JPH02245186A - Human monoclonal antibody reactive with g serum-type pseudomonas aeruginosa, cell capable of producing same, its production and pharmaceutical - Google Patents

Abstract

PURPOSE:To provide human-human hybridoma and cell strain stemmed therefrom, capable of stably producing in relatively large quantities human monoclonal antibody reactive with G serum-type Pseudomonas aeruginosa. CONSTITUTION:For example, using, as parent cell strain, infinitely reproductive cell strain having only human chromosome and resistant to both 8-azaguanine and ouabain, and using, as human antibody-productive cells, Epstein-Barr virus transformant cells reactive with G serum-type Pseudomonas aeruginosa, the objective human-human hybridoma (FERM P-2269, 2274) is prepared.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to the use of serotype G Pseudomonas aeruginosa (Pseudomonas aeruginosa).
A human-human hybridoma cell line capable of stably supplying a large amount of human monoclonal antibody against a), the human monoclonal antibody produced by the cell line, and prevention of Pseudomonas aeruginosa infection using the same as an active ingredient , relating to therapeutic formulations.

BACKGROUND OF THE INVENTION Pseudomonas aeruginosa infection is an opportunistic infection that often occurs in patients with various underlying diseases or patients receiving drugs with immunosuppressive effects. Currently, Pseudomonas aeruginosa infection is considered the most difficult infection to treat. That is, Pseudomonas aeruginosa not only exhibits resistance to almost all antibiotics that have been commonly used, but also has a strong tendency to easily induce resistance to antibiotics developed in recent years. Therefore, research is being conducted on preventive and therapeutic methods aimed at increasing the host's ability to treat Pseudomonas aeruginosa.

In recent years, globulin preparations containing human immunoglobulin purified from the serum or plasma of healthy individuals or chemically modified products thereof as active ingredients are often used to treat Pseudomonas aeruginosa infections. However, among the antibodies contained in these preparations -3=, the amount of antibodies that have affinity for Pseudomonas aeruginosa and are effective for treatment is not constant, and because the content is small, these preparations Many people question the effectiveness of prevention and treatment. Therefore, there is an urgent need to develop human monoclonal antibodies that are effective at low doses.

Pseudomonas aeruginosa contains lipopolysaccharides (LPS) present on the outer membrane.
Classification by serotype is performed using an immune antibody that recognizes the 〇-polysaccharide side chain on the molecule (abbreviated as ), that is, an antibody against the serotype-specific 0 antigen of Pseudomonas aeruginosa. There is still much debate regarding the classification of Pseudomonas aeruginosa by serotype, but in Japan, the classification of Pseudomonas aeruginosa into 13 serotypes from type A to type M [Homma, Japan J, Exp, Me
d, 329-336 (1976)'] is widely used. The proportion of Pseudomonas aeruginosa serotypes isolated from patients infected with Pseudomonas aeruginosa in clinical settings is almost constant, with 5 of the 13 serotypes: A, B, E, G, and ■. It is known that bacteria account for a high proportion of the bacteria.

On the other hand, mouse monoclonal antibodies against Pseudomonas aeruginosa are produced using the mouse-mouse hybridoma technology developed by Köhler and Milstein (Ki5hler and Milstein).
in, Nature, 256.495-497 (1
975)] [for example, Hanc
ock et al., Infect.

Immun, 37.166-171 (1982)
), application to type-based diagnosis [e.g. Meiji Seika, EP 1
01039) and basic research to search for monoclonal antibodies useful for preventing infection.

Sadoff et al.
reported that mouse monoclonal antibodies against polysaccharide side chains had high protective activity against lethal infection by bacteria of the corresponding serotype in mouse infection experiments [5a
Doff et al., Abstracts of the 198
2 Interscience Conference
onAntimicrobial Agents a
nd Chemotherapy, N(1253(1
982) ). Subsequent reports have also demonstrated the efficacy of mouse or human monoclonal antibodies against Pseudomonas aeruginosa serotype-specific antigen in in vivo and in vitro test systems [e.g., Sawada et al., J. Infect, D.
is, 150.570-576 (1984),
Keibo Nakamura et al., Japanese Journal of Bacteriology, 39°337 (198
4), Pennington, Infect, Im
mun, 54゜239-244 (1986), 5
uzuki et al., Microbiol, Immunol
.

31, 959-966 (1987), Zwerni
K et al., Infect.

Immunity, 56.1873-1879 (1
988) ). In addition, regarding the use of serotype-specific human monoclonal antibodies for the prevention and treatment of Pseudomonas aeruginosa infections,
The present inventors disclosed an antibody that solely recognizes the 〇-polysaccharide side chain on serotype-specific LPS molecules as described in the patent application specification
-248626] and an antibody that commonly recognizes multiple 〇-polysaccharide side chains is disclosed in the patent application specification [International Publication No. u08
8104669) and several other patent application specifications [
Genetic Systems Corporation, E
P 163493 and BE 905890, Teijin Ltd., 11086103754, Yukinaga Pharmaceutical Co., Ltd., JP 61-091134, Merck End Company Inc., EP 256713).

