JPH0428280B2 - - Google Patents

Description

[Detailed description of the invention]

a Industrial Application Field The present invention relates to human α ₂ -plasmin inhibitor (α ₂
-plasmin inhibitor; α ₂ -antiplasmin), in particular recognizes the fibrinolytic action inhibiting site (reactive site) of human α ₂ -plasmin inhibitor as an antigen, and as a result, the fibrinolytic activity of plasmin of human α ₂ -plasmin inhibitor It relates to a portion of a monoclonal antibody molecule that has the function of suppressing the inhibitory activity against lytic action and promoting fibrinolysis. b. Prior Art Human α ₂ -plasmin inhibitor was first isolated and purified by Aoki and Moroi, and is a strong plasmin inhibitor that instantly inhibits the esterase activity of the fibrinolytic enzyme plasmin. , is known to be a single-chain glycoprotein with a molecular weight of approximately 67,000 containing 11.7% sugar [Moroi &Aoki; The Journal of Biological
Chemistry, 251, 5956-5965 (1976)]. On the other hand, human α ₂ -plasmin inhibitor has 3
It is known that there are different types of active sites. 1st
is a site that inhibits the fibrinolytic action of plasmin (hereinafter referred to as the “reactive site”)
[B. Wiman & D. Collen; The Journal of
Biological Chemistry, 25419291−9297 (1979)
The second is the plasmin binding site on the carboxyl terminal side [B. Wiman & D. Collen;
European Journal of BioChemistry, 84, 573
-578 (1978)], and the third is the fibrin binding site at the amino terminal end [Y. Sakata, et al.
al., Thrombosis Research, 16, 279−282.
(1979)]. If we can provide a portion of a monoclonal antibody protein that selectively recognizes the reactive site as an antigen among these three types of active sites in human α ₂ -plasmin inhibitor, we can use this to develop human α ₂ -plasmin inhibitor. Since it can directly suppress the fibrinolysis-inhibiting effect of and promote fibrinolysis, it can be used as a thrombolytic promoter. Furthermore, by using this antibody fragment, it is also possible to measure α ₂ -plasmin inhibitor or α ₂ -plasmin inhibitor/plasmin complex in a solution. c Structure of the present invention According to the present invention, a monoclonal antibody against human α ₂ -plasmin inhibitor, especially this α ₂ -
Parts of highly specific monoclonal antibodies are provided that recognize reactive sites of plasmin inhibitors as antigens and have the ability to suppress the fibrinolysis-inhibiting effects of α ₂ -plasmin inhibitors. The hybridoma cells of the present invention can be prepared by the method of Koehler and Milstein.
Nature 256, 495-497 (1975)]. That is, after immunizing a mouse with human α ₂ -plasmin inhibitor, the spleen cells of this mouse were fused with mouse myeloma cells, and the resulting hybridoma cells were immobilized on human α ₂ − plasmin inhibitors in microtiter plates. Systematically tested and selected for antibodies that react with plasmin inhibitors. In this way, hybridoma cells that synthesize and secrete antibodies against human α ₂ -plasmin inhibitor are selected. The obtained hybridoma cells were cultured in a serum-free medium, and the antibody against human α ₂ -plasmin inhibitor secreted into the culture supernatant was
Human _α2 on fibrin plates
- The activity of suppressing the fibrinolytic inhibitory effect of plasmin inhibitors will be tested. the result,
A hybridoma cell that produces a monoclonal antibody that has the activity of specifically suppressing the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor has been isolated. Monoclonal antibodies of the invention are obtained from the products produced by such hybridoma cells. The monoclonal antibody thus obtained is human α ₂ −
It acts monospecifically on the reactive site of plasmin inhibitor. Next, the monoclonal antibody of the present invention and the method for producing the same will be explained in detail. A. Isolation and purification of antigen; Human α ₂ -plasmin inhibitor used as an antigen was isolated and purified from human plasma by the method of Aoki and Moroi. B. Immunization of mice with human α ₂ -plasmin inhibitor; male Balb/c mice are used, but mice of other strains can also be used.
The immunization regimen and the concentration of human α ₂ -plasmin inhibitor should then be chosen so that a sufficient number of antigen-stimulated lymphocytes are formed. For example, mice were immunized intraperitoneally several times at certain intervals with a small amount of α ₂ -plasmin inhibitor, and then intravenously administered several times. Spleen cells are removed for fusion several days after the final immunization. C Cell fusion; The spleen is removed aseptically and a single cell suspension is prepared from it. The spleen cells are fused with mouse myeloma cells from an appropriate line by use of an appropriate fusion promoter. The preferred ratio of splenic pulp cells to myeloma cells is about 20:1 to about 2:1.
is within the range of 0.5 for approximately 10 ⁸ splenocytes
The use of ~1.5 ml of fusion medium is appropriate. Many myeloma cells used for cell fusion are known, but in the present invention, P3-X63-Ag8-U1 cells (hereinafter abbreviated as P3-U1) [Yelton, DEet
al., Current Topics in Microbiology and
