JPH06339372A - Transformed cells - Google Patents

Abstract

PURPOSE:To obtain a new EB virus transformed cell capable of producing a human monoclonal antibody having affinity which is specific to antigen every serum type of Pseudomonas aeruginos and capable of providing a medicine having excellent preventing and treating effects on infectious disease of Pseudomonas aeruginos. CONSTITUTION:A heparin-added peripheral blood collected from a person having high anti-Pseudomonas aeruginos antibody value is diluted with a physiological saline, etc., and then, the blood layer is superposed on lymphoprep and centrifuged to collect a cloud cell layer of interface layer and the layer is made to refloat on a buffer and put on a lymphoprep and centrifuged and T lymphocyte is removed and B lymphocyte is collected. Then, this B lymphocyte is mixed with a liquid containing EB virus and the mixture is cultured in carbon dioxide incubator at 37 deg.C and whether monoclonal antibody of Pseudomonas aeruginos exist or not in the culturing serum is measured and a positive cell is cloned using a rosette forming method and a soft agar colony forming method to provide the objective EB virus transformed cell capable of producing human monoclonal antibody having affinity specific to antigen every serum of Pseudomonas aeruginos.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a transformed cell, and more specifically, the present invention is a trait for producing a human-derived monoclonal antibody which is effective in the preventive treatment of Pseudomonas aeruginosa infection. It concerns a transformed cell.

[0002]

Background of the Invention and Background of the Invention Pseudomonas aeruginosa (hereinafter sometimes referred to as Pseudomonas aeruginosa) exists widely in nature and is found at high rates in water, sewage, etc., and also in the oral cavity and intestine of humans, animals. . This bacterium exerts pathogenicity rather than in a general infection patient due to the bactericidal force of the bacterium itself, that is, a patient with reduced infection resistance, that is, a cancer patient, a person under immunosuppressive therapy, a transplant patient, a burn patient and a newborn baby, etc. Is.

At present, Pseudomonas aeruginosa infection is considered to be the most difficult to treat infection. In other words, Pseudomonas aeruginosa is not only resistant to almost all of the antibiotics that have been commonly used until now, but it is also easy to induce resistant strains of Pseudomonas aeruginosa to recently developed antibiotics. This is because there are many cases. Further, in view of the limit of antibiotic therapy, development of an infection preventive agent, that is, a vaccine with bacterial components aiming at enhancing the host side Pseudomonas aeruginosa treatment capacity, for example, a table of 13 or more types of Pseudomonas aeruginosa by serotype Pseudomonas aeruginosa multivalent vaccine obtained by purifying and mixing existing antigens, OEP mainly composed of protein as a serological common antigen of Pseudomonas aeruginosa, or protease toxoid, elastase toxoid or exotoxoid prepared from OEP in OEP 3 or 4 with added
There are mixed vaccines. However, it is no exaggeration to say that all of the vaccines have insufficient protection against infection or safety, and excellent ones with practicality are desired. Furthermore, immunoglobulin therapy by passive immunization of human serum immunoglobulin preparations produced from healthy human serum has also been studied, but many of the actual preparations have low antibody titers against Pseudomonas aeruginosa and their preventive and therapeutic effects. There are quite a few people who question

[0004]

DISCLOSURE OF THE INVENTION The object of the present invention is to newly develop a transformed cell producing a human monoclonal antibody having a specific affinity for a serotyped antigen of Pseudomonas aeruginosa. It is what

[0005]

The present invention has been made to solve the above problems. Hereinafter, the present invention will be described in detail.

I. Summary of the invention

1. The present invention has been made for the purpose of creating a transformed cell that has a specific affinity for a Pseudomonas aeruginosa serotype antigen and produces a human monoclonal antibody whose globulin class is IgG or IgM. By using the transformed cell according to the present invention, the novel human monoclonal antibody described above can be efficiently prepared, and a prophylactic and / or therapeutic agent for Pseudomonas aeruginosa can be prepared either alone or in combination. Can be created.

