JPH05168493A - Monoclonal antibody against pseudomonas aeruginosa elastase - Google Patents

Abstract

PURPOSE:To obtain a novel monoclonal antibody which has activity specifically linking to Pseudomonas aeruginosa elastase to neutralize the toxicity, thus can be used in determination of a trace amount of Pseudomonas aerufinosa elastase and as a diagnostic, prophylactic and therapeutic agent for the infectious diseases caused by Pseudomonas aeruginosa. CONSTITUTION:Purified Pseudomonas aeruginosa elastase is dialyzed against a phosphate buffer solution of pH7.2 and the product is used as an immunogen by mixing with Freund's complate adjuvant and the mixture is subcutaneously given to BALB/c mouse for immunization. The mouse is given the booster and the splenic cells are collected after the final immunization. The spleen cell is fused with a mouse myeloma cell using polyethylene glycol and cultured in HAT medium to obtain the hybridoma cells. The clones producing anti- Pseudomonas aeruginosa elastase antibody are selected from the hybridoma cells, then the cells are cloned by the limiting dilution analysis and the monoclonal hybridoma is cultured to produce the monoclonal antibody against Pseudomonas aeruginosa elastase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a monoclonal antibody that specifically binds to Pseudomonas aeruginosa elastase and has its toxicity-neutralizing activity, a cell line producing the monoclonal antibody, and Pseudomonas aeruginosa containing the monoclonal antibody. A preventive / therapeutic agent for bacterial infections. The monoclonal antibody of the present invention is characterized by exhibiting a strong inhibitory activity by directly interacting with the enzyme active site of Pseudomonas aeruginosa elastase, and its binding is inhibited by a metalloprotease inhibitor. Further, the monoclonal antibody of the present invention is characterized in that it specifically binds to Pseudomonas aeruginosa elastase but does not bind to toxoidized Pseudomonas aeruginosa elastase. Since the monoclonal antibody has the activity of neutralizing the toxicity of Pseudomonas aeruginosa elastase, it provides an effective therapeutic agent for Pseudomonas aeruginosa infection. Furthermore, the present monoclonal antibody provides a method for detecting and quantifying a trace amount of Pseudomonas aeruginosa elastase, and is applied as a diagnostic agent for Pseudomonas aeruginosa infection and a reagent for quantifying elastase.

[0002]

BACKGROUND OF THE INVENTION Pathogenic bacteria which are problematic in the treatment of bacterial infections are changing with the development of antibiotics. That is, with the transition of the types of clinically used antibiotics, bacteria causing bacterial infections, so-called pathogenic bacteria, have changed. Conventionally, the number of cases of infection with low pathogenic or weakly virulent bacteria, especially Pseudomonas aeruginosa has increased, and Pseudomonas aeruginosa has become one of the major pathogenic bacteria in recent years. Pseudomonas aeruginosa infection causes serious symptoms and causes death in immunocompromised patients who have been treated with immunosuppressive drugs, or in immunodeficient or immunocompromised patients such as cancer patients, burn patients and newborns. It is known as so-called opportunistic infection, which often leads to. Examples of the virulence factor of Pseudomonas aeruginosa include endotoxin, which is a bacterial cell component, and exotoxin / exoenzyme produced from Pseudomonas aeruginosa. About 90% of P. aeruginosa clinical isolates produce the protease, Pseudomonas aeruginosa elastase. Pseudomonas aeruginosa elastase not only destroys biological tissues at the site of Pseudomonas aeruginosa infection, but also decomposes serum components such as immunoglobulins and complements which are bioprotective substances It is a pathogenic factor. Chemotherapy using antibiotics and synthetic antibacterial agents is the first method for preventing and treating bacterial infections. To date, various antibiotics classified into β-lactams (penicillins, cephalosporins) aminoglucosides, macrolides, etc. have been developed,
Most of them, most Gram-positive cocci represented by Staphylococcus and Gram-negative bacteria such as Escherichia coli show sensitivity,
It has shown remarkable clinical effects. However, despite a lot of research and development to date, there are still few drugs that are sensitive to Pseudomonas aeruginosa, and the high frequency of resistant bacteria still overcomes the infectious disease of this bacterium. It has become a major factor that has not yet reached. In addition, although antibiotics suppress the growth of bacteria, they do not have the activity of neutralizing the toxicity of pathogenic factors produced by bacteria that exacerbate the symptoms of infectious diseases, that is, exotoxins and enzymes. It is one of the major limitations.

Incidentally, an elastase inhibitor therapy is considered as a therapy for the purpose of treating infections and reducing symptoms by neutralizing Pseudomonas aeruginosa elastase. Pseudomonas aeruginosa elastase is known to be a zinc metalloprotease whose zinc ion is an essential component for enzymatic activity (M
orihara et al, Agricultural and Biological Chemist
ry 39, 1123-1128 (1975)). Metalloproteases are a type of protease widely distributed in microorganisms and animals. Inhibitors belonging to the metalloprotease group include ethylenediaminetetraacetic acid (EDTA), which is a chelating agent for divalent metal ions, O-phenanthroline, and phosphoramidon (Suda et al, Journal, which is composed of a functional group having a metal chelating function and a peptide moiety. of Antibiotics 26, 621-
623 (1973)) and HO-NHCO-CH (CH2 _- CH- (CH
₃ ) ₂ ) -CO-Ala-Gly-NH ₂ (Hudgin et al, L
ife Sciences 29,2593-2601 (1981)) and the like are known. The latter metalloprotease inhibitor is a so-called active site-directed type in which the metal chelate moiety coordinates to a zinc ion, and the peptide portion of the inhibitor interacts with the enzyme active site existing in the vicinity thereof to inhibit the enzyme activity. It is an inhibitor.