Immunology, 81, 1 (1978)] was used.
This is a cell line resistant to 8-azaguanine and is associated with the enzyme hypoxanthine-guanine phosphoribosyltransferase (hypoxanthine-guanine phosphoribosyltransferase).
guanine phosphoribosyl transferase) and therefore HAT (hypoxanthine,
(aminopterin, thymidine) does not survive in medium. Moreover, this cell line itself does not secrete antibodies, and is a so-called non-secreting type. A preferred fusion promoter is, for example, polyethylene glycol having an average molecular weight of 1000 to 4000, although other fusion promoters known in the art may also be used. In the examples of the present invention, polyethylene glycol with an average molecular weight of 1540 was used. D. Selection of fused cells; in a separate container (e.g., a microtiter plate), a mixture of unfused spleen cells, unfused myeloma cells, and fused cells is grown in a selective medium that does not support unfused myeloma cells. Dilute and culture for sufficient time (about 1 week) to kill unfused cells. The medium should be drug resistant (e.g. 8-
A medium (such as the HAT medium described above) that is resistant to azaguanine and does not support unfused myeloma cells is used. Unfused myeloma cells die in this selective medium. These unfused spleen cells are non-neoplastic cells, so after a certain period of time (about 1 week)
die out. Cells fused to these cells can survive in selective media because they have both the neoplastic properties of myeloma parent cells and the properties of parent spleen cells. E. Confirmation of antibodies against human α ₂ -plasmin inhibitor in each container; after hybridoma cells are thus detected, their culture supernatants are collected and tested for antibodies against human α ₂ -plasmin inhibitor by enzyme linked immunoassay (Enzyme Linked Immunoassay). Sorbent
Assay). F. Selection of hybridoma cells producing antibodies with activity against human α ₂ -plasmin inhibitor; hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor were cultured in a serum-free medium. The containing culture supernatant was concentrated and incubated with human α ₂ -plasmin inhibitor for a certain period of time. Further, plasmin was added to this human α ₂ -plasmin inhibitor mixture, and the mixture was placed on a fibrin plate to measure the area of fibrin dissolution. In this way, hybridoma cells producing antibodies with activity against human α ₂ -plasmin inhibitor are selected. G. Cloning of hybridoma cells producing antibodies of interest; When hybridoma cells producing antibodies of interest are cloned by an appropriate method (e.g. limited dilution method), antibodies are produced in two different ways. According to the first method, monoclonal antibodies produced by hybridoma cells can be obtained from the culture supernatant by culturing hybridoma cells in an appropriate medium for a certain period of time. According to a second method, hybridoma cells can be injected into the peritoneal cavity of isogenic or semi-isogenic mice. Monoclonal antibodies produced by the hybridoma cells can be obtained from the blood and ascites of the host animal after a certain period of time. The thus obtained monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor was tested using the proteolytic enzyme papain using Porter's method [RRPorter,
Biochemical Journal 73 , 119–126 (1959)
[Reference]] and isolated as a partial structure of the part (Fab) surrounded by the dotted line in the attached drawing. The Fab partial structure of this monoclonal antibody was examined for its activity in suppressing the fibrinolytic inhibitory effect of human α ₂ -plasmin inhibitor on a fibrin plate. As a result, human α ₂ −
It was confirmed that it has the activity of specifically suppressing the fibrinolytic inhibitory effect of plasmin inhibitor. The portion of the monoclonal antibody having at least the Fab structure of the present invention acts monospecifically against the reactive site of human α ₂ -plasmin inhibitor. The present invention will be described in detail below with reference to Examples. Example 1 (1) Preparation of human α ₂ -plasmin inhibitor Human plasma was prepared according to the method of Aoki and Moroi described above.
Human _α2 -plasmin inhibitor from 2360ml
Obtained 7.7 mg. (2) Immunization of mice Male Balb/c mice were immunized with 100 μg of human α ₂ -plasmin inhibitor and Complete Freund's adjuvant.
The patient was immunized intraperitoneally twice with an interval of 21 days using an emulsion of . human α ₂ -plasmin inhibitor after an additional 7 and 88 days.
An additional 30 μg was 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 were treated with L-glutamine 0.39g/, kanamycin sulfate 0.2g/, and NaHCO ₃ 2.0g/
RPMI-1640 medium (manufactured by GIBCO) supplemented with
Moved to. Proliferated cells were washed three times with RPMI-1640 and resuspended in RPMI-1640 medium. (4) Preparation of myeloma cells Mouse myeloma cells P3-U1 were prepared using 0.39 g of L-glutamine, 0.2 g of kanamycin sulfate,
RPMI- ₁₆₄₀ medium (10% FCS-
The cells were cultured in RPMI-1640 (abbreviated as RPMI-1640). Myeloma cells were in the log phase of cell division at the time of cell fusion. (5) Cell fusion Suspend spleen cells and myeloma cells in serum-free RPMI-1640 medium at a ratio of 10:1, and