While the light is about to be determined, the production of human monoclonal antibodies - inside the shell is human B.
Epstein-Barr virus (Epstein) in cells
-Barr virus (hereinafter abbreviated as EB virus) to produce EB virus-transformed cells, but human antibody-producing cells such as B cells are fused with a parent cell line that has unlimited proliferation ability to create human-mouse heterozygous cells. This is done by creating a hybridoma or a human-human hybridoma.

EB virus-transformed cells produced by the EB virus transformation method generally have low antibody production, poor passage stability, and relatively high auxotrophy, so they are not suitable for mass culture production using serum-free medium. Not suitable. When mouse myeloma is used as a parent cell line and fused with human antibody-producing cells, the created human-mouse heterohybridoma synthesizes and secretes mouse proteins along with human antibodies, making it difficult to produce human monoclonal antibodies to be administered to humans. It is not necessarily suitable for use as a strain. Furthermore, there are some problems when a human-human hybridoma is produced by using a parent cell line containing only human chromosomes and capable of infinite proliferation and fusing it with a human antibody-producing cell. For example, the fusion efficiency between a parental cell line derived from a human myeloma and a human antibody-producing cell is low.

Furthermore, although the fusion efficiency of the parent cell line derived from EB virus-transformed cells and human antibody-producing cells is relatively high, the produced human-human hybridomas may simultaneously produce antibodies without antigen specificity. In many cases, the amount of antibodies produced is low. Human-human hybridomas produced by fusion of human antibody-producing cells and parental cell lines derived from hybridomas of human myeloma and EB virus-transformed cells produce relatively high amounts of antibodies, but antibodies with unknown antigen specificity. The property of producing both at the same time has not been resolved.

The present inventors have discovered that it is possible to produce human-human hybridomas that produce human monoclonal antibodies that are reactive with G serotype Pseudomonas aeruginosa, and that the human-human hybridomas can be used in various species. The human monoclonal hybridoma is capable of stably growing in a culture medium and producing a relatively large amount of antibody for a long period of time. We discovered that antibodies can be prepared.

Based on these results, the present inventors determined that the human-human
The present invention was completed by testing the protective activity of antibodies produced by hybridomas against Pseudomonas aeruginosa infection.

The term "human-human hybridoma" as used in the present invention refers to a hybridoma having only human chromosomes, which is produced by fusion of a parent cell line with unlimited proliferation potential and a human antibody-producing cell.

The selection characteristics as used in the present invention refer to chemical or physical characteristics of the parent cell line that enable selection of prepared hybridomas from unfused cells. For example, when using a parent cell line resistant to 8-azaguanine or 6-thioguanine and ouabain as selection characteristics, only hybridomas with human antibody-producing EB virus-transformed cells are present in culture medium containing hypoxanthine, azaserine, and ouabain. Survive with.

A parent cell line having the above-mentioned selection characteristics is appropriately selected. In addition, human antibody-producing cells reactive with Pseudomonas aeruginosa are appropriately selected from human B cells and cells derived therefrom.

Hereinafter, human antibody-producing EB virus-transformed cells that are reactive to G serotype Pseudomonas aeruginosa will be used as the parent cell line, which is a cell line that has only human chromosomes, unlimited proliferation ability, and resistance to 8-azaguanine and ouabain. The present invention will be explained by taking as an example the case where human-human hybridomas are produced using human antibody-producing cells.

(Specific explanation) ■ Pseudomonas aeruginosa used In the present invention, for convenience, the classification of Pseudomonas aeruginosa used is based on the serotype classification determined by the serotype review committee sponsored by the Pseudomonas aeruginosa research group, and from type A to A strain belonging to type M is used.

Strains belonging to types A to M are available from American Type Culture Collection (ATCC), Institute for Fermentation (IFO), and the Institute of Medical Science, the University of Tokyo.

2. Preparation of human-human hybridoma The human hybridoma according to the present invention, which produces human monoclonal antibodies reactive with G serotype Pseudomonas aeruginosa, is a parent cell line for hybridoma production. MP 4109 or its subcultures and antibody-producing cells were transferred to human 1 using known methods.
MONOcLONAL ANTIBODIESJ p3
63. Published by Plenum Press (1980), etc.]
It can be produced by cell fusion according to . Human antibody-producing cells can be found in the peripheral blood, lymph nodes, tonsils, lungs, and cord blood at delivery of healthy individuals who produce antibodies against Pseudomonas aeruginosa or patients with a history of Pseudomonas infection. B cells obtained by the method described above can be used;
After infecting with virus, transforming, and culturing for a certain period of time,
It is preferable to use EB virus-transformed cell colonies in which secretion of antibodies reactive with Pseudomonas aeruginosa has been detected in the culture supernatant, or a single cell line selected from these EB virus-transformed cell colonies. be.

Next, we will add detailed explanations for each step.

