JPH02492A - Novel monoclonal antibody - Google Patents

Abstract

PURPOSE:To enable measurement of the amount of a tissue type plasminogen activator (t-PA) antigen, etc., excellent in specific aftinity against antigens by culturing a mouse hybridoma, etc., in a culture medium. CONSTITUTION:The splenic cell of BALB/C strain mouse sensitized with t-PA derived from a human melanoma cell is fused to a mouse myelomatous cell (e.g., P3X63Ag8.U1) to provide (A) a mouse hybridoma clone sp-322, which is then cultured in a culture medium containing bovine fetal blood serum, etc., at about 37 deg.C in a vapor phase of 5wt.% CO2 and about 95wt.% air for 2-4 days to afford a culture. The resultant culture is then dialyzed and purified to isolate a monoclonal antibody having the specificity for the n-PA and following properties. Molecular weight; 153000+ or -10000. IgG subclass) IgG1. L-chain amino acid sequence within the L-chain variable region; amino acid sequence expressed by the formula. Binding part to antigen; part with affinity for fibrin or vicinity thereof, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel monoclonal antibody having specificity for tissue-type plasminogen activator (hereinafter referred to as t-PA).

More specifically, 1) the present invention has excellent specific affinity for antigens, and can be more advantageously used for the purification of t-PA and measurement of the amount of t-PA antigen; - It relates to a novel monoclonal antibody that can also be used as a method for measuring PA activity.

(Prior Art) t-PA is a type of serine protease that is involved in the fibril undissolution mechanism, and is associated with the destruction of two tissues and the formation of fibrin due to the contraction and expansion of blood vessels.

It is secreted and released from vascular endothelial cells, adsorbs to fibrin, decomposes plasminogen which also has affinity for fibrin, and converts it into plasmin which has fibrinolytic ability. Unlike commercially available plasminogen activators such as urokinase and streptokinase, t-PA has an affinity for fibrin and can be concentrated at the site of thrombus, so it can be used as a non-fibril dissolving agent in place of these commercially available products. Attention has been paid.

In addition, measurement of blood t-PA levels is necessary for screening for t-PA and other non-fibrolytic agents, confirming the clinical effects of 1-PA administration, and for diagnosing thrombosis and other diseases, as well as for monitoring medical conditions. It is also known to be useful in monitoring.

Monoclonal antibodies with t-PAK specificity are being used as an alternative to polyclonal antibodies due to the need to establish a simple purification method for obtaining higher purity t-PA or a method for measuring blood t-PA levels. It has been studied in JP-A-59-5]21 (706 et al.) and JP-A-61-148200 (FTP 1163.FT
P 217 ), and are reported in JP-A-61-181964 and JP-A-61-183299 (X-21, X-23) and are well known. In addition, commercially available products include ESR-1~
Examples include ESP-7 (t-pA monoclonal antibody manufactured by Bioscotto).

(Problem) However, these monoclonal antibodies have poor affinity for antigens, so they are still insufficient for improving the degree of purification or improving the precision and sensitivity of measurement methods, and improvements are desired. was.

(Means for Solving the Problems) In order to overcome these problems, the present inventors have developed a new method using a cell fusion method that has specificity for t-PA and excellent affinity for antigens. We succeeded in obtaining a monoclonal antibody, which made it possible to purify t-PA and its derivatives and to apply it to measuring the activity and antigen amount of t-PA.

The anti-t-PA monoclonal antibody of the present invention has specificity for t-PA and has a molecular weight of 153,000±10,0
00. The IgG subclass is IgG1. The specific amino-terminal amino acid sequence of the chain variable region (described below) is specified in that the antigen-binding site is at or near the fibrin affinity site, and in particular, anti-t-PA monoclonal antibodies having the above amino acid sequence have been completely unknown. It is a novel monoclonal antibody. The anti-t-PA monoclonal antibody of the present invention includes any antibody having the above-mentioned physical and chemical properties and structure.

In particular, anti-t-
Examples include PA monoclonal antibody SP-322. Monoclonal antibody SP-322 is produced by culturing mouse hybridoma SP-322 in a culture medium or mouse ascites. Mouse hybridoma clone 5P-322 is a t-P derived from human melanoma cells.
BALB/C mouse lung cells sensitized with A and mouse myeloma cells 2 such as P3X63Ag8・Ul (P3
For example, Köhler and Milstein (K'
It can be obtained by fusion using the cell fusion method of Cihler and Milstein (see Examples below).