[0004] However, these inhibitors also show inhibitory activity against many proteases belonging to many metalloproteases such as enzymes that produce enkeferin and endothelin which are important physiologically active peptides in Honshu, It is easily inferred to show toxicity and side effects, and there is a limit as a drug for treating bacterial infections. On the other hand, immunoglobulin therapy (antibody therapy) is one of the therapies aimed at preventing and treating bacterial infections by neutralizing and inhibiting toxins and enzymes derived from bacteria. It is attracting attention as an alternative. Not only bacteria but also toxins / enzymes can be produced by actively immunizing animals such as horses and rabbits.
Antibody therapy in which a serum having a high antibody titer against an enzyme is obtained and the serum is administered is well known to humans as examples of diphtheria toxin and snake venom, and further, experimental treatment using various animals. It has been proved in many experiments that it exhibits a remarkable therapeutic effect on infectious diseases. Furthermore, in view of the specificity of the antibody, it goes without saying that the target bacterial-derived enzyme is neutralized without affecting metalloproteases in vivo. Pseudomonas aeruginosa common antigen (OE
P), Pseudomonas aeruginosa elastase, and an immunoglobulin preparation comprising an antiserum (polyclonal antibody) having a relatively high antibody titer against Pseudomonas aeruginosa protease are known. Even if antisera obtained from animals other than humans have high titers, this method of transferring so-called heterologous proteins other than humans into the human body causes serious side effects such as anaphylaxis and other allergic reactions. There is a possibility of causing it. On the other hand, a conventional human immunoglobulin preparation is obtained by collecting blood from a healthy person or a patient who has already been infected with bacteria, and fractionating and purifying the immunoglobulin fraction by a known method, followed by polyethylene glycol addition, proteolytic enzyme treatment, It is formulated not only for intramuscular injection but also for intravenous injection by a method of removing aggregates such as sulfonation and DEAE-column chromatography. These human immunoglobulin preparations have several drawbacks. First, bacteria and toxins derived from bacteria
The antibody titer to the enzyme is low, and a sufficient therapeutic effect cannot always be expected. Second, it is difficult to stably supply a large amount of high titer immunoglobulin. It is made from blood collected from healthy volunteers and patients,
It is extremely difficult to constantly obtain a high titer serum, and the antibody titer may vary from production lot to production lot. Thirdly, by using human blood as a material, immunoglobulin preparations can be added to hepatitis viruses such as HBs virus, ATLV (Adult T cell leukaemia virus), and HIV (Hum).
an immunodeficiency virus), or contamination with pathogens such as non-A non-B hepatitis virus. Thus, development of a safe immunoglobulin preparation having a high antibody titer against bacteria and toxins / enzymes derived from bacteria, especially elastase derived from Pseudomonas aeruginosa, and having a large therapeutic effect on bacterial infections is desired.

[0005]

[Problems to be Solved by the Invention] Pavlovskis and Wretlind
Reported that the antiserum against Pseudomonas aeruginosa elastase was significantly effective in experimental mouse Pseudomonas aeruginosa infection
(1979), Vol.24, p.181-187), the development of an immunoglobulin preparation having a strong neutralizing activity against Pseudomonas aeruginosa elastase is also desired. Monoclonal antibodies with neutralizing activity against Pseudomonas aeruginosa elastase may be used to stably produce immunoglobulin preparations that are highly effective against infectious diseases, but they inhibit the proteolytic enzyme activity of elastase. So far, no monoclonal antibody having so-called neutralizing activity has been obtained. Lagace and Frechette reported a monoclonal antibody against Pseudomonas aeruginosa elastase (Infect. Immun. (1991), Vol.59, p.7.
12-715), they do not show that the antibody has a neutralizing activity against elastase, nor do they show any efficacy in treating infection of elastase-producing Pseudomonas aeruginosa. The difficulty in obtaining a monoclonal antibody having elastase-neutralizing activity is predicted from the fact that elastase is a proteolytic enzyme and can also degrade and inactivate the monoclonal antibody itself. That is,
Monoclonal antibodies that simply bind elastase are unsuitable. That is, of the epitopes (antigenic determinants) that are considered to exist innumerably on the elastase molecule,
Only monoclonal antibodies that recognize an epitope that can contribute to neutralizing activity, which truly blocks proteolytic enzyme activity, will be suitable. From the current knowledge of elastase, it is difficult to predict a monoclonal antibody having a strong neutralizing activity against Pseudomonas aeruginosa elastase. On the other hand, in addition to antibody therapy, there is active immunotherapy in which a so-called vaccine in which toxins and enzymes are detoxified is administered. In Pseudomonas aeruginosa, the effectiveness of a multi-component vaccine comprising Pseudomonas aeruginosa common antigen (OEP), Pseudomonas aeruginosa elastase toxoid, and Pseudomonas aeruginosa protease toxoid has been shown. (Hommma et al., Japanese Journal of Experime
ntal Medicine 48,111-133 (1987))

Pseudomonas aeruginosa elastase toxoid means formalin treatment (Homma et al. Japanese Journal of Experime
ntal Medicine 48, 111-133 (1987)) or ClCH ₂ -CO-HO which is a chloroacetyl peptide derivative.
_{Leu-Ala-Gly-NH 2} (HOLeu is N- hydroxy -L- leucine) (Morihara et al, Journal of Clini
Cal Microbiology 23,53-55 (1986)), which was prepared for the purpose of retaining the antigenicity and lacking the enzyme activity. Even in this case, elastase itself has lost the enzyme activity, and only the antibody titer to the protein in the state where the essential enzyme active site is destroyed rises, and it truly binds to the active center of the enzyme and neutralizes the activity. It is conceivable that an increase in antibody titer cannot be expected. However, enzymatically active elastase cannot be administered because of its toxicity. From these points as well, the usefulness of the immunoglobulin preparation consisting only of the antibody having the property of neutralizing the enzyme activity of elastase, that is, its toxicity is high.