Isolation and concentration of B cells from blood and the above tissues, etc.
This can be carried out efficiently by combining specific gravity centrifugation using a cell fraction solution such as Ficoll-Conray solution, E rosette formation method, panning method, and the like. Furthermore, B cells can be cultured for several days in a culture medium supplemented with Hawkelidomy 1-diene (PWM) to allow the B cells to proliferate, and then subjected to cell fusion.

The method for transforming B cells with EB virus is a known method [
For example, 5jeinitz et al., Nature, '26
9.420422 (1977)). B958 cells (marmoset leukocyte-derived cells that produce infectious EB virus) were grown in RPM 1640 medium containing 20% fetal calf serum (hereinafter abbreviated as FC3).
(hereinafter sometimes abbreviated as culture solution), and the culture supernatant obtained by centrifugation on day 7, which is close to the stationary phase, is used as the virus solution [Ono et al., Records of the 4th Annual Meeting of the Japanese Society of Immunology, 3994
01 (1974)). After centrifuging the B cells and removing the supernatant by aspiration, add the virus solution to the resulting pellet I and disperse it.
Incubate for an hour. After culturing, centrifuge, remove the supernatant by aspiration, and reduce the cell density to the pellet.
Add culture medium and disperse the cells so that the concentration ranges from 105 cells/ml to 5 x 10'' cells/ml. Dispense the cell dispersion into each well of a 24-well culture plate or a 96-well culture plate, and incubate at 37°. Cultivate for 2 to 4 weeks in the presence of C15% carbon dioxide gas.During this period, it is desirable to replace half of the culture medium with fresh culture medium every 3 to 4 days.

Detection of antibodies reactive with Pseudomonas aeruginosa can be carried out using methods such as general radioimmunoassay or enzyme-linked antibody assay (hereinafter abbreviated as ELISA) [monoclonal antibody J p144,
Kodansha Publishing (]983) etc.). The present invention uses the ELISA method. That is, 0.3% formalin-treated Pseudomonas aeruginosa cells were fixed in advance on a membrane filter, reacted with the cell culture supernatant for a certain period of time in a container, and then reacted with an enzyme-labeled rabbit anti-human antibody. A dot immunobinding assay method (hereinafter abbreviated as DIBA method) that measures the presence or absence and amount of production of a target antibody based on the rate of coloration of a substrate caused by an enzymatic reaction.
(Anal,.

Biochem, 119.142-147 (198
2)) is used as a simple assay method.

The culture supernatant of each well in which a proliferating colony of EB virus-transformed cells was observed was analyzed by the above ELISA method.
After selecting the wells containing the target antibody, cells in these wells were collected using the soft agar method
89, Rough 1 ~ Published by Science Publishing (1985), etc.] or limiting dilution method [Naritai "Monoclonal Antibodies" p73, Kodan 1
(1983) etc.]. Further, after cell proliferation is observed by cloning, assay by ELISA is performed again.

Through one to several rounds of cloning, a single cell line that secretes only the antibody of interest can be obtained.

Fusion of MP 4109 and human antibody-producing cells can be carried out using in-shell fusion reagents such as polyethylene glycol (hereinafter abbreviated as PEG) or Sendai virus (Hemagglutinating virus o).
This can be done using virus particles such as f Japan: HVJ). For example, average molecular weight 1000 to 600
Approximately 0 PEG is added to 30% to 50% of PEG in RPMI 1640 medium or Dulbecco's modified Eagle's medium (DMEM).
% (W/V) is recommended as a fusion solution. It is also desirable to add dimethyl sulfoxide (DMSO) to increase the fusion efficiency. It can also be done by a physical method using an electric fusion device or the like.

For example, cells in wells in which production of the target antibody was observed after transformation with MP 4109 and CB virus, or antibody-producing cells isolated from peripheral blood, etc., from 1 = 1], :10
Mix in appropriate ratios, add a cell fusion medium (such as RPMI 1640 medium containing 50% PEG and 10% DMSO), and fuse the cells. Next, a culture medium suitable for the growth of only the fused hybridomas (hereinafter abbreviated as selective medium) is added at a cell density of I x 10' cells/ml to 5 x 10 cells/ml.
Distribute the cells so that the number of cells/ml is 1. Dispense the cell dispersion into a 24-well or 96-well culture plate,
Culture at 37°C in the presence of 5% carbon dioxide for 2 to 4 weeks. During this time, it is desirable to replace half of the selective medium with fresh selective medium every 3 to 5 days. At this time, if mouse peritoneal exudate cells or the like are allowed to coexist as feeder cells, the proliferation of the hybridoma can be accelerated. If the human antibody-producing cells are cells that do not have the ability to proliferate indefinitely (B cells), use a selective medium containing hypoxanthine, aminopterin, and thymidine (hereinafter referred to as IIAT medium) or a medium containing hypoxanthine and azaserine (hereinafter referred to as IIAT medium). Below, H.A.
medium) can be used. In addition, if the human antibody-producing cells are cells that have unlimited proliferation ability, such as EB virus-transformed cells, the selection medium may be HAT medium supplemented with ouabain (HAT-0 medium) or HA medium supplemented with ouabain (HAT-0 medium). HA-0 medium) can be used. After selecting the culture supernatant of each well in which a proliferating hybridoma colony was found by the above ELISA method to select the wells in which the target antibody is present, cloning is performed by the limiting dilution method.