The medium for culturing the above-mentioned hyperdomas is Dulbecco's modified Eagle's minimum essential medium (Da-Ibeceo').
s modified mjnjmum essence
tal medium (hereinafter abbreviated as DMEM), fetal bovine serum, L-glutamine, crucose, and sodium pyruvate.

A medium containing 2-mercaptoethanol and an antibiotic (eg, halogenicillin G, streptomycin, gentamicin, etc.) is used.

The hybridoma of this invention is usually cultured in a medium with a
BALB/C system pretreated in a gas phase of 5% carbon dioxide and 95% air for 2 to 4 days or with 2,6,10.14-tetramethylpentadecane (Pristane, trade name, manufactured by Aldrich) at °C. It is carried out intraperitoneally in mice for about 20 days, and antibodies are produced in amounts that can be purified.

The monoclonal antibody thus produced can be separated and purified from the culture supernatant or ascites by conventional methods for protein isolation and purification. Such methods include, for example, centrifugation, dialysis, salting out with ammonium sulfate, DEAE
- Column chromatography using cellulose, hytroxylapatite, Flotin A-agarose, Sephadex (trademark, manufactured by Pharmacia Fine Chemicals), etc.

(Effect of the invention) The anti-t-PA monoclonal antibody SP-322 thus obtained showed high affinity for t-PA,
Agarose gels activated with 322, such as bromine cyanide-activated Sepharose 4B (trademark), can be used as immunoabtubants for the purification of t-PA and its derivatives, or as reagents for measuring the antigen amount or activity of t-PA.
5P-322-bound agarose is prepared by reacting with Pharmacia Fine Chemicals (M) or Affigel 10 (trademark, manufactured by Bio-Rad Laboratories) by a conventional method. Using this, you can use the column method or patch method.
-PA may be purified by a conventional method.

Anti-t-PA monoclonal antibody SP-322 is t-
It can be used as a reagent in enzyme immunoassay (F:IA) and radioimmunoassay (RIA) of PA. In the enzyme immunoassay method, after blocking SP-322 on a solid phase such as a microtiter plate with coated bovine serum albumin, etc., a known or unknown amount of t is added.
- PA is estimated.

In order to use the anti-t-PA monoclonal antibody of the present invention for the purification of t-PA and its derivatives, trout, anti-t-PA
Monoclonal antibody SP - unlabeled or piotin-conjugated anti-t-PA antibody is added, and a chromogenic luminescent substrate of the enzyme or enzyme-labeled avidin and its substrate are used to generate color and luminescence. 1-PA quantitative determination is performed by comparing the t-PA concentration in the subject with a standard curve prepared in advance using a t-PA standard.

Furthermore, the S P-322 anti-t-PA monoclonal antibody does not target the fibrinolytic activity of t-PA.

t-PA is adsorbed onto a microtiter plate etc. coated with SP-322, and its activity can be determined by a direct method, i.e., by interacting with a chromogenic substrate specific for t-PA, or by an indirect method, That is, first, plasminogen is converted to plasmin.

A chromogenic substrate specific to plasmin is decomposed from the generated plasmin 9 to cause a color reaction.

t-PA is quantified by comparing the t-PA concentration in the sample with a standard curve prepared in advance using a t-PA standard (Separate application by the present applicant, Japanese Patent Application No. 1723-1983)
(See No. 00).

(Example) EXAMPLES The present invention will be described in detail below with reference to Examples.

Example 1 Hybridoma SP-3220 regulation a) Preparation of immunized tile cells BAL B/C female mouse (8 weeks old at the start of immunization)
After emulsifying human melanoma cell-derived t-PA (manufactured by Bioscotto) purified by mixing with the same volume of Freund's complete adjuvant at a dose of 30 μg/mouse or 7.5 μg/mouse, It was administered intraperitoneally (first immunization). Thereafter, 30 μg/mouse or 7.5 μg/mouse of t-PA was mixed and emulsified with the same volume of Freund's incomplete adjuvant and injected intraperitoneally twice at intervals of 3 to 4 weeks. 6 weeks after the third immunization, t-PA at 30μg/mouse or 7.5μg
0.4 ml of physiological saline solution containing 50% of each animal was administered intraperitoneally (
final immunization). Three days after the final immunization, lung cells were collected from two mice and suspended in DMEM medium. Red blood cells were disrupted by treatment with 0.17M ammonium chloride for 10 minutes at 0°C and removed by centrifugation.