[0007]

[Means for Solving the Problems] In view of these circumstances, by using elastase retaining enzyme activity as an immunogen, a number of monoclonal antibodies against Pseudomonas aeruginosa elastase were prepared, and the properties of each monoclonal antibody were analyzed. The invention was completed. Specifically, as a method for screening a specific antibody against elastase, a method for determining the neutralizing effect of the enzyme activity was developed instead of the enzyme immunoassay method or the radioimmunoassay using the conventionally used antigen as a solid phase. Adopted. By this method, conventionally, a monoclonal antibody is screened for which the reactivity disappears due to a change in the three-dimensional structure (conformation) of the antibody protein generated when the antigen is immobilized or labeled with a radioisotope. It has become possible. Using this method, a number of monoclonal antibodies against Pseudomonas aeruginosa elastase were obtained. While examining the therapeutic effect of each monoclonal antibody, detailed analysis of physicochemical, immunochemical and biological properties revealed that it directly interacts with the enzyme active center site of Pseudomonas aeruginosa elastase and We succeeded in obtaining a monoclonal antibody with a strong inhibitory effect.

The monoclonal antibody of the present invention is characterized by having a toxicity neutralizing activity against Pseudomonas aeruginosa elastase.
Further, the monoclonal antibody of the present invention is characterized in that it specifically binds to Pseudomonas aeruginosa elastase and the binding is inhibited by a metalloprotease inhibitor. Furthermore, the monoclonal antibody of the present invention is characterized by specifically binding to Pseudomonas aeruginosa elastase, but not toxoidized Pseudomonas aeruginosa elastase. The present invention relates to a monoclonal antibody having the above-mentioned immunochemical / biochemical properties, a cell line producing the monoclonal antibody, and an immunoglobulin preparation for treating infections containing the monoclonal antibody.

A cell line that specifically reacts with Pseudomonas aeruginosa elastase and continuously produces the monoclonal antibody having the above characteristics is obtained by the following method. The hybridoma of the present invention consists of five steps: immunization, cell fusion, fused cell selection, assay and cloning. The immunization process is a process of sensitizing animals (excluding humans) with a target antigen. As an antigen, a mixture obtained by mixing a purified product of Pseudomonas aeruginosa elastase with Freund's complete or incomplete adjuvant is sensitized by injecting it into an animal such as a mouse 1 to 6 times at intervals of 1 week to several months. In humans, natural infection with elastase-producing Pseudomonas aeruginosa corresponds to this immunity. The route of administration of the antigen may be intraperitoneal administration, subcutaneous administration, or intravenous administration, but subcutaneous administration is particularly preferable. Cell fusion 2
It is desirable to additionally sensitize Pseudomonas aeruginosa elastase purified product 5 days before. The cell fusion step comprises the step of fusing spleen cells or lymphocyte B cells extracted from the immunized (sensitized) animal with myeloma cells. Human peripheral blood, spleen, which has high serum anti-pseudomonas elastase antibody titer as lymphocytes
It is also possible to use lymphocytes derived from tonsils and lymph nodes. As myeloma cells, HPRT (hypoxanthine-guanine
phosphoribosyltransferase) -deficient mouse myeloma cells P3-X63-Ag8-U1 (ATCCCRL1597), P3-NS-1 / 1-Ag4-1 (ATC
C TIB 18), P3-X63-Ag8-653 (ATCC CRL1580), SP2 / 0-Ag
14 (ATCC CRL8287), SHM-D33 (ATCC CRL 1668), etc. are used as the representative, but in addition, animal and human origin usually used in the field of cell engineering, or heterologous fused cells such as human / mouse heteromyeloma Non-producing or non-secreting immunoglobulin-derived myeloma cells or B lymphoblastoid cells can also be used. HVJ as a fusion method
The method (method using Sendai virus), the polyethylene glycol method (PEG method), the electrical fusion method and the like are used. In the PEG method, PEG 1,000-6,000 is 30-50% (w /
Use at the concentration of v). The fusion efficiency can be increased by adding lectin, poly-L-lysine, DMSO or the like. The fusion method is the method of Koeler and Milstein (Natur
e 256, 495-497. (1975)).

The fused cell selection step is a step of selecting the fused cells by culturing in an appropriate selective medium in which only the fused cells can grow. When HGPRT-deficient myeloma cells were used as fusion parent cells, HAT (Hypoxanthi
ne, Aminopterin, Thymidine) medium and HAz (Hypoxanthin
e, Azaserine) medium is used. Change to fresh selection medium every 2 to 4 days and incubate for 10 to 20 days. In addition, known methods such as the actinomycin D / emetine combination method, the dimethylpyrocarbonate method, and the ouabain method can be used. The assay step is a step of selecting a target specific antibody-producing cell from the fused cells. Antibodies are selected by their ability to neutralize the activity of proteins that act as antigens, that is, in the present invention, the activity of inhibiting the hydrolysis of proteins and peptides such as elastin that serves as a substrate for Pseudomonas aeruginosa elastase. The cloning step is a step of forming the specific antibody-producing cells of interest into a uniform cell population derived from one clone. Ultra dilution method, soft agar method, single cell manipulation
cell line which produces an antibody that reacts with Pseudomonas aeruginosa elastase derived from a monoclone (single clone), a so-called hybridoma.