Further, after cell proliferation is observed by cloning, assay by ELISA is performed again. Through one to several rounds of cloning, a single cell line that secretes only the antibody of interest can be obtained.

The human-human hybridoma of the present invention can be cultured using a conventional medium. For example, after dispersing in a culture medium to a cell density of 5 x 10' cells/ml to 2 x 10' cells/ml and seeding them in an appropriate cell culture container, at 37°C, 5%
Can be cultured in the presence of carbon dioxide. Examples of culture solutions include R
A basal medium such as PMI 1640 or DMEM to which an appropriate amount of Fe2 is added is suitable. Furthermore, various low serum or serum-free media can be used as appropriate. For example, NYS
F 404 serum-free medium alone or NYSF 404
A serum-free medium supplemented with an appropriate amount of bovine serum albumin is recommended. For subculture, it is preferable to repeat cell collection and seeding at intervals of 3 to 7 days.

The human hybridoma of the present invention can be cryopreserved by conventional techniques. For example, cells are dispersed in an appropriate cell cryopreservation solution at a cell density of I x 10' cells/ml to 5 x 107 cells/ml, and then incubated in liquid nitrogen or liquid nitrogen gas, or at -20°C. It can be stored frozen in a freezer at 80°C. For the cell cryopreservation solution, it is recommended to use animal serum, albumin, methyl cellulose, glucose, dimethyl sulfoxide, etc., as appropriate, added to the above-mentioned basal medium, neutral buffer, etc.

``Frozen cells can be restored using standard techniques. for example,
Rapidly thaw the storage solution containing frozen cells in warm water,
It is preferable to wash the cells with a culture solution or the like to wash out DMSO contained in the storage solution, and then disperse the cells in the culture solution and culture them.

The amount of immunoglobulin in the culture groove can be measured by general ELIS.
This can be done by method A. For example, when using the ELISA method, an anti-human immunoglobulin antibody is immobilized on a solid phase (the antibody used at this time is hereinafter abbreviated as immobilized antibody),
A portion of the culture supernatant is reacted. Next, an enzyme-labeled anti-human immunoglobulin antibody is reacted, a substrate is added, and the amount of immunoglobulin in the culture supernatant can be measured from the coloring ratio produced by the enzyme reaction. The amount of human IgM can be measured by using an anti-human IgM (mu chain specific) antibody as the homologous antibody and a peroxidase-labeled anti-human IgM (mu chain specific) antibody as the enzyme-labeled antibody.

3. Production of human monoclonal antibodies The human-human hybridoma of the present invention synthesizes and secretes only heavy chains derived from antibody-producing cell lines, and can be stably maintained for long periods in normal animal cell culture media. It is possible to proliferate for several generations. In addition, it has the ability to produce antibodies even in serum-free medium without the risk of contamination with unknown impurities derived from the medium when purifying antibodies from culture, and is useful for the prevention and treatment of Pseudomonas aeruginosa infections. It is ideal for obtaining human monoclonal antibodies, which are raw materials for composition preparation.

The human-human hybridoma of the present invention is cultured in a serum-free medium, and the culture solution is extracted by any conventional method such as gel filtration, ion exchange chromatography, adsorption chromatography using hydroxyapatite, etc. Physicochemical purification methods, affinity chromatography methods using carriers immobilized with substances that have affinity for antigens or human monoclonal antibodies (e.g. protein A, anti-human immunoglobulin antibodies, etc.), electrophoresis methods,
By combining precipitation methods such as ammonium sulfate salting out, a single human monoclonal antibody having reactivity with G serotype Pseudomonas aeruginosa can be purified to a high degree relatively easily.

4. Production of human monoclonal antibody preparation The human monoclonal antibody of the present invention that is reactive with G serotype Pseudomonas aeruginosa has high protective activity against infection with the corresponding serotype Pseudomonas aeruginosa. The human monoclonal antibody of the present invention can be used alone,
Alternatively, by adding commonly used additives, excipients, etc., it can be used as a liquid or lyophilized preparation for the prevention and treatment of Pseudomonas aeruginosa infections. Additives and excipients are generally selected from natural products and compounds used in biological preparations, but animal proteins such as albumin, polysaccharides such as dextran, amino acids, and saccharides are used to maintain antibody stability. The use of gives good results. Furthermore, the human monoclonal antibody of the present invention can also be used to prepare a preparation by mixing it with other monoclonal antibodies or polyclonal antibodies that are reactive with Pseudomonas aeruginosa or microorganisms other than Pseudomonas aeruginosa.

5. Prevention and treatment of infectious diseases using human monoclonal antibodies For the actual prevention and treatment of Pseudomonas aeruginosa infections, the human monoclonal antibodies of the present invention or preparations containing them may be used alone or in combination of two or more, or by using Pseudomonas aeruginosa. It may be used in combination with other monoclonal antibodies having reactivity with or preparations containing the same or globulin preparations.

