test using human plasma (III) Healthy human plasma 1
- to a final concentration of 100,000 cpm/mj, δI-labeled fibrinogen was added to the fibrinogen to a final concentration of 100,000 cpm/mj. The cells were incubated for 1 minute to allow thrombus formation. Blood clots were treated with 5 ml of tPA.
and into healthy human plasma containing various concentrations of P, l-34471 monoclonal antibody. Additionally, 30 units/d and 60 units/- of tPA were added to the plasma. Thrombolysis was measured according to the method shown in Example 12.

The results are shown in FIG. 0.4μM PI3447
-1 monoclonal antibody and 5 units/ml of tPA
When used in combination with tPA, 30 units/d of tPA produced similar fibrinolytic activity, and 1.2 μM of PI-
3447-1 monoclonal antibody and 5 units/snake
When tPA was used in combination, fibrinolytic activity of 60 units/d was shown, which was similar to that when P△ was used.

Example 16 Thrombolysis test using tPA-depleted plasma Thrombus was formed using tpΔ-depleted plasma 1d according to the method of C1 Example 13 and Example 15. Thrombi were treated with 5 units/me of tPA and various concentrations of Pl-344.
It was added to healthy human plasma containing 7-1 monoclonal antibody. Thrombolysis was measured according to the method shown in Example 12. The results are shown in FIG. P l-3447'
-1 monoclonal antibody is dependent on exogenous t[)A;
Fibrinolytic promoting activity was observed in proportion to its addition.

Example 11 Binding test of plasminogen to fibrin Thrombus was formed according to the method of Example 14 using healthy human plasma or tPA-depleted plasma 1d. Blood clots derived from healthy human plasma are (a:
GILI-plasminogen (1
Glu-plasminogen labeled with +b151 (100,000 cpm/mf+)
), 5 uniyy h/mf! II) tl-]A and H 1.2μMl! The cells were incubated at 37° C. for 30 minutes in plasma containing iN degree of PI-3447-1 monoclonal antibody.

In addition, thrombi derived from tPA-depleted plasma are (C) Glu-Blastmi21-gen (100,000 C1) m/ld), 51
Nitsuh/rtd! of tPA, and 1.2 μMi of 1 degree P
The cells were placed in plasma containing I-3447-i monoclonal antibody and incubated at 37°C for 30 minutes. The thrombus was then rolled up with a bamboo skewer and treated with 1% raw serum albumin.
After washing in Tris buffer containing units/ml of aprotinin, measurements were made with a γ-counter. The results are shown in FIG. It was found that in the presence of the pl-3447-1 monoclonal antibody, Qlu-plasminogen bound to the thrombus approximately twice as much as normal.

[Brief explanation of drawings]

Attached FIG. 1 is a diagram showing the partial structure of Fab of the monoclonal antibody of the present invention when it is degraded using papain. In the figure, C indicates a variable region, and C indicates a constant region. Attached Figure 2 shows the results of the fibrin clot dissolution test using the purified system. The horizontal axis shows the incubation time (hours),
The vertical axis shows 1251-labeled fibrin degradation product (FDP
) elution amount (%) is shown.Various concentrations P I-34
When using 4L-1 monoclonal antibody (・),
When 5 μM Pl-3447-2 monoclonal antibody was used (■), when α2PI was not added and the monoclonal antibody was also added, it was shown as (Δ). Attached Figure 3 shows the results of examining the effects of urokinase and tPA in a thrombolytic test using human plasma. The horizontal axis shows the incubation time (hours), and the vertical axis shows the elution amount (%) of +751-labeled fibrin degradation product. Venous embolization patient plasma (△), healthy person plasma (・), t
PA-depleted plasma (■), double-chain urokinase-depleted plasma (×
) showed the results. The results of adding blood clots derived from healthy person's plasma to healthy person's plasma are shown as (0). Attached FIG. 4 shows the results of a binding experiment of anti-αzP monoclonal antibody to a plasma plug. The horizontal axis shows the amount of antibody, and the vertical axis shows the binding needle of +251-labeled antibody to the thrombus. 1-' f -3447-1 monoclonal antibody-added plasma (•), PI-3447-2 monoclonal antibody-added plasma (■), α2P■-depleted plasma (O)
, and non-crosslinked thrombus (mu). Attached FIG. 5 shows the results of a thrombolytic test using exogenous ℃PA using human plasma. The horizontal axis shows the incubation time (hours), and the vertical axis shows the elution amount (%) of the ll'il-labeled fibrin decomposition agent. When using 5 units l-/d of tPA (mu), 1.2 μM Pl-3447-1 monoclonal antibody (0), 30 units [·/- of tPA (b)
. Figure 6 shows the results of a thrombolytic test using tPA-depleted plasma. 3 hours (hours)
The vertical axis shows the elution amount (%) of the ts■-labeled fibrin degradation product. PI-3447-1 at various concentrations
When monoclonal antibody and 5 units/- of tPA were used together (△), 1.2 μM of PI-3447-1
The case with only monoclonal antibody (◯) and the case with neither antibody nor tPA (·) are shown. Attached Figure 7 shows the results of a plasminogen binding test to fibrin. The vertical axis shows the amount (%) of 1251-labeled plasminogen bound to the fibrin thrombus. Patent Applicant Teijin Co., Ltd. Ceremony Attorney Patent Attorney 1) Jun Hiroshi Figure 1 T-2j 1 1incubation time (h) Spear 31 1 1incubation Time (h) Chili packet 19G ddded (μM) -F (-7 request) 1ncubation Time 1251-FDP Release (%) O meeting ω Takashi・71 too

Claims (1)

[Scope of Claims] 1. A thrombolytic agent containing a protein having tissue plasminogen activating protein activity and a monoclonal antibody that suppresses the plasmin inhibitory activity of α_2PI. 2. The ratio of the protein with tissue plasminogen activating protein activity to the monoclonal antibody that suppresses the plasmin inhibitory activity of α_2PI is 0.01 to 20 (mol/mol
) The thrombolytic agent according to claim 1. 3. The thrombolytic agent according to claim 1 or 2, which is an aqueous solution of a protein having tissue plasminogen activation protein activity and a monoclonal antibody that suppresses the plasmin inhibitory activity of α_2PI. 4. The thrombolytic agent according to claim 1 or 2, wherein the tissue plasminogen activation protein is double-chain tPA. 5. The thrombolytic agent according to claim 1 or 2, wherein the tissue plasminogen activation protein is single-chain tPA. 6. A monoclonal antibody that suppresses the plasmin inhibitory activity of α_2PI as a pre-administration agent for administration of tissue plasminogen activating protein. 7. Thrombus/plasminogen binding promoter consisting of monoclonal antibodies that suppress the plasmin inhibitory activity of α_2PI.