By subjecting this hybridoma to a known method, for example, chemical treatment with a DNA mutagen and physical treatment such as UV irradiation, hybridoma progeny cells having improved proliferative ability and antibody producing ability can be obtained. it can. It goes without saying that this progeny cell retains the biological / physical properties described in the present invention, like the parent hybridoma. In addition, a method known from this hybridoma, for example, Molecular Cloning, (Cold Spring Har
The same biological / physical properties as the antibody of the present invention can be obtained by cloning the antibody gene or antibody variable region gene by the method described in (Bor Laboratory), preparing an expression plasmid containing the gene, and expressing it in an animal cell as a host. Antibodies with biological properties can be obtained. Also, known methods such as Morrison's method (Proc. Natl. Acad. Sc
i. USA. Vol. 81, pp. 6851-6855 (1984)) to construct a novel gene in which the antibody variable region gene is linked to another appropriate gene such as human antibody constant region gene, and an expression plasmid containing the gene is constructed. A hybrid molecule having the same variable region as that of the antibody of the present invention and having the same biological activity as that of the antibody of the present invention can be obtained by expressing the animal cell or microorganism as a host. The antibody of the present invention is
A culture supernatant obtained by culturing the hybridoma in a test tube in a normal animal cell medium containing serum or serum, or pristane (2,6,10,14-tetramethylpentadecane, Aldrich)
Alternatively, it is purified from a biological exudate obtained by inoculating an animal such as a mouse pre-administered with Freund's incomplete adjuvant and culturing in vivo. That is, the obtained culture supernatant or bioexudate is used in a conventional biochemical method for protein purification, for example, ammonium sulfate precipitation method, ion exchange chromatography method, gel filtration method, and antibodies represented by protein A and protein G. It is purified by subjecting the bound protein to a resin-immobilized affinity chromatography method.

The purified monoclonal antibody is formulated by a method usually used for formulation of biological products. Basically, it is filled in a sterile vial after filtering and sterilizing operation using a membrane filter or the like. It may be freeze-dried. Further, a stabilizer is added if necessary. This monoclonal antibody preparation
The preventive agent may consist of one kind of monoclonal antibody against Pseudomonas aeruginosa elastase, but more preferably at least two or more kinds of monoclonal antibodies capable of recognizing different antigenic determinant sites of Pseudomonas aeruginosa elastase molecule. Used as a mixture with antibodies. Alternatively, it is used as a mixture with an antigen derived from Pseudomonas aeruginosa other than Pseudomonas aeruginosa elastase, for example, a conventional antibody that recognizes Pseudomonas aeruginosa exotoxin such as exotoxin and protease, and outer membrane protein / endotoxin components. Furthermore, a monoclonal antibody against Pseudomonas aeruginosa elastase obtained by the present invention can be added to an antibody against bacteria, viruses, fungi, protozoa, and cancer cells other than Pseudomonas aeruginosa. The monoclonal antibody obtained by the present invention is added to a conventional immunoglobulin preparation to give a high titer immunoglobulin preparation against elastase. The monoclonal antibody obtained by the present invention mainly belongs to class IgG but is not limited thereto.
The present monoclonal antibody includes all mouse antibodies, human antibodies, mouse-human chimera antibodies, class / subclass conversion antibodies, etc., as long as they have the above-mentioned characteristics. Furthermore, Fab fragments and Fv fragments of antibodies having a neutralizing effect on Pseudomonas aeruginosa elastase are also included. As the quantification of Pseudomonas aeruginosa elastase using the monoclonal antibody of the present invention and the diagnosis of elastase-producing Pseudomonas aeruginosa infection, immunochemical quantification methods and diagnostic methods using ordinary antibodies are considered, and the monoclonal antibody can be used. If so, there is no limit. For example, an enzyme immunoassay method, a radioimmunoassay, an agglutination method and the like are exemplified.

[0013]

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to these. The conventional ELISA method used for the assay of Pseudomonas aeruginosa elastase antibody is shown below. (1) Preparation of ELISA plate Pseudomonas aeruginosa elastase was added to phosphate buffer (pH 7.2;
Cl (8g / 1), KCl (0.2g / 1), Na ₂ HPO ₄・ 12H
₂ O (2.99 g / 1) and KH ₂ PO ₄ · (0.2 g / 1)): hereinafter abbreviated as PBS) and dissolved at a concentration of 10 μg / ml in a 96-well microplate (Falcon # 3912) (abbreviated as microplate) ), 100 μl / well, 4 ℃
Incubated overnight. After removing the elastase solution from the microplate, a 3% bovine serum albumin (BSA) PBS solution was added to the wells at a rate of 12 per well.
A microplate in which 0 μl of each of them was dispensed and incubated at 37 ° C. for 30 minutes to block the unadsorbed portion of elastase was used as an antigen adsorption plate in the subsequent operations.