The human monoclonal antibody reactive with G serotype Pseudomonas aeruginosa of the present invention or a preparation containing the same can be directly administered to humans for the prevention and treatment of Pseudomonas aeruginosa infection. dose,
Although the administration route is appropriately selected, the dose is preferably 0.01 to 10 mg per body weight (kg), and the administration route can be appropriately selected from intradermal, subcutaneous, intramuscular, intravenous administration, etc.

Therefore, the present invention makes it possible to provide a cell line for industrial production of human monoclonal antibodies that can be used in a wide range of fields such as prevention, treatment, and diagnosis of Pseudomonas aeruginosa infections.

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these.

The parent cell line MP 4109 for producing hybridomas has been deposited with the FIKEN Co., Ltd. since October 27, 1988 under the deposit number FIKEN Co., Ltd. No. 2129. The human monoclonal IgM-producing EB virus-transformed cell line MP 5050 cross-reactive with G and H serotypes Pseudomonas aeruginosa has been deposited with the Microtech Institute since December 9, 1988 under the deposit number 160.
It has been deposited as No. 0. As the standard serotype of Pseudomonas aeruginosa classified by the Pseudomonas aeruginosa research group, G serotype Pseudomonas is classified as ATCC2758.
4 (IID 1008) and ATC for H serotype Pseudomonas aeruginosa.
C27585 (IID 1009) was used.

Example 1. Preparation of anti-Pseudomonas aeruginosa antibody-producing hybridoma-1 (1) Cell fusion MP 4109 and MP 5050 were grown in RPMI 1640 medium containing 10% FC5 (hereinafter sometimes abbreviated as 10% FC3 culture medium), and each Collect RPMI
Washed with 1640 medium. 1.5×10′ cells each were mixed in a 50 ml plastic centrifuge tube. After centrifugation (175×g, 10 min), aspirate the supernatant and add 0.5 ml of 50% P directly to the cell pellet.
EG (M, W, 1500, Wako Tsumugi Pharmaceutical) and 10
RPMI 1640 medium containing % DMSO was gently added and rotated slowly to fuse the cells. 2 minutes later, ], O
After adding 1 ml of RPMI 1640 medium and stirring gently, the mixture was centrifuged (175×g, 10 minutes). Aspirate the supernatant and incubate the cell pellet with 20% FC3, 2X 10-'M
Hyboxanthin (Sigma), 1 μg/ml Azaserine (
RPMI 1640 medium (hereinafter sometimes abbreviated as HA-0 medium) containing 5 x 10-'M ouabain (Sigma) was added to suspend the cells to a density of 1 x 10 cells/ml. , 0.1 ml per well of a 96-well flat bottom culture plate was seeded (288 wells in total).

The cells were statically cultured at 37°C in the presence of 5% carbon dioxide gas. 4
After one day, 0.1 ml of HA-0 medium was added, and then half of the HA-0 medium was replaced with fresh HA-0 medium every 3 to 5 days. After 4 to 5 weeks, cell proliferation was observed in a total of 10 wells.

(2) Detection of anti-Pseudomonas aeruginosa antibodies The presence or absence of anti-G serotype P. aeruginosa human antibodies in the culture supernatant is determined by DIB.
I investigated using method A. 0.1 ml of the culture supernatant of each well was added to 0.4 μg of G serotype Pseudomonas aeruginosa ATCC275 per dot.
A nitrocellulose membrane filter containing 84 formalin-treated dried bacterial cells (3
, 1 mm square) in a 96-well U-bottom microplate. After reacting for 2 hours at room temperature and then reacting with peroxidase-labeled rabbit anti-human immunoglobulin antibody (Dako) for 2 hours, color was developed using 4-chloro l-naphthol as a substrate, and the mixture was transferred onto a nitrocellulose membrane filter on which the antigen had been immobilized. Those in which color development was observed by visual observation were judged to be positive for antibody production.

(3) Out of the culture supernatants of 10 wells in which cloning cell proliferation was observed, production of anti-G serotype P. pyogenes antibody was observed in the culture supernatants of 8 wells. Cells in 8 wells were individually collected, and the number of cells was accurately counted using a hemocytometer. The cells were dispersed in HA-0 medium to form a cell suspension at a cell density of 20 cells/ml. After removing the supernatant from each well of a 96-well flat bottom culture plate (hereinafter referred to as a feeder plate) in which I x 10" mouse lung cells were seeded per well, 0.1 ml of the cell suspension was added to each well. The cells were seeded and cultured statically at 37°C in the presence of 5% carbon dioxide.