The target human monoclonal antibody-producing cells against Pseudomonas aeruginosa are produced by transforming human-derived B cells with EB virus (Epstein-Barr Virus), and A human monoclonal antibody against Pseudomonas aeruginosa is produced by selecting an antibody that reacts specifically with serotype-specific antigens and has a globulin class of IgG or IgM class.

2. EFFECTS The human monoclonal antibody obtained by the transformed cells according to the present invention achieves an excellent preventive and therapeutic effect against Pseudomonas aeruginosa infection by combining it with a single agent or a combination of two or more agents.

It should be noted in the present invention that a transformed cell producing a human-derived monoclonal antibody rather than an animal such as a mouse is used as the monoclonal antibody which specifically reacts with the serotype antigen of Pseudomonas aeruginosa. Among the human-derived monoclonal antibodies produced by the cells, which are very characteristic in that they are first successfully produced, antibodies whose globulin classes are IgG and IgM are particularly effective. Globulin class is IgG, IgM
And an antibody having a direct aggregation activity on Pseudomonas aeruginosa cells,
The present inventors have for the first time found that Pseudomonas aeruginosa that reacts with the antibody exhibits prophylactic and therapeutic effects at extremely low concentrations and doses.

II. Detailed explanation of the invention

1. Pseudomonas aeruginosa used In the present invention, the classification of Pseudomonas aeruginosa used was in accordance with the serological classification determined by the serotype review committee sponsored by the Pseudomonas aeruginosa Research Group (1975) (Table 1 shown in Table 1 below). ).

[0013]

[Table 1]

2. Preparation of human monoclonal antibody-producing cells Human monoclonal antibody-producing cells against Pseudomonas aeruginosa according to the present invention are produced by transforming human B cells with EB virus.

In general, as a method for producing a human monoclonal antibody, there are a human-human hybridoma method, a mouse-human hybridoma method, an EB virus transformation method and the like.

Up to now, the method of (1) is a technique with a low degree of perfection because of lack of excellent hybridoma proliferability and lack of gene because excellent human myeloma cells do not exist. The method of (1) has a problem that it is difficult to establish a stable strain because the gene deletion phenomenon occurs immediately after the gene is integrated because of mouse-human. Therefore, the present inventors produced the human-derived monoclonal antibody of the present invention by the EB virus transformation method, which is said to be capable of producing a relatively stable antibody-producing cell line. As the material for human B cells, use peripheral blood of healthy individuals in which serum production of anti-Pseudomonas aeruginosa antibody is found, or isolated lymph nodes (for example, tonsils) of unnecessary patients, and increase B cell concentration as much as possible. Therefore, the mononuclear cell fraction containing B cells was concentrated and used by the File Call / Conray gravity centrifuge method or the like.

The basic method of transformation of human B cells by EB virus is based on a known method (for example, Nature 267, 52, 1977, the 4th Annual Meeting of the Japanese Society of Immunology, 399, 1974), and EB virus persistence. The target antibody-producing cell population is screened by infecting human B cells with the culture supernatant of the producing cell line as a virus source and detecting a monoclonal antibody from the cell population mutated to a proliferative type according to a known assay method. Furthermore, the monoclonal antibody-producing clonal cell line of the present invention, which became a monoclone, was obtained by the known rosette formation method using antigen-sensitized sheep erythrocytes and soft agar colony formation method.

As the assay method for detecting the monoclonal antibody, there are the following methods. One is a method using red blood cells bound with protein A, which is a method showing excellent sensitivity and specificity among the methods for detecting a specific antibody. In addition, the agglutination reaction method that determines the antibody titer by observing the degree of agglutination, or the simplest method for determining a globulin class or subclass, generally requires that the culture supernatant be concentrated approximately 10-fold. It is an Ouchterlony method that can be confirmed by precipitating the supernatant with an antibody and developing it on an SDS-polyacrylamide gel. Further, the antigen or cells are fixed on a U-shaped microtiter plate made of polyvinyl chloride, and the culture supernatant is added thereto and incubated to cause an antigen-antibody reaction, and then ¹²⁵ I or an enzyme-labeled anti-mouse antibody or Pr
There is also a radioimmunoassay method or an enzyme immunoassay method (EIA method) for examining radioactivity or enzyme activity in each well after washing with the addition of protein A. A method suitable for the purpose may be selected from various assay methods. Basically, it is important that the sensitivity is high and the cost is short. The present inventors use the following EIA method.