(2) ELISA Before assaying the antigen adsorption plate with 5 mM EDTA and 0.05% Twee
It was washed 3 times with PBS containing n 20 (hereinafter abbreviated as PBST). Then, 50 μl of PBST was dispensed per well, and 50 μl of a sample (serum or culture supernatant) appropriately diluted with PBST was added per well, if necessary, and incubated at 37 ° C. for 2 hours. The sample was then removed and washed 3 times with PBST. Subsequently, the second antibody solution was added in an amount of 100 μl per well and incubated at 37 ° C. for 2 hours. For the measurement of the antibody titer, a phosphatase-labeled affinity-purified anti-mouse immunoglobulin antibody (Kirkegaard & Perry Lab. Inc.), which was diluted 500 to 1,000 times with PBST, was used as the second antibody. After removing the second antibody and washing three times with PBST, a color-developing substrate solution (3 mg of p-nitrophenylphosphoric acid-2-sodium salt was added to 1 ml of NaN ₃ (0.2 mg / ml), MgCl ₂ .6H ₂
10% diethanolamine buffer containing O (0.1 mg / ml)
An aqueous solution dissolved in pH 9.1) was added to each well in an amount of 100 μl and reacted at 37 ° C. After reacting for 30 minutes, the absorbance at 405 nm (A ₄₀₅ ) was measured by multiscan (Titertek). Further, the detection of the anti-elastase antibody by neutralizing the elastin-degrading activity in the present specification follows the method shown below. FITC (fluorescein isothiocyanate) -labeled elastin (Elastin Products Company, Missouri) at a concentration of 10 mg / ml in 0.1 M Tris-maleic acid buffer (pH 7.0) containing 1 mM CaCl ₂ (hereinafter referred to as buffer I) ) Suspended substrate solution 60 μl, elastase solution 60 μl dissolved in buffer I at a concentration of 0.3 μg / ml and hybridoma culture supernatant (medium was used for control) 60 μl in V-bottom 96-well micro Dispense into a plate, 37 ℃,
Incubate for 2 hours. Plate 3 000 rpm, 5 m
After centrifuging in, 100 μl of supernatant is collected and transferred to a 96-well flat bottom plate. The amount of free FITC in this supernatant was determined by corona microplate reader MTP-32 (Corona Elect).
ric Co., Ltd., Ibaraki, Japan). The elastin degradation inhibitory activity is [(fluorescence intensity when adding culture supernatant)-(background)] / [(fluorescence intensity when adding only medium)-(background)]. However, the background represents the fluorescence intensity in the absence of elastase.

Example 1. Production of mouse monoclonal antibody
Ltd. (1) antigen Preparation commercial Pseudomonas aeruginosa elastase purified product (Nagase Biochemical) was used. Pseudomonas aeruginosa elastase in phosphate buffer (pH 7.2; composition
NaCl (8 g / l), KCl (0.2 g / l), Na ₂ HPO ₄・ 12H ₂ O (2.99 g
/ l)) and KH ₂ PO ₄ (0.2 g / l): hereinafter abbreviated as PBS) and used as an immunogen. (2) Preparation of immunized spleen cells BALB / c mice (female, 4 weeks old at the time of initial immunization, body weight about 15 g) were used for immunization. 0.2 ml of a mixture containing 1 volume of PBS containing Pseudomonas aeruginosa elastase at a concentration of 1 mg / ml and 1 volume of Freund's complete adjuvant according to a conventional method, per mouse)
Primary immunization was carried out by subcutaneous administration (containing 0.5 μg of Pseudomonas aeruginosa elastase). After that, 4 times at 2 week intervals,
100 μg of Pseudomonas aeruginosa elastase mixed with Freund's incomplete adjuvant was subcutaneously administered and booster immunization was performed. About 1 month after the last booster, 100 μg of Pseudomonas aeruginosa elastase
Was administered intravenously for final immunization. Three days later, the spleen was removed to obtain spleen cells suspended in Eagle's MEM medium (Hanai Kagaku). (3) Cell fusion 10% of mouse myeloma cells P3X63-Ag8-653 (ATCC CRL1580)
The cells were cultured in RPMI-1640 medium (Hansui Kagaku) supplemented with fetal bovine serum FCS (Gibco), and the cells were collected in the logarithmic growth phase and used for cell fusion. The fusion method is described by Koehler et al. (Nature, 256, 495-497,
1975). That is, spleen cells and myeloma cells were suspended in serum-free RPMI-1640 medium at a ratio of 5: 1,
Centrifugation was performed for 7 minutes at 1,000 rpm (Tomy Seiko CD-100R). To the precipitation, add 1 ml of 45% (W / W) polyethylene glycol (PEG) 4000, 10% (V / V) dimethylsulfoxide solution kept at 37 ° C over 1 minute, and then at 37 ° C for 1 minute. I let it stand.

Then, 2 ml of serum-free RPMI-1640 medium was added to 1 m.
It was added and suspended at a ratio of l / 1 min. Furthermore, serum-free RPMI-
7 ml of 1640 medium was gradually added to dilute PEG. After standing at 37 ° C for 20 minutes, centrifuge at 1,000 rpm for 7 minutes, and then resuspend in RPMI-1640 medium containing 10% FCS to obtain a cell concentration of 6 × 10 ⁴ cells / ml as mouse myeloma cells. Cloudy, 0.2 in a 96-well microplate (Sumitomo Bakelite MS-3096F)
Dispensed in ml / well. The fused cells were cultured at 37 ° C. in 5% CO ₂ . One day after cell fusion, half the volume of medium was replaced with fresh HAT medium (10 ^-4 M hypoxanthine, 4 × 10
RPMI containing ^-7 M aminopterin and 1.6 × 10 ^-5 M thymidine
1640 medium). After that, half-volume exchange with HAT medium was performed 3 times every 2 days. After 10 days, cell proliferation was observed in 70% of the microplate wells. As a result of the assay by ELISA or elastin degradation neutralizing activity,
Production of specific antibody was observed in 42 wells. Table 1 shows typical positive examples.