Centrifuged at 200g. After removing the supernatant medium, the sediment was incubated with 1 ml of a 50% polyethylene glycol solution (PH 8.2) with an average molecular weight of 1540 for 2 minutes.
Incubation was performed at 37°C. then serum free
9 ml of RPMI-1640 medium was added and the cells were carefully resuspended for 5 minutes. The suspension was then centrifuged at approximately 200 g for 5 min, followed by 8 x 10 ⁶ cells/ml.
10% FCS−RPMI− to obtain a concentration of
1640 medium and then distributed onto 96 microwell plates (approx.
100μ). This fused cell is 5% at 37°C.
Cultured using _CO2 . (6) Selection and culture of hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor One day after cell fusion, 100μ of HAT medium was added to each well. Thereafter, half of the medium was replaced with fresh HAT medium at 2-day intervals and cultured. Eight days later, the culture supernatant of the hybridoma cells was screened for antibodies against human α ₂ -plasmin inhibitor by enzyme immunoassay. The antigen used in the screening was human α ₂ -plasmin inhibitor, and the second antibody was alkaline phosphatase (alkali phosphatase).
phoshatase) labeled rabbit anti-mouse. All of the 349 wells in total were positive by enzyme immunoassay, indicating that they produced antibodies against α ₂ -plasmin inhibitor. When active cell proliferation was observed, HT medium was added. HT a total of 4 times at 1-day intervals
The medium was replaced with a new medium, and then cultured using a normal 10% FCS-RPMI-1640 medium. Example 2 (Selection of hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor) Fibrinolytic inhibitory activity of human α ₂ -plasmin inhibitor was determined from among the hybridoma cells producing antibodies against human α ₂ -plasmin inhibitor. The following method was used to screen hybridoma cells that produce antibodies that have the effect of suppressing cancer. 10% FCS-PRMI for hybridomas in each well
-1640 medium, and the number of cells was approximately 2×10 ⁷ . The cells were centrifuged at approximately 200g for 5 minutes,
After removing the culture supernatant, cells were placed in serum-free RPMI−
Washed with 10 ml of 1640 medium. Approximately 200g for another 5 minutes
Centrifuge the cells at
ml/, transferrin 5.0ml/, ethanolamine 5.0ml/, sodium selenite 5.0
ml/, L-glutamine 0.39g/, kanamycin sulfate 0.2g/, Hepes 2.38g/, and
RPMI−1640 supplemented with NaHCO ₃ 1.5 g/:
Dulbecco's MEM: Ham's F-12 (2:1:1)
Mixed serum-free medium (hereinafter referred to as “MITES medium”)
) was suspended in 10 ml and cultured for 3 days. The culture supernatant was collected and concentrated to 25 medium.
0.4 μg of human α ₂ -plasmin inhibitor was added to 25 μg of this concentrated solution and incubated at 37° C. for 30 minutes. Next, add 0.025 units of plasminogen and 0.031 units of urokinase and adjust the volume.
It was set to 40μ. 10μ of this was placed on a fibrin plate. The fibrin plate was left 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 the fibrinolytic inhibitory activity of human α ₂ -plasmin inhibitor in addition to the antibody produced by 1D10 hybridoma cells was completely suppressed. Example 3 Cloning of hybridoma cells; A hybridoma cell (1D10) that showed a positive result in an antibody activity test against human α ₂ -plasmin inhibitor was cloned by the following method. Dilute 1D10 cells to 0.9 cells per well of a 96-well microtiter plate.
Add Balb/c mouse thymocytes as feeder cells and distribute into plates using 10% FCS-RPMI-1640.
Cultured in medium. Observation under a microscope confirmed that it was definitely a single cell colony. Antibodies against human α ₂ -plasmin inhibitor in the culture supernatant of hybridoma cells were screened by enzyme immunoassay. A total of 26 wells were positive by enzyme immunoassay and produced monoclonal antibodies against human α ₂ -plasmin inhibitor. Purification of monoclonal antibodies: In order to produce large quantities of monoclonal antibodies against human α ₂ -plasmin inhibitor, approximately
10 ⁷ hybridoma cells were injected intraperitoneally into pristane-pretreated Balb/c mice. Approximately 1
Using the ascites fluid collected after a week, the method of Ey et al. [PL
Ey, SJProwse and CRJenkin, I.
See mmunochemistry, 15, 429-436 (1978)]
According to Protein A-Sepharose 4B (protein
The antibody was purified using a Sepharose 4B) column. 20 mg of monoclonal antibody against human α ₂ -plasmin inhibitor was obtained from 2.5 ml of ascites fluid. Characteristics of the purified monoclonal antibodies; The specific class of the purified monoclonal antibodies was determined by Ochterlony gel diffusion test using class-specific anti-mouse antiserum. The results are shown in Table 1 below. Here antibodies each 1D10C1,
1D10F10, 1D10−1F5, 1D10B11 and 1D10−
2H8 is a monoclonal antibody obtained by cloning from 1D10 hybridoma cells. In addition, antibody names 1B10C4 and 1B10G11 are from Example 1
One of the hybridoma cells obtained in
This is a monoclonal antibody obtained by cloning from 1B10 hybridoma cells. Most of the antibodies against human α ₂ -plasmin inhibitor were H chain r ₁ and L chain K.