One feeder plate was used for each cell. 4
After 1 day, add 0.1 ml of 11 A-0 medium, then add half of the HA-0 medium every 3 to 5 days to fresh HA-0 medium.
The medium was replaced with 0 medium. After 2 to 4 weeks, the presence or absence of anti-G serotype P. pyogenes human antibody in the culture supernatant of wells in which growth was observed was examined by DIBA method. Cells from wells that were determined to be positive for producing antibodies that react with serotype G Pseudomonas aeruginosa were re-cloned as described above. Through two rounds of cloning, two hybridoma strains, MP 5142 and MP 5151, which produce human monoclonal antibodies that cross-react with serotypes G and H of Pseudomonas aeruginosa classified by the Pseudomonas aeruginosa research group were obtained.

The fully grown hybridomas were gradually expanded in 96-well flat bottom culture plates. Cells are 75% FC3,
The cells were suspended in a cell preservation solution consisting of 10% DMSO, 115% RPMI 1640 medium at a density of 1X10 cells/ml, and then dispensed into 2D11 freezing tubes. After cooling to -20°C at a rate of 1°C per minute, it was stored frozen in liquid nitrogen.

(4) Measurement of antibody production amount The amount of IgM secreted by the hybridoma to the outside of the cells was measured as follows. Collect cells in logarithmic growth phase and 10% FC.
The cells were suspended in 3 culture solution at a density of 1×10′ cells/ml, and 1 ml was inoculated into each well of a 6-well culture plate, followed by static culture at 37° C. in the presence of 5% carbon dioxide gas. After 24 hours, the culture supernatant was separated by centrifugation (250×g, 10 minutes), and the amount of human IgM in the supernatant was determined by ELISA. Hybridoma HP 5142, MP 5
151 means that 106 cells each have 16 cells in 24 hours.
μg, 18 μg of human IgM was secreted into the culture supernatant.

(5) Measurement of passage stability of cell lines The passage stability of cell lines was investigated from cell proliferation performance and antibody production ability.

Growth stability was examined by measuring the growth curve of the cells at the start of culture and 3 months after the start of subculture. The stability of antibody production is determined at the start of culture, 1 month after culture start, 2 months after culture start,
The amount of IgM antibody produced by the cells of Trichigetsu was investigated by measuring by ELISA method in the same manner as in (4).

Subculture was performed as follows. Cells were suspended in two independent lines in 10% FC3 culture medium at a density of 5 x 10' cells/ml, then seeded in 4 cells in flasks with a bottom area of 25 ci, and incubated at 37°C in the presence of 5% carbon dioxide. Cultured statically.

Cells were collected every 3 to 4 days, resuspended in fresh 10% FC3 culture medium to the same density, and then statically cultured for 3 months continuously.

The growth curve was measured as follows. Collect cells in logarithmic growth phase that were independently cultured in two lines, suspend them in 10% FC5 culture medium at a density of 5 x 104 cells/ml, and add 1 ml to each well of a 6-well culture plate for a total of 6 wells. The seeds were seeded and cultured stationary at 37°C in the presence of 5% carbon dioxide gas. Every day, the culture in one well was collected, live and dead cell densities were measured, and the culture supernatant was
The gM amount was measured by ELISA method.

MP 5151 showed almost the same growth curve as the cells at the start of culture and 3 months after the start of culture. The doubling time calculated from the growth curve was 23.5 hours. Also, 106
The IgM secreted by each cell in 24 hours is 18 μg at the start of culture, 15 μg after 1 month, 15 μg after 2 months, 13 μg after 3 months, and during subculture for 3 months. There were no significant changes in antibody production.

MP 5151 was deposited with the Institute of Fine Technology as Article No. 2274.

Example 2. Cell culture and antibody purification-IMP'51
51 cryopreserved cells were recovered, expanded and cultured using 10% FC3 culture medium, and further expanded and cultured in NYSF 404 serum-free medium [Noritsugu Yabe, Tissue Culture, 11.458 (1985)]. They were collected, suspended in 500 ml of NYSF 404 serum-free medium to a density of 5 x 10' cells/ml, and seeded in 10 flasks (bottom area 175a#). The cells were statically cultured at 37° C. in the presence of 5% carbon dioxide for 5 days.

4 by centrifugation (400xg, 20 minutes) from the culture.
80 ml of supernatant was obtained and filtered through a membrane filter with a pore size of 0.22 microns.

Add 480 ml of saturated ammonium sulfate solution to the filtrate,
It was left at 4°C. The next day, centrifuge it 10. OO
OXg for 30 min), and the precipitate was collected. 5 ml PBS (
-) to dissolve the precipitate and thoroughly dialyze against PBS (-) to obtain a crude IgM fraction.

For purification, fractionation was performed using a hydroxyapatite-packed column for high-performance liquid chromatography at a flow rate of 1 ml and 17 minutes. 0CA-column (guard column; 4mm
10mm, main column; 7.6mm x 100mm
, Mitsui Toatsu Chemical Co., Ltd.) was thoroughly washed in advance with 0.01M sodium phosphate buffer (pH 7.0) containing 0.15M sodium chloride (hereinafter abbreviated as solution A),
2 ml of crude IgM fraction was added. Add liquid A for 10 minutes, and then add 0.0.
25M sodium phosphate buffer (pH 7.5) (hereinafter referred to as
The column was washed for 15 minutes with a solution to which B solution was added. Thereafter, linear concentration gradient elution was performed in which the proportion of solution B ranged from 25% to 100% over a period of 20 minutes. The IgM fraction eluted as a single peak was thoroughly dialyzed against PBS(-). 1.3.7 mg of IgM from 480 ml of culture supernatant
(N7-2) solution was obtained.