[0017]

[Table 1]

(4) Ascites 4 weeks ago, pristane (2, 6, 10, 14-tetramethylpentadecane; Aldrich Chemical Co. Inc., Wisconsin)
Hybridomas grown in in vitro culture after cloning in Balb / C mice treated with 0.5 ml per mouse
5 × 10 ⁶ cells were inoculated into the abdominal cavity, and 7 to 14 days later, ascites was collected from the abdominal cavity of the mouse. (5) Purification Mouse ascites was purified by Protein A monoclonal antibody purification system MAPSII (Bio-Rad; New York). That is, ascites was treated with a lipid lipid solution (Repligen Corp.), and
Perform ultracentrifugation at 00,000xg, 1h, 15 ℃. Obtained ascites and M
APSII binding buffer pH 9.0 (Bio-Rad) was mixed 1: 1 and applied to an Affigel Protein A column (Bio-Rad), followed by washing with the above-mentioned binding buffer. After elution with MAPSII elution buffer (pH 3.0; Bio-Rad), it was immediately dialyzed against PBS to give a purified sample.

Example 2. Characterization of monoclonal antibody (1) In vitro elastase neutralization test 1) Qualitative analysis by SDS-PAGE using bovine serum albumin as a substrate Table 1 shows Pseudomonas aeruginosa elastase and bovine serum albumin (BSA).
React in the presence of the mouse monoclonal antibody shown in
The effect of antibodies on the degradation of bovine serum albumin by Pseudomonas aeruginosa elastase was analyzed by SDS-PAGE. Mouse monoclonal antibody (10 μl of ascites) was added to Pseudomonas aeruginosa elastase (0.1 μg / ml, 10 μl), pre-incubated at 37 ° C for 30 minutes, and then BSA (5 mg / ml, 100 μl) was added.
The reaction was performed at 7 ° C for 2 hours. The reaction mixture is 2% SDS, 5% 2-
125 mM with mercaptoethanol and 10% glycerol
Heat-treat in Tris-HCl buffer pH 6.8 at 100 ° C for 5 minutes at 10-20% linear gradient acrylamide concentration.
SDS / polyacrylamide gel electrophoresis was performed. The protein on the gel was stained with Coomassie Brilliant Blue and the degradation of BSA was observed. The BSA band observed under the condition without addition of Pseudomonas aeruginosa elastase was completely decomposed by the addition of Pseudomonas aeruginosa elastase. Degradation of BSA by Pseudomonas aeruginosa elastase was inhibited by the addition of ET-4D3.
Band A was recognized. On the other hand, addition of other monoclonal antibodies did not affect BSA degradation.

(2) Quantitative analysis using FITC-elastin as a substrate The enzymatic activity of Pseudomonas aeruginosa elastase was used in a reaction system in which FITC-labeled elastin (Elastin Products Co., Missouri) was used as a substrate to coexist with a monoclonal antibody. We examined the effect of this. Degradation of elastin by elastase solubilized FITC
Was used as an index. Pseudomonas aeruginosa elastase (10,30 μg / ml; 7
5 μl) mouse monoclonal antibody ET-4D3 or ET-2B
1 (100 μg / ml; 75 μl) was added and pre-incubated at 37 ℃ for 30 minutes, then FITC-labeled elastin (5 mg / ml;
150 μl) was added and reacted at 37 ° C. for 3 hours in the dark. The labeled elastin was removed by centrifugation from the reaction mixture, and 100 μl of the supernatant was collected and dispensed into a 96-well plate. The amount of FITC contained in the supernatant was measured using an automatic photometer for 96-well plates (Corona MTP-32). The results are shown in Table 2. Both monoclonal antibodies inhibited proteolysis by Pseudomonas aeruginosa elastase.
ET-4D3 inhibited more than 50% of one molecule of Pseudomonas aeruginosa elastase with one or more antibody molecules. On the other hand, the inhibition of degradation by ET-2B1 was weaker than that of ET-4D3.

[0021]

[Table 2]

3) Neutralizing Activity of Monoclonal Antibody ET-4D3 with Pseudomonas aeruginosa Elastase Enzymatic Activity Using Synthetic Substrate Furyl acryloyl-glycyl-leucine amide (hereinafter, F
Abbreviated as AGLA; measured using Peptide Institute (Osaka). ET-4D3 was added to this reaction system to examine the enzyme activity neutralizing ability. FAGL dissolved in 0.1 M Tris-maleic acid buffer (pH 7.0) at a concentration of 2.5 mM
Pseudomonas aeruginosa elastase (purchased from Nagase Biochemical) 0.5 μg in 200 μl of A, and PB of ET-4D3 at various concentrations
After mixing 100 μl of S (−) solution and incubating at 37 ° C. for 1 hour, 0.7 M sodium phosphate buffer (pH
The reaction was stopped by adding 400 μl of 6.0). A reaction mixture containing no enzyme was prepared and used as a control.
The decrease in the absorbance at m ((absorbance of control)-(absorbance after reaction)) was measured, and the enzyme activity was defined as 1 μg of enzyme and the change in absorbance per hour (−ΔA ₃₄₅ / h · μg protein). ET
The neutralizing ability of -4D3 was expressed as a relative residual enzyme activity with the enzyme activity when not added as 100%. Mouse anti-elastase antibody ET-2B1 as a comparison target of the antibody,
Pseudomonas aeruginosa elastase and a metalloprotease with a similar enzyme active site (Beve)
r and Iglewski, Journal of Bacteriology 170, 4309-431
4 (1988)) is a thermolysin (Bacillus thermoprot
Derived from the eolyticus rokko strain. , Sigma) 0.3 μg was used. The results are shown in Table 3. When using a synthetic substrate, ET
Neutralizing activity was found only in -4D3, and no neutralizing activity was found in ET-2B1. Furthermore, ET-4D3
Neither ET-2B1 nor neutralizing activity against thermolysin was observed.