That is, a certain amount of Pseudomonas aeruginosa cells was used as an antigen on a membrane filter, and a culture supernatant of a hybridoma was reacted with this, and then a peroxidase-labeled goat anti-human immunoglobulin antibody (Kappel) was applied. Dot immuno-binding assay method (DIBA that can measure the amount of antibody produced by the coloring ratio of enzyme reaction)
Method, Analytical Biochem. , 11
0, 142, 1082) is used.

In this way, a target human monoclonal antibody-producing clonal cell line, that is, an EB virus-transformed cell producing a human monoclonal antibody against Pseudomonas aeruginosa was established.

3. Production of Human Monoclonal Antibody Thus, the transformed cell of the present invention can be obtained. By using the cell, a human monoclonal antibody against Pseudomonas aeruginosa can be efficiently produced.

That is, the large-scale preparation of human monoclonal antibody from the obtained antibody-producing clonal cell line is carried out by collecting a culture solution by, for example, a large-scale culture using a spinner flask, a roller bottle, etc. By purifying by the specific purification method and / or the specific purification method, the target human monoclonal antibody can be obtained in a purified state.

Non-specific purification: The desired monoclonal antibody can be purified from the culture supernatant or the like by a combination of known methods such as gel filtration, ion exchange cellulose chromatography, and salting out with ammonium sulfate.

Specific purification: Sepharose 4B activated by cyanogen bromide (Pharmacia) or Affigel-
Affinity chromatography in which the immunoglobulin fraction or culture supernatant is allowed to flow into a column in which an antigen is bound to 10 (Bio-Rad) and insolubilized to bind the antibody, and then the adsorbed antibody is eluted with a urea solution or the like for purification Can be used as needed. Further, when the target human monoclonal antibody is IgG, it is recommended that an affinity column on which protein A is immobilized is used because purification is easy.

When the target human monoclonal antibody is IgM, for example, a purification method by gel filtration using Sephacryl S300 (Pharmacia) or a purification method for further increasing the purification degree by using a hydroxyapatite column is also available. Used.

The monoclonal antibody obtained from the cells of the present invention is of human origin as described above,
Has a specific affinity for Pseudomonas aeruginosa serotyping antigens,
In addition, the globulin class having direct agglutination activity against Pseudomonas aeruginosa is IgG or IgM, and these properties can be defined by the methods described below. That is, the specific affinity of Pseudomonas aeruginosa for serotyping antigens means that only the strains belonging to one group of the Pseudomonas aeruginosa serotyping bacteria of groups A to M in Table 1 react with each other, It means that it does not react with the strains belonging to the group (for example, it reacts only with the bacteria of the group A and does not react with the other groups B to M).

Further, the direct agglutination activity of the human monoclonal antibody obtained from the cells of the present invention against Pseudomonas aeruginosa cells is, as shown in Example 1, treated with formalin of Pseudomonas aeruginosa in a 96-well U-bottomed microplate. Alternatively, it means the antibody activity of directly aggregating dead cells by heat treatment.

Further, the globulin class of the human monoclonal antibody obtained from the cells of the present invention is the gel filtration pattern of the purified antibody with Sephacryl S-300 and the DIBA method with the peroxidase-labeled goat anti-human IgG or IgM antibody (Kappel). Means those identified as IgG or IgM, respectively.

The human monoclonal antibody obtained from the cells of the present invention shows excellent preventive and therapeutic effects against Pseudomonas aeruginosa infection, as shown in Test Examples. When used as a prophylactic or therapeutic agent, it is preferably used in the form of an injection. The dosage for humans is 0.01 to 20 mg / kg, and administration methods such as subcutaneous injection, intramuscular injection and intravenous administration can be selected. Hereinafter, the present invention will be described more specifically by illustrating Examples, Production Examples, Application Examples and Test Examples.