[Table] Example 4 Inhibition of human α ₂ -plasmin inhibitor activity by antibody against human α ₂ -plasmin inhibitor 1 μg of human α ₂ plasmin inhibitor and 5 μg of each monoclonal antibody were mixed in 0.05 M phosphate buffered saline (hereinafter abbreviated as PBS). ) Dissolve in 50μ and at 37℃
Incubation was carried out for 30 minutes. Next, 0.025 units of plasminogen and 0.031 units of urokinase were added to make the liquid volume 60 μg. 10μ 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 Table 2 below. The values in the table below are relative values when the area dissolved by 0.025 units of plasminogen and 0.031 units of urokinase is taken as 100%. Here, the antibody name 1D10-1H2 is a monoclonal antibody obtained by cloning from 1D10 hybridoma cells.

[Table] Example 5 Effect of monoclonal antibody against human α ₂ -plasmin inhibitor on binding between human α ₂ -plasmin inhibitor and fibrin I ¹²⁵ -labeled human α ₂ -plasmin inhibitor
0.01 µM and 0.05 µM monoclonal antibody against _α2 -plasmin inhibitor in 2% bovine serum albumin - 0.05 M Tris buffer (PH7.4) - 0.15 M NaCl
After incubation at 37℃ for 30 minutes,
It was left at 4°C overnight. To this antigen-antibody reaction mixture, add 2.5mMCaCl ₂ , 7μM fibrinogen fraction,
Add 2 units/ml thrombin to make a total volume of 100μ
The mixture was then incubated at 37°C for 30 minutes. Formation of a coagulum (fibrin clot) was observed. After 30 minutes, 100μ of 200mM MEDTA was added to remove calcium ions, and the coagulum was rolled up with a bamboo skewer and washed three times for 5 minutes with a washing solution [2% BSA, 0.05M
Tris buffer (PH7.4), 0.15M NaCl, 2m
I washed it with [MEDTA]. 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 common commercially available mouse IgG as a comparative antibody.

[Table] This result shows that the monoclonal antibodies against each human α ₂ -plasmin inhibitor are monoclonal antibodies that do not recognize the fibrin binding site of human α ₂ -plasmin inhibitor. Example 6 Search for a monoclonal antibody that recognizes the reactive site of human α ₂ -plasmin inhibitor This example investigated the effect of a monoclonal antibody against α ₂ -plasmin inhibitor on the inactivation of plasmin by human α ₂ -plasmin inhibitor. It is something that α ₂ -plasmin inhibitor 0.6 μg and monoclonal antibody against α ₂ -plasmin inhibitor
6.6μg was dissolved in 60μ of 2% bovine serum albumin solution [0.05M Tris buffer (PH7.4), 0.15M NaCl], incubated at 37°C for 30 minutes, and left at 4°C overnight. This reaction mixture and 20μ of plasmin solution (0.47μM)
Mix 0.05M Tris buffer (PH7.4),
Two bottles of each sample were prepared by adding 0.15M NaCl to make a total volume of 500μ, and incubation was performed at 37°C for 2 or 20 minutes. Next 3.5m
Add 200μ of an aqueous solution of M synthetic substrate S-2251 (H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide dihydrochloride), and measure at 405 nm using a spectrophotometer (Beckman, DU-8). The change in absorbance per unit time at wavelength 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 α ₂ -plasmin inhibitor and plasmin were reacted without addition of a monoclonal antibody. The results are shown in Table 4 below.