[0023]

[Table 3] From Table 3, it was found that at least one molecule of the antibody inhibited the proteolytic enzyme activity of one molecule of Pseudomonas aeruginosa elastase by 50%.

Example 3. Anti-elastase antibody ET-4D
Preparation of Fab fragment of 3 and elastase by the fragment
Somatic activity (1) Preparation of Fab fragment of monoclonal antibody ET-4D3 Monoclonal antibody ET-4D3 (PBS (-) solution)
10 mg was dialyzed against 20 mM phosphoric acid / 10 mM EDTA buffer (pH 7.0) and then concentrated to about 0.5 ml with an ultrafiltration device. The obtained IgG solution (0.
5m) and 20m added with 20mM cysteine hydrochloric acid
0.5 ml of M phosphate buffer (pH adjusted to 7.0) is mixed, and papain-immobilized Agarose (PIERCE, Rockfo
rd, Illinois USA) suspension (1 ml) was added, and the mixture was incubated at 37 ° C. for 16 hours with vigorous stirring. After the reaction, the immobilized papain was filtered off. Affi-P was added to the obtained filtrate.
rep Protein A MAPS II Binding buffer (Bio-Rad Labo
ratories, Richmond, CA USA) in an equal amount
A-Cellulofine (Seikagaku) column (φ0.8cm x 2cm)
I went to Wash the column with Affi-Prep Protein A Binding buffer and concentrate the flow-through fraction to an appropriate volume.
perose 6 column (φ1cm x 30cm; PharmaciaLKB, Uppsal
a, Sweden), and the protein fractions detected by the absorbance at 280 nm were collected and used as the ET-4D3 Fab fragment preparation. (2) Neutralization of Pseudomonas aeruginosa elastase enzyme activity using a synthetic substrate by the Fab fragment of the monoclonal antibody ET-4D3 Whether or not the Fab fragment of ET-4D3 was neutralized according to Example 2- (3) was furylacryloyl. -Glycyl-
It was examined using leucine amide (FAGLA). The examination conditions were carried out according to Example 2- (3). The results are shown in Table 4.

[0025]

[Table 4]

(3) Neutralization of Pseudomonas aeruginosa elastase activity using fluorescence-labeled elastin as a substrate with Fab fragment of ET-4D3 Neutralization of elastase activity of Fab fragment of ET-4D3 was carried out in Example 3- (2). Similarly, FITC-labeled elastin was used for the study. 10 mg / m of ET-4D3 or Fab fragment of ET-4D3 in 100 µl of PBS (-) solution and 0.1 M tris-maleic acid buffer (pH 7.0).
FITC-elastin suspension 100 suspended at a concentration of 1
μl, and elastase solution (200 μg / ml) 5 μ
1 was mixed and incubated at 37 ° C. for 2 hours with vigorous stirring. After the reaction, the mixed solution was centrifuged at 10,000 rpm for 3 minutes, and 100 μl of the obtained supernatant was recovered and FI in the supernatant was collected.
The amount of TC was measured according to Example 2- (2). The results are shown in Table 5.

[0027]

[Table 5]

From the above results, ET-4D3 was Fab
It was shown that the fragment also has a high neutralizing activity against Pseudomonas aeruginosa elastase as well as intact IgG. That is, the enzyme-neutralizing activity is determined by ET regardless of whether a synthetic peptide substrate or a fluorescently labeled elastin is used
-4D3 and the Fab fragment of the antibody showed similar neutralizing activity.

Example 4. Effect of various metalloprotease inhibitors on the binding between monoclonal antibody and elastase (1) Analysis of antigen-antibody binding reaction by gel filtration The binding between antigen elastase and monoclonal antibody was analyzed by gel chromatography. Immediately after mixing the monoclonal antibody (10 μg) and elastase (4.3 μg) to a final volume of 500 μl with PBS, immediately
It was applied to a se 6 column (φ1 cm × 30 cm). At this time, the molar ratio of the monoclonal antibody and elastase is 1: 2.
The elution position of the protein was known by absorption at 280 nm. The results are shown in Fig. 1. 29.3 minutes for elastase alone (a),
Monoclonal antibody (b) is 24.
At 9 minutes, an antigen-antibody complex was observed at an elution time of 23.8 minutes in the mixture (c) of the elastase and the anti-elastase monoclonal antibody in a molar ratio of 2: 1. (2) Effect of metalloprotease inhibitor on antigen-antibody binding Elastase (4.3 μg), monoclonal antibody (1
0 μg) and a metalloprotease inhibitor at a concentration capable of inhibiting 100% of the enzyme activity of elastase to give a final volume of 500 μl according to Example 4- (1).
It was applied to a se 6 column. Incidentally, phosphoramidon than Peptide Institute, HO-NHCO-CH (CH 2 -CH- (CH
_{3) 2) -CO-Ala-} Gly-NH 2 is Calbiochem Cor
Ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline were purchased from Dojindo Chemical Co., Ltd. from p. (SanDiego, CA, USA). The results are shown in Figure 2. The binding of ET-4D3 to elastase was 100 μM phosphoramidon or 1
0μM of _{HO-NHCO-CH (CH 2} -CH- (CH 3) 2)
It was inhibited by the addition of _{-CO-Ala-Gly-NH 2} ( Fig. 2 b, b). On the other hand, EDTA and O-phenan
Almost 100% elastase activity for throline
ET-4D even when 10 times the amount of inhibition (1 mM) is added
No effect was observed on the binding between 3 and elastase (Fig. 2-C, D). In addition, ET-4D3 shown in Table 1
With respect to the anti-elastase monoclonal antibodies other than the above, no effect was observed on the antigen-antibody reaction by adding any inhibitor. The above results are ET-4D3
The ability to bind the antigen to the active site of elastase (here, phosphoramidon, HO-NHCO-
_{CH (CH 2 - H- (CH} 3) 2) -CO-Ala-Gly-
It is shown to be specifically inhibited by NH ₂ ). On the other hand, by removing the zinc ion essential for the enzymatic activity of elastase with a metal chelating agent, no influence was observed on the antigen-binding ability of any monoclonal antibody including ET-4D3.