b) Preparation of hybridoma SP-322 The tile cells (5 x 10' cells) prepared above were transformed into mouse myeloma cells p3x5.
3Ag8・Ul (P3U1) (IX 10' pieces, manufactured by Dainippon Pharmaceutical Co., Ltd.) and the method of Koehler and Milstein [Natsu
RE, Vol. 256, p. 495 (1975)]. That is, after washing the tile cells and P3U1 cells several times with DMEM, they were placed in a 50 ml plastic test tube and thoroughly mixed. The medium was then removed by centrifugation, and 50% (w/v) polyethylene glycol (manufactured by Sigma, average molecular weight 3640) kept at 37°C was added to the medium.
1 ml of DMEM was added to the bodies under stirring over a period of 1 minute. Next, 10 mt of DMEM kept at 37°C was added dropwise to terminate the cell fusion reaction.

After centrifuging the reaction solution and removing the supernatant.

HAT medium [hyboxanthin (IX 10-'M),
Aminopterin (4X 10-'M), thymidine (1,
A DMEM medium containing 10% fetal bovine serum supplemented with 6X 10-'M) was added to the residue until the tile cell concentration was 5X10-'M.
'The number of cells/ml was adjusted. This suspension was dispensed into a 24-well plastic plate at 2 rnl (1×10 6 tile cells) per hole. Every 4 to 5 days, half of the medium was removed by suction and the above HAT medium was added.

Seven to ten days after cell fusion, proliferation of hybridomas was observed in all the wells, so anti-t-PA activity in the culture supernatant was measured by the immunoprecipitation method described below.

C) Measurement of anti-t-PA activity in culture supernatant by immunoprecipitation method t-PA labeled with radioactive iodine (+251) (by chloramine T method) [100 μl, 0.1% bovine serum albumin, 0.3M chloride Sodium, 0.05%
It was dissolved in 25mM ToIJ buffer containing (w/v) Tween 80 (pH 7.4). Below +251- t-
PA] and 20 μl of hybridoma culture supernatant were mixed and left in a plastic tube at room temperature overnight.
1 μt of rabbit anti-mouse IgG antiserum was added, and the mixture was left at room temperature for an additional 3 hours. To this, 10 μl of Pansorbin (trademark, manufactured by Calbiochem) suspension was added and mixed. After 30 minutes, 2 rnl of Tamaki Tris buffer was added and immediately centrifuged (3000 rpm, 15 minutes). Remove the supernatant.

The radioactivity of +251tpA in the washings was measured using an Autowell Gamma Counter ARC300 manufactured by Aloka. Hybridoma growth wells showing significant +25I-t-PA immunoprecipitation were grown in DMEM medium and cloning was performed.

BALB/C mouse thymus cells (106 cells/well)
Cloning was performed twice by the limiting dilution method using a 96-well microplate as a feeder layer, and the resulting hybridoma clone was named 5P-322. In addition, anti-t-P produced by hybridoma 5P-322
The A monoclonal antibody was also named 5P-322.

Example 2 Production and Purification of Monoclonal Antibodies BAL B/C male mice were incubated at 6-8 weeks of age.
6゜10,14-tetramethylpentadecane (Primethane.

After intraperitoneal injection of 0.5 ml (manufactured by Aldrink).

107 hyperdomas 5P-322 after 7-14 days
was suspended in physiological saline 0.5+n4 and inoculated intraperitoneally.