[Table] The monoclonal antibodies obtained from the results of Examples 5 and 6 above specifically recognize the reactive site of human α ₂ -plasmin inhibitor, and do not recognize either the plasmin binding site or the fibrin binding site. I found out that there isn't. Example 7 Cleavage of a monoclonal antibody that recognizes the reactive site of human α ₂ -plasmin inhibitor with papain Dissolve 1 mg of the monoclonal antibody 1D10C1 described in the above example that specifically recognizes the reactive site of human α ₂ -plasmin inhibitor. Solution [2mM
Dissolve in 300μ of EDTA, 12.5mM cysteine, 50mM Tris buffer (PH7.4), 0.15M NaCl, add 100μ of papain solution with a concentration of 1mg/ml,
The reaction was carried out at 37°C for 18 hours. This reaction solution was subjected to liquid chromatography,
Fab components were fractionated. The molecular weight of this was measured by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions.
It was confirmed to be a Fab component consisting of a fragment of 23,000 and the entire L chain with a molecular weight of about 23,000. Example 8 Suppression of human α ₂ -plasmin inhibitor activity by Fab component of antibody against human α ₂ -plasmin inhibitor 1 μg of human α ₂ -plasmin inhibitor and 3 μg of each monoclonal antibody were dissolved in 50 μg of 0.05 M PBS and inked at 37°C for 30 minutes. I was educated. Next, 0.025 units of plasminogen and 0.031 units of urokinase were added to make the liquid volume 60μ.
10μ of this was placed on a fibrin plate.
Fibrin plates are processed under conditions of 37℃ and 95% humidity or higher.
The solution was allowed to stand for 18 hours, and the dissolved area was measured. The results are shown in Table 5 below. The values in the table below are 0.025 units of plasminogen and 0.031 units of urokinase.
This is a relative value when the area dissolved by the unit is taken as 100%.

[Table] In addition, in the same manner as in Example 6, the inhibition of human α ₂ -plasmin inhibitor activity by the Fab component of this monoclonal antibody was investigated. α ₂ -plasmin inhibitor 0.6 μg (9 pmol)
and 40 pmol of monoclonal antibody against α ₂ -plasmin inhibitor in 2% bovine serum albumin solution [0.05M Tris buffer (PH7.4), 0.15M NaCl] 60μ
The mixture was dissolved in water, incubated at 37°C for 30 minutes, and left at 4°C overnight. This reaction mixture and 20μ of plasmin solution (0.47μM)
Mix 0.05M Tris buffer (PH7.4)
0.15M NaCl was added to bring the total volume to 500μ. next
Add 200μ of 3.5mM synthetic substrate S-2251 aqueous solution,
The change in absorbance per unit time at a wavelength of 405 nm 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 α ₂ -plasmin inhibitor and plasmin were reacted without adding a monoclonal antibody. The results are shown in Table 6 below.

【table】

[Table] From the above results of Example 8, the monoclonal antibody having Fab of the present invention specifically recognizes the reactive site of human α ₂ -plasmin inhibitor and inhibits fibrinolysis of human α ₂ -plasmin inhibitor. I found out that it can be suppressed.

[Brief explanation of drawings]

The attached drawing shows the monoclonal antibody of the present invention when it is degraded using papain.
FIG. 3 is a diagram showing a partial structure of Fab. In the figure, V indicates a variable region and C indicates a constant region.

Claims (1)

[Scope of Claims] 1. A monoclonal antibody fragment against human α ₂ -plasmin inhibitor, which recognizes a site that inhibits the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor, and which recognizes a site that inhibits the fibrinolytic action of plasmin in human α 2 -plasmin inhibitor, and which inhibits a plasmin binding site and a fibrin binding site. A monoclonal resistance Fab that suppresses the fibrinolytic inhibitory effect of human α ₂ -plasmin inhibitor that does not recognize any of the following:
A monoclonal antibody fragment having at least a portion. 2. The monoclonal antibody fragment according to item 1 is a monoclonal antibody fragment against human α ₂ -plasmin inhibitor, which recognizes a site for inhibiting the fibrinolytic action of plasmin in human α ₂ -plasmin inhibitor, and recognizes a plasmin binding site and a plasmin binding site. A monoclonal antibody fragment consisting only of the Fab portion of a monoclonal antibody that suppresses the fibrinolysis-inhibiting effect of human α ₂ -plasmin inhibitor that does not recognize any of the fibrin binding sites.