(3) Binding of monoclonal antibody ET-4D3 to elastase toxoid The elastase toxoid was prepared by the following method. Commercially available Pseudomonas aeruginosa elastase (Nagase Biochemical) was dialyzed against 0.1 M Tris buffer (pH 7.5; containing ₂ mM CaCl ₂ ) overnight, and then the protein concentration was adjusted to 0.5 mg / ml. The method of Morihara et al. (Journal of Clinical Microbiol
The esterase toxoid prepared according to Ogy 23, 53-55 (1986)) is called toxoid-I. Specifically, (Cl
_{CH 2 -CO-HOLeu-Ala-} Gly-NH 2, the Peptide Institute) was reacted overnight at room temperature was added to a concentration of 1 mg / ml, to prepare a toxoid -I preparation. Furthermore, using formalin, the method of Homma (Japanease Jour
The toxoid prepared according to nal of Experimental Medicene 48, 111-133 (1978) is called toxoid-II. Specifically, elastase (final concentration 2 mg / ml) was added to 4
React with 4% formalin / 0.2 M borate buffer (pH 9.0) at 4 ° C. overnight. The product obtained is sephadex G-5
0 (Pharmacia LKB column chromatography,
The protein fractions having a molecular weight of about 25,000-50,000 were pooled and used as a toxoid-II preparation. The binding properties of ET-4D3 to the two toxoids were investigated by Superose 6 gel filtration chromatography. Experimental conditions are Example 3-
Followed (1). The results are shown in Figure 2. ET-4D3 showed no binding property to either toxoid-I or toxoid-II (Fig. 2-e, g). On the other hand, all the anti-elastase antibodies other than ET-4D3 shown in Table 1 quantitatively bound to toxoid-I. (Fig. 2
-F)

[Brief description of drawings]

FIG. 1 Analysis of binding of anti-elastase monoclonal antibody and elastase by Superose 6 gel chromatography. (A) Elastase, (b) ET-4D
3, (C) is a molar ratio of ET-4D3 and elastase 1:
Mixture of 2.

[FIG. 2] Effects of various metalloprotease inhibitors on the binding of monoclonal antibody ET-4D3 and elastase,
In both analysis of the binding of ET-4D3 to elastase toxoid by Superose 6 gel chromatography, ET-4D3 and elastase (or its toxoid) were mixed at a molar ratio of 1: 2. (I)
Is phosphoramidon 100 μM, (b) is HO-N
_{HCO-CH (CH 2 -CH- (} CH 3) 2) -CO-Ala-
Gly-NH ₂ ) at 10 μM, (c) at 1 mM of EDTA, (d) at o-phenanthroline at 1 mM. (E) is ET-4D3 and toxoid-I, (f) is ET-2B1 and toxoid-I,
(G) is a chromatogram when ET-4D3 and toxoid-II were mixed at a molar ratio of 1: 2. Dashed line 1,
2 and 3 are the monoclonal antibody /
The elution positions of elastase complex, monoclonal antibody, and elastase are shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C07K 15/28 7731-4H C12N 5/20 G01N 33/573 A 9015-2J 33/577 B 9015- 2J // C12N 15/06 G01N 33/569 D 9015-2J (C12P 21/08 C12R 1:91) (72) Inventor Hiroshi Noguchi 4-2-1 Takashi, Takarazuka-shi, Hyogo Sumitomo Chemical Co., Ltd.

Claims (14)

[Claims] 1. A monoclonal antibody which specifically binds to Pseudomonas aeruginosa elastase and has toxicity-neutralizing activity. 2. A monoclonal antibody which specifically binds to Pseudomonas aeruginosa elastase, and the binding is inhibited by a metalloprotease inhibitor. 3. A metalloprotease inhibitor is phosphoramidon.
Or HO-NHCO-CH (CH _2-CH- (CH₃)₂)
-CO-Ala-Gly-NH₂Is characterized by
The monoclonal antibody according to claim 2. 4. A monoclonal antibody which specifically binds to Pseudomonas aeruginosa elastase but does not bind to toxoidized Pseudomonas aeruginosa elastase. 5. The monoclonal antibody according to claim 1, 2, 3 or 4, wherein the monoclonal antibody is IgG. 6. An antibody fragment containing a site involved in antigen binding in the monoclonal antibody according to claim 1, 2, 3, 4 or 5. 7. The antibody fragment according to claim 6, wherein the antibody fragment is a Fab site. 8. The antibody fragment according to claim 6, wherein the antibody fragment is an Fv site. 9. A cell line which produces the monoclonal antibody according to claim 1, 2, 3 or 4. 10. The cell line ET-4D3 (122)
88) 11. A monoclonal antibody produced by the cell line ET-4D3. 12. A method for producing a monoclonal antibody, which comprises culturing a cell line producing the monoclonal antibody according to claim 1, 2, 3 or 4. 13. A cell line ET-4D3 (122)
88), a method for producing a monoclonal antibody, which comprises culturing 14. Claims 1, 2, 3, 4, 5, 6, 7, 8
Alternatively, a prophylactic / therapeutic agent for Pseudomonas aeruginosa infection comprising the monoclonal antibody or antibody fragment described in 11 above.