After 10 to 20 days, the ascites produced was collected from the sacrificed mice that underwent laparotomy. 5 to 10 ml of monoclonal antibody-containing ascites was obtained from one mouse. The ascites was centrifuged to remove insoluble matter, and 9 equivalent volumes of saturated ammonium sulfate solution was added thereto, and the mixture was left at 4°C for 1 hour to overnight. The resulting precipitate was centrifuged and added to 0.1 MI containing a small amount of 0.9% sodium chloride.
It was dissolved in J acid buffer (pH 8) and then dialyzed against 100 times the volume of the same buffer overnight (gamma globulin fraction). From this fraction, IgG was purified using MAPS-n mouse monoclonal antibody purification kit (trademark, manufactured by Bio-Rad Laboratories). That is, after adding the same volume of binding buffer to the gamma globulin fraction and mixing, Affigel Protein A (manufactured by Bio-Rad Laboratories) or Protein A Sepharose CL 4, which had been sufficiently flattened using the same binding buffer, was added.
Column packed with B (manufactured by Pharmacia Fine Chemicals) (gel ped volume 5 or 10 m1)
) and washed with 10 column volumes of binding buffer. Next, add approximately 3 mL of the elution buffer included in the kit.
The column volume eluted the IgG. The eluted IgG was concentrated by salting out with ammonium sulfate and dialyzed against a 0.01-0.1M phosphate buffer containing 09% sodium chloride, etc., and was used as an IgG standard of 5P-322. Normal ascites 1 rn
An rgc of 111-18ff1 per t was obtained.

Example 3 Physicochemical Properties of Monoclonal Antibodies The following physical properties, except for item b), were measured using the IgG fraction of monoclonal antibody 5P-322 purified in Example 2.

a) Molecular weight: 153,000 (±10,000) SD
S polyacrylamide gel electrophoresis was performed using Laemli [
Nature+ 227+ 680 (1970)]
Performed on 9% or 12% plate gel using buffer system.

The molecular weight of the antibody was estimated. The molecular weight marker is the Electrophoresis Molecule i Calibration Kit (low molecular weight kit) manufactured by Pharmacia Fine Chemicals.

Phosphorylase b, 94000; Bovine serum albumin, 67000; Ovalbumin, 43000;
Carbonic anhydrase, 30000; Trypsin inhibitor 20100; α-lactalbumin,
14400) was used.

b) rgG subclass: IgGI Using the hybridoma culture supernatant, 5P-
322 IgG subclasses were determined. This law is 0uch
Using Terlony's double immunodiffusion method, add 75μ of culture supernatant to the center hole.

Add 10 various anti-mouse immunoglobulin antibodies to the outer hole.
After adding μL and leaving it at room temperature for a day and night, the formation of a sedimentation line due to antigen-antibody reaction was observed.

C) Amino acid sequence (amine terminal): L chain: Asp (Ie-Val-Leu-Thr-Gl
n-8er-Pro-Ala-8et-Leu-Ala
- Sequence analysis of the L chain and H chain amino termini of Vat-8er IgG was performed using Edman degradation [Edman et al.
european+ J, Bio-chem,, I.
The IgG fraction was subjected to 5DS-polyacrylamide electrophoresis under reducing conditions to separate L chains and H chains, and each type was recovered from the gel by electroelution.

After purification by reverse-phase high-performance liquid chromatography, each product was introduced into a gas-phase sequencer (Model 470A, manufactured by Aglide Biosystems), and automatically subjected to coupling reaction, pegtide bond cleavage reaction, and PTH (phenylhydantoin). Amino acid formation reactions were performed. The obtained PTH-amino acids were separated using an Applied Biosystems model 120 APTH analyzer directly connected to a sequencer, and each PTH-amino acid was identified by comparison with the retention time of a standard PTH-amino acid.

As a result, 14 amino acid residues from the amino terminus having the above sequence were identified for the L chain. On the other hand, for the H chain, it is thought that the amine terminus may have been blocked in some way, and the above method did not lead to determining the sequence directly from the amine terminus.

d) Affinity with the antigen The affinity with the antigen is the 5P-
In the immunoprecipitation reaction with 322, unlabeled t-PA
was added to various concentrations to cause antagonism, and an inhibition curve (Figure 1) was created. The approximate dissociation constant was estimated from the degree of unlabeled t-PAg giving 50% inhibition, and that of monoclonal antibody 5P-322
Approximately 20-4 compared to A monoclonal antibody ESP-2
It was estimated that the affinity was 0 times higher. Note that this affinity is comparable to that of the polyclonal antibody +387 (manufactured by American Diagstica), which has the highest affinity for antigens.