Example 3. Preparation of anti-Pseudomonas aeruginosa antibody-producing hybridoma-2 (1) Preparation of EB virus solution B95-8 cells producing and releasing EB virus were
The cells were suspended in RPMI 1640 medium containing 20% FC3 (hereinafter sometimes abbreviated as 20% FC5 culture medium) to a density of 105 cells/ml, and in the presence of 5% carbon dioxide.
It was statically cultured at 37°C. Culture on day 7 near stationary phase] 1
The supernatant was collected by centrifugation (800×g, 10 min),
Membrane filter with pore size of 0.45 microns (
Millipore) to obtain an EB virus solution.

(2) Preparation of human lymphocytes 50 ml of heparinized peripheral blood was collected from a healthy individual whose serum antibody activity against serotype G Pseudomonas aeruginosa showed a positive reaction up to a 1000-fold dilution using the DIBA method. An equal volume of RPMI 1640 medium was added to dilute the mixture to 2 times, and the mixture was layered on a half volume of Ficoll Paque (Pharmacia) so as not to disturb the interface, and centrifuged at room temperature (400x for 30 minutes). After centrifugation, the interfacial layer was removed using a Pasteur pipette, an equal volume of 20% FC5 culture solution was added, and centrifugation was performed at room temperature (250×g, 10 minutes). After suspending the precipitated cells in a 20% FC3 culture medium, the centrifugation operation was repeated once more to obtain a human lymphocyte pellet (cell number: 4.8 x 107 cells).

(3) For 4.8 x 107 human antibody-producing cells transformed by EB virus (1)
48ml of the virus solution prepared above was added and incubated at 37°C for 1 hour. After incubation, cells were collected by centrifugation (250×g, 10 minutes).

The cells were dispersed in 20% FC3 culture medium and adjusted to a density of 5 x 10' cells/ml, then added in 0.1 ml portions to a 96-well flat-bottomed culture plate and cultured stationary at 37°C in the presence of 5% carbon dioxide gas. did. After 4 days, 0.1 ml of 20% FC3 culture solution was added, and then half of the culture solution was replaced with fresh culture solution every 3 to 5 days. For wells in which cell proliferation was observed, Example 1. In the same manner as in (2), the presence or absence of anti-G serotype P. pyogenes human antibody in the culture supernatant was determined using DIBA@, which is an enzyme immunoassay. Cells in wells that were determined to be positive for antibody production were expanded and cultured in a 24-well culture plate.

(4) Cells in wells in which reactivity with G serotype Pseudomonas aeruginosa was observed by the cloning antibody detection method were transferred to a 6 cm petri dish. Cells grown in a 6 cm petri dish were subjected to a loning operation once using the soft agar method. First, after accurately measuring the number of cells using a hemocytometer, a cell suspension with a density of 1 x 10 r' cells/ml was prepared, and 0.1 ml of this cell suspension was added to 0.3% agarose (G-Plaque agarose, F.・MC Co., Ltd.) was added to the culture solution 30 resistant and mixed. Next, 3 ml of culture solution containing cells and 0.3% agarose was dispensed into a 6 cm Petri dish, which had been previously dispensed and solidified with 4 ml of culture solution containing 0.5% agarose (10 plates per cell). ). The 6 cm petri dish into which the cells were dispensed was cultured stationary at 37° C. in the presence of 5% carbon dioxide gas. After 3 to 5 weeks, when the cells have proliferated in the soft agar and colonies are visible to the naked eye, each colony is preliminarily pipetted with 0.1 ml of 20% F per well using a Pasteur pipette.
The C5 culture solution was transferred to each well of a 96-well flat bottom culture plate and cultured. 20% FC3 culture solution 0 after 2 days
．． 1 ml was added, and two days later, the presence or absence of anti-Pseudomonas aeruginosa monoclonal antibodies in the culture media was measured using the DIBA method for wells in which cell proliferation was observed.

Cells in wells determined to be positive for antibody production were expanded and cultured in a 24-well culture plate. Three days later, the presence or absence of anti-Pseudomonas aeruginosa antibodies in the culture supernatant was determined using the DIBA method in the wells of the 24-well culture plate. Among these, the cells in the wells with high anti-Pseudomonas aeruginosa antibody activity in the culture supernatant were sequentially expanded and cultured to form an anti-G serotype P. aeruginosa hIgM-producing EB virus-transformed cell colony N19N1.7N19N2.7N1.
9N3.7N19N4.7N19N5.7N19N6 was obtained.