[0030]

Example 1 Preparation and properties of human monoclonal antibodies against each serotype Pseudomonas aeruginosa

1) EB virus B95 that produces and releases EB virus (hereinafter EBV)
-8 cells were cultured in a medium in which 20% FCS was added to RPMI-1640, and the culture supernatant on day 7 near the stationary phase was separated and used as a virus source.

2) Separation of lymphocytes Peripheral blood supplemented with heparin, which was collected from a healthy person who had a high anti-Pseudomonas aeruginosa antibody titer, was physiological saline or PBS (-).
After being diluted 2-fold with, the cells were slowly overlaid so that the interface was not squeezed on the 1/4 to 1/3 amount of Lymphoprep. After overlaying, the mixture was centrifuged at 1550 rpm for 30 minutes at room temperature. After centrifugation, the white cloudy cell layer of the interface layer was taken out with a Pasteur pipette, and the cells were PBS (-) at 1400 rpm and 10
Minute (first time), then 1000 rpm, 10 minutes (2,
It was centrifugally washed at the third time). The cells were resuspended in RPMI 1640 solution at 5 × 10 ⁶ cells / ml.

3) Removal of T lymphocytes (concentration of B lymphocytes) 1 volume of SRBC treated with 0.5% AET, 1 volume of mononuclear cell suspension (5 × 10 ⁶ cells / ml), SRBC absorption FCS
0.2 volume was mixed in a centrifuge tube (bottom), left at room temperature for 5 minutes, and then centrifuged at 1000 rpm for 5 minutes at 4 ° C. After centrifugation, the plate was left standing on ice for 1 hour. After the reaction, the cell pellet was gently stirred with a Pasteur pipette to prepare a suspension, which was placed on Lymphoprep and centrifuged at 1550 rpm for 30 minutes at 4 ° C. After centrifugation, cells that did not form rosettes on the interface (B lymphocytes) were taken out, and PBS (-)
In the same manner as in 2 above, centrifugal washing was performed 3 times. 1 x 10 ⁷ cells
The cells were suspended in the culture solution so that the number of cells / ml (when the number was high) to 2.5 × 10 ⁶ cells / ml (when the number was low).

4) Establishment of cell line by EBV infection EBV was added to 0.1 ml of B lymphocyte suspension (1 × 10 ^{7 to} 2.5 × 10 ⁶ cells / ml) per well of a culture plate (24 wells). Add 0.1 ml of the liquid and in a carbon dioxide incubator at 37 ° C,
Incubated for 60 minutes. After incubation, add 0.8 ml of culture solution to make the total volume 1 ml, and
Static culture was performed at 37 ° C. in a ₂ gas incubator. 1 ml of culture medium was added 4-5 days after EBV infection. Then 4-5
The culture medium is exchanged by ½ the amount of the culture medium every day, and after 6 weeks, the culture plate in which vigorous cell growth is observed is examined for the presence or absence of anti-Pseudomonas aeruginosa monoclonal antibody in the culture supernatant by enzyme immunization. It was measured by the dot immunobinding assay, which is a measurement method.

The DIBA method uses a 96-well U-bottom multi-plate and a membrane filter (3.1 mm) in which 0.4 μg of 0.3% formalin-treated per dot treated with all the serotype Pseudomonas aeruginosa cells is used as an antigen. Horn) and 100 μl of the above-mentioned culture supernatant at room temperature for 30 minutes and then with peroxidase-labeled goat anti-human immunoglobulin antibody (manufactured by Kappel) for 30 minutes, followed by color development using chlornaphthol as a substrate and an antigen-immobilized membrane filter. Those in which color development was visually observed were determined to be positive for antibody production.

The specific affinity of Pseudomonas aeruginosa to serotype-specific antigens was tested on each membrane filter immobilized with 13 serotypes A to M in Table 1 as antigens. It was confirmed by reacting only with the serotype antigen.