Furthermore, differences in the affinity of the monoclonal antibody of the present invention due to differences in the armor type of t-PA were investigated using t-PA derived from single-chain and double-chain human melanoma cells manufactured by American Diagstica. Differences in affinity were observed in the inhibition curves when 1 to 100 ng/tube (7) single-chain or double-chain t-PA was added to the above-mentioned 125r-t-PA immunoprecipitation reaction. As shown in the figure, 5P-322 is 1
It was estimated that it has approximately the same affinity for both the single-chain and double-chain types of t-PA.

e) Effect of monoclonal antibody 5P-322 on t-PA fibrinolytic activity 5P-32 on in vitro fibrinolytic activity of t-PA
The influence of 2 was investigated using the 12'x-standard R fibrin film dissolution method. 15 - Fibrin film dissolution method is Hoy
The method of raerts et al. [J, Biol, Chem,
, 257 +2912 (1982)]. That is, 125I-fibrinogen (Commsariat A Lene) was added to 1.8 μM fibrinogen.
9 by mixing an appropriate amount of
50 in a 6-hole microtiter plate (Limbro)
Pg/well was added and dried at 40°C overnight.

To this, add 1.6u/mt thrombin (manufactured by Kaori Pharmaceutical Co., Ltd.).
00 μL each and left at 37°C for 4 hours to form fibrin. This plate was washed twice with 10 mM phosphate buffer containing 0.2% town serum albumin and 0.9% sodium chloride, and then subjected to activity measurement.

Add 50 μt of 200 nM plasminogen to each formula, and add 50 μt of t-PA standard or t-PA.
An unknown sample containing . Take 50μ of each formula and measure the dissolved 125 I-fibrin using an Aloka autowell gamma counter.
-Fibrinolytic activity of PA was quantified.

The t-PA standard product used was International t-P.
A Star/Dirt [Gaffney and Curtj
g+ Thromb.

Haemostas, 53+134 (1985)
] This is human melanoma cell-derived t-PA manufactured by Bioscotto, which was standardized as follows.

S P of 0.8-80 a glols at room temperature overnight in advance.
t-PA incubated with -322 (5u/ra
When the activity value of t-PA was measured, a slight (approximately 15%) decrease in activity was observed as shown in Figure 3. There was no significant difference from the factor antibody). On the other hand,
Polyclonal anti-t-PA antibody (goat, manufactured by American Diagstica) or monoclonal antibody ES P-2 manufactured by Bioscotto showed significant inhibition of activity even at the lowest dose.

f) Antigen binding site After incubating t-PA and 5P-322 at a weight ratio of 1:1000 at room temperature for 10 hours, Verhe
The method of ijen et al. [EMBO, J, 5, 3525
(1986)] investigated the affinity of t-PA for nophipurin. Add 1 u of thrombin to a mixture of t-PA (final concentration 2 μg/mt) and fibrinogen (final concentration 1-125 μg/mt), stir, and allow fibrin to form at room temperature for 3 minutes, then centrifuge the tube. (1
(6000 rpm, 8 minutes), and non-fibrin-bound t-PA activity in the supernatant was measured by fibrin plate method. In addition, in the fibrin plate method, 5P-322 does not significantly inhibit t-PA activity. As shown in Figure 4, pretreatment with 5P-322 significantly inhibits the binding of t-PA to fibrin, indicating that 5P-322 has a binding site at or near the fibrin affinity site of t-PA. t-P
It is thought that it does not bind to the active site of A.

g) Cross-reactivity with urokinase 2 The cross-reactivity between SP-322 and urokinase (Midori Juji) was investigated by the above-mentioned immunoprecipitation method and 125-fibrin film lysis method. In the immunoprecipitation method, “5I-t
- Cross-reactivity was observed in the inhibitory effect of urokinase on the immunoprecipitation of PA by 5P-322, but urokinase 1
44% inhibition was observed only when 50 u/1 ube was added (Figure 5).

When this was compared with the inhibition curve by t-PA (Fig. 1), the cross-reactivity of urokinase was judged to be about 0.1% of that of t-PA. In addition, 5P-322 did not inhibit urokinase activity (3 u/m7) at concentrations up to 80 μg/ml as measured by the 125 neef fibrillin film method.

Example 5 was added to each well of a 96-well flat-bottomed microplate with 0.3 mg of N-hydroxysuccinimide biotin.
200 μt of monoclonal antibody 5P-322 dissolved in M sodium carbonate (pH 9,6) at 1 μg/rnl was added.

After incubating for 4 hours at room temperature or overnight at 4 °C, discard the solution and add 10 mM containing 1% BSA)! J
200μ of TBS buffered saline (TBS)* was added and left to stand at room temperature for 2 hours to block unattached components in each well.

Thereafter, washing was performed several times for several minutes with a TBS (TBS/Tween) solution containing 005% Tween 720 (Teken 20 trade name).

t-PA or sample in TBS containing 0.25% BSA
/Tween (BSA/TBS/Tween) solution, add 200 pl to each well, and incubate for 2 hours at room temperature.
The reaction was carried out for 1 hour or overnight at 4°C. TB as before
After washing with S/Tween solution, BSA/TBS/T
Biotinylated goat anti-human melanoma t-PA antibody (5 mg of goat anti-human melanoma t-PA antibody (Chuo Kagaku Kogyo) diluted to 1 μg with ween solution was added, and the reaction was performed at room temperature for 2 hours. After the reaction, TBS /Tween solution, and then BSA/TBS/Tween solution.
Streptavidin-pyotinated horseradish peroxidase complex (Amersham) diluted with
was added and reacted at room temperature for 2 hours.

After washing with TBS/Tween solution, 0.006% hydrogen peroxide as enzyme substrate as well as 0.1 rTlg
Anl 3.3:5.1M acetic acid-sodium citrate buffer (pH 5) containing 5'-tetramethylbenzidine
, 8) was added in 200 μL portions and incubated at room temperature for 15 minutes. After incubation.

Add 50μt of 3M sulfuric acid to stop the reaction,
Absorbance in μm was measured.

*Tris buffer (10mM) Lis-HCl, sodium chloride 9g/l, pH 7.5) t-PAO, it was possible to quantify up to 3ng/mt.

Quantification of t-PA antigen amount in human plasma 9 Normal blood collection for normal people and 100 mmHg.

Blood collected after 5 minutes of loading, ELISA for 5 cases each
t-PA was quantified by the method.

The specimen was prepared using the method of Bergadorf et al. [Thromb,
Haemoitas,, 5Q.

740-744 (1983)]. 50 μt of plasma obtained by centrifuging the surface layer of blood collected with citric acid
Add 50μ of 1M sodium acetate (pH 3,9) to
Incubated for 15 minutes at 37°C. After incubation, 0.1M disodium hydrogen phosphate/0.4
A sample was prepared by adding 10μ of M sodium hydroxide aqueous solution and further adding 20μ of 0.5% Tween 20.

The results are shown in Table 1. From this result, t-
The average PA concentration is 6.1 ng/ml in the case of normal blood collection.
In the case of loading blood collection, it was 10.4 ng/ml.

Table 1. t-PA concentration in normal human plasma Normal blood collection Load blood collection

[Brief explanation of the drawing]

Figure 1 shows inhibition curves of immunoprecipitation reactions of I-t-PA with various antibodies and various concentrations of unlabeled t-PA.
- Inhibition curve of immunoprecipitation reaction with -322 by single-chain or double-chain non-labeled t-PA, Figure 3 is a diagram showing the inhibitory effect of various antibodies on t-PA activity in +251-fibrin film lysis method, Figure 4 shows t without fibrin binding.
Figure 5 shows the influence of monoclonal antibody 5P-322 on the fibrin affinity of t-PA. Urokina patent applicant for immunoprecipitation reaction
Yamanouchi Pharmaceutical Co., Ltd. Representative Patent Attorney Seiya Fujino

Claims (1)

[Scope of Claims] 1. A monoclonal antibody that is specific for a tissue-type plasminogen activator and has the following properties. (a) Molecular weight: 153,000±10,000 (b) IgG subclass: IgG1 (c) L chain variable region amino-terminal amino acid sequence L chain: [Gene sequence is available] (d) Antigen binding site: Fibrin affinity Sexual site or its vicinity. 2. The monoclonal antibody according to claim 1, wherein the tissue-type plasminogen activator is derived from human melanoma cells. 3. The cell line is mouse hybridoma clone SP-
322. The monoclonal antibody according to claim 1 or 2, which is No. 322.