The cell population in which the antibody production was observed was cloned by the rosette forming method and the soft agar colony forming method. A clone having a strong growth ability by cloning is expanded and cultured with a Stirling system (manufactured by Intermet), and the culture supernatant is affinity column (for IgG) using each type of Pseudomonas aeruginosa as an antigen and Sephacryl S3000.
Purified (for IgM). The obtained antibody culture solution was filtered through a membrane filter having a pore size of 0.22 micron and stored at 4 ° C until use in animal experiments.

The present inventors have found 14 strains of transformed cells that produce human monoclonal antibodies that specifically react with each serological type of Pseudomonas aeruginosa. Of these, E type, B type,
Transformed cells that specifically react with G type and I type were 4 strains, 5 strains, 2 strains, and 3 strains, respectively.

Of the antibodies obtained from each cell,
The properties of the E serotype Pseudomonas aeruginosa-specific reactive antibody are shown in Table 2 below, and the properties of the B serotype P. aeruginosa-specific reactive antibody are shown in Table 3 below. The properties of the Pseudomonas aeruginosa-specific reactive antibody are shown in Table 4 below, and the properties of the I serotype Pseudomonas aeruginosa-specific reactive antibody are shown in Table 5 below.

[0040]

[Table 2]

[0041]

[Table 3]

[0042]

[Table 4]

[0043]

[Table 5]

The direct agglutination activity of human monoclonal antibody against Pseudomonas aeruginosa was measured by the following method.

A 96-well U-bottomed multiplate (Falcon) was treated with 0.3% formalin of Pseudomonas aeruginosa of each serotype or a suspension of dead cells (PBS) by heat treatment at 120 ° C.
Suspended in (-), OD ₅₄₀ 0.4) was dispensed in 50 μl aliquots. This is followed by human monoclonal antibody PBS
The dilution series by (-) was dispensed by 50 μl each, the plate surface was covered with a sealer (Limbro's plate sealer), shaken at room temperature for 10 minutes, and allowed to stand at 37 ° C. for 1 hour. The plate was allowed to stand at 4 ° C. to 10 ° C. for 15 hours or longer, then the plate was returned to room temperature, and the image of the agglutination reaction of the bacterial cells was observed through the back surface of the plate and judged.

[0046]

[Test Example 1] Protective activity of human monoclonal antibody specifically reacting with each serotype Pseudomonas aeruginosa against Pseudomonas aeruginosa infection

The protective effect against the Pseudomonas aeruginosa infection of the human monoclonal antibody specifically reacting with each type obtained in Example 1 was examined.

First, 5 groups of 10-week-old BALB / C female mice were each provided with a human monoclonal antibody which specifically reacts with each bacterium type at 50 ng and 500 ng per mouse.
5 μg, 50 μg and 5 mg were intraperitoneally administered, respectively.

After 2 hours, strains specific to each type (IID1130 (E type), IID1002 (B type)
Type), ATCC10145 (G type), IID1010
(Type I)) 1 × 10 ⁶ intraperitoneal challenge was performed. In the control group, only physiological saline was administered instead of the monoclonal antibody. On day 7 after infection with Pseudomonas aeruginosa, the 50% effective dose (ED ₅₀ value) was determined from the survival rate of the mice in each administration group. The results are shown in Tables 2 to 5, and both the human monoclonal antibodies whose globulin class is IgG and IgM are effective, and from the above results, among the human-derived monoclonal antibodies, the globulin class is IgG. , An antibody with strong agglutinating activity was found to have an extremely strong protective effect against Pseudomonas aeruginosa infection.

[0050]

Production Example 1 Cells producing monoclonal antibody I and monoclonal antibody Y were cultured according to the method of Example 1 and purified by a hydroxyapatite column.
The obtained IgM solution was filtered through a membrane filter having a pore size of 0.22 micron and used until the time of use in animal experiments.
It was stored frozen at 80 ° C. The infection preventive activity against Pseudomonas aeruginosa was examined according to the method of Test Example 1. However, a solution containing 0.4 μg, 2 μg, and 10 μg of human monoclonal antibody I, 1.25 μg and 5 μg of human monoclonal antibody Y
g, 20 μg of the solution was administered.

The results are shown in Table 6 shown in Table 6 below.

[0052]

[Table 6]

As is clear from the above, the purification method was changed from that of Example 1 to further raise the degree of purification of the antibody to remove contaminants, and at the same time, the storage temperature of the purified antibody until animal experiments was further lowered. When the antibody activity was prevented from decreasing by setting the temperature to −80 ° C., IgM also showed a very strong Pseudomonas aeruginosa protective effect as compared with the case of Test Example 1, and the value was also observed when it was higher than IgG.

[0054]

[Application Example 1] Liquid and lyophilized preparation of human monoclonal antibody that specifically reacts with E serotype Pseudomonas aeruginosa

1) Large scale preparation of human monoclonal antibody

(A) Large-scale culture Monoclonal antibody No. 1 obtained in Example 1 The strain producing 3 was added to the serum-free medium (H
B101TM medium (manufactured by Fujirebio Inc.) was acclimated. Next, the acclimated cells were cultured in a 5 l Stirling system (manufactured by Intermed) and used for the following purification.

(B) Large-scale purification About 15 mg of globulin fraction was obtained using Protein A-Sepharose 4 B (Pharmacia) or DEAE Sephacel.

2) Solution The monoclonal antibody obtained as described above was dissolved in a 3/10 M aminoacetic acid solution to give a globulin fraction concentration of 100 μg /
It was made to be ml. The thus obtained product was used as a membrane filter (Nuclepore No. 2).
0, manufactured by Nukuripore Co., Ltd.) and aseptically dispensed in 1 ml aliquots into vials to obtain a liquid preparation of the substance of the present invention.

3) Lyophilized preparation The globulin fraction obtained by large-scale purification by the above method was dissolved in physiological saline containing aminoacetic acid to a final concentration of 3/10 M to 100 μg / ml, and mannitol was further added. It is added to a concentration of 50 mg / ml, sterile filtered using a membrane filter (Nuclepore No. 20, manufactured by Nukuripore), and aseptically dispensed in 2 ml aliquots into vials and freeze-dried to obtain the substance of the present invention. A lyophilized formulation was obtained.

[0060]

[Test Example 2] Therapeutic effect using each serotype Pseudomonas aeruginosa-specific human monoclonal antibody

Monoclonal antibody No. having a preventive effect in Test Example 1 3, IV, A, and X were tested for their therapeutic effects.

That is, 5 groups of 10-week-old BALB / C female mice were challenged intraperitoneally with 1 × 10 ⁶ bacteria which react with each monoclonal antibody in the same manner as in Test Example 1 and 2 hours later, 100 ng to 10 mg of each monoclonal antibody was intraperitoneally administered, and ED ₅₀
The value was calculated. The results are shown in Table 7 below.

[0063]

[Table 7]

As is clear from the above, it was confirmed that all of these monoclonal antibodies have a strong preventive effect. Further, although the therapeutic effect of each monoclonal antibody which showed a strong preventive effect in Test Example 2 was reduced to about 1/10, a therapeutic effect was also recognized.

No change was observed in the behavior of the mouse even at the maximum dose of 70 mg / kg.

[0066]

Industrial Applicability According to the present invention, transformed cells producing human monoclonal antibodies having specific affinity for serotyped antigens of Pseudomonas aeruginosa were obtained for the first time.

By culturing the cells, a novel human monoclonal antibody against Pseudomonas aeruginosa can be efficiently obtained, and the antibody can be used for prevention and / or treatment of Pseudomonas aeruginosa infection. .

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Claims (3)

[Claims] 1. An EB which produces a human monoclonal antibody having a specific affinity for a serotyped antigen of Pseudomonas aeruginosa.
Virus transformed cells. 2. The cell according to claim 1, wherein the globulin class of the human monoclonal antibody is IgG. 3. The cell according to claim 1, wherein the globulin class of the human monoclonal antibody is IgM.