(5) Cell fusion anti-G serotype P. aeruginosa human 1g-produced EB virus transformed cell colony 7N19N1.7N19N3.7N19N
4 mixed 1. OX 107 and the same number of MP41
Example 1.09 was used. Cell fusion was performed in the same manner. The fused cells were seeded in HA-0 medium as a cell suspension at a density of 0.6 cells per well (total of 192 wells) in an amount of 0.1 ml per well of a 96-well flat bottom culture plate. The cells were statically cultured at 37°C in the presence of 5% carbon dioxide gas. After 4 days, 0.1 ml of HA-0 culture medium was added to the crabs, and then half of the HA-0 medium was replaced with fresh HA-0 medium every 4 to 5 days. For 37 wells in which cells had grown, the presence or absence of anti-Pseudomonas aeruginosa antibodies in the culture supernatant was determined by the DIBA method.

Antibody activity was observed in the culture supernatant of 37 wells. Hybridoma HP 5148 was cloned and produced a human monoclonal antibody that reacts only with G serotype Pseudomonas aeruginosa.
, MP 5114, MP 5115, MP5116, N
Six strains, P5117 and MP5118, were obtained.

(6) Measurement of IgM production amount Regarding the hybridoma obtained by cell fusion in (5), Example 1. The amount of antibody produced was measured in the same manner as in (4).

Hybridoma MP 5148, MP 5114, HP
5115, MP5116, NP5117, MP511
8, 10″ cells each received 10 μl in 24 hours.
g, 18 μg, 10 μg, 12 μg, 11 μg, 15 μg
g of human IgM were secreted into the culture supernatant.

(7) Passage stability of the cell line Example 1 except that MP 5114 was used. The proliferation of the cell line and the stability of antibody production were measured in the same manner as in (5).

MP 5114 showed almost the same growth curve as the cells at the start of culture and 3 months after the start of culture. The doubling time calculated from the growth curve was 24 hours. In addition, the IgM secreted by 106 cells in 24 hours was 18 μg at the start of culture, 12 μg after 1 month, 14 μg after 2 months, and 15 μg after 3 months, and continued for 3 months. There was no significant change in antibody production during subculture.

MP 5114 was deposited with the Institute of Fine Technology as Article No. 2269.

Example 4. Cell culture and antibody purification-2MP 51
14 cryopreserved cells were raised, expanded using 10% FC5 culture medium, further expanded in NYSF 404 serum-free medium, and then cultured in 50 resistant NYSF to a cell density of 5 x 10 cells/resistant. 404 serum-free medium.

After seeding in one flask (bottom area 175 aff), 5%
The cells were statically cultured at 37° C. for 5 days in the presence of carbon dioxide gas. Collect the cells and 50
The cells were suspended in 0 ml of NYSF 404 serum-free medium and seeded in one star rolling culture flask (Techne). Culture was carried out with stirring at 20 rpm for 5 days at 37° C. in the presence of 5% carbon dioxide gas. Centrifuge (400 x g, 2
0 min) to obtain 480 ml of supernatant with a pore size of 0.
It was filtered through a 22 micron membrane filter.

Antibodies were purified from the filtrate by the same procedure as in Example 2.

From 480 ml of culture supernatant, 9 mg of IgM (N
8-2) A solution was obtained.

Example 5. Test of protective activity of human monoclonal antibodies against Pseudomonas aeruginosa infection The human monoclonal antibodies N7-2 and N8-2 obtained in Examples 2 and 4 were examined for their protective activity against Pseudomonas aeruginosa infection. Mice (Balb/c, female) aged 8 to 12 weeks old - Groups of 5 to 10 mice received 50 ng of human monoclonal antibody per mouse, 50
0.2 ml of a solution containing 0 ng, 512 g, and 50 μg was administered intraperitoneally, and 2 hours later, a bacterial solution of G serotype Pseudomonas aeruginosa (P-28) was challenged intraperitoneally. In the control group, only physiological saline was administered instead of human monoclonal antibody. P
-28 was seeded on a heart infusion agar plate medium and cultured at 37°C overnight. Scrape the grown bacterial colonies, dilute with physiological saline, add 5% mucin,
The challenge bacteria amount was prepared to be 8 times the 50% lethal dose (LD, value) per mouse, and used as a bacterial solution. The 50% effective dose (ED, o value) was determined from the survival rate of mice in each administration group on day 7 after Pseudomonas aeruginosa challenge. N7-
2. The ED5o values of N8-2 are 0.11 μg and 0.1 μg, respectively.
It was 11 μg. Each human monoclonal antibody had high protective activity against infection with G serotype Pseudomonas aeruginosa.

Example 6. Preparation of liquid N7-2 obtained in Example 2 and N8 obtained in Example 4
-2 at 1 mg/ml. Human serum albumin (Calbio) was prepared at 0.2% (w7v) using PBS (-), and sterilized and filtered using a membrane filter with a pore size of 0.22 microns. A liquid preparation was prepared by separately dispensing each antibody in an amount of 1 ml per vial aseptically.

It was left at a temperature of 4°C or 37°C for one month. The storage stability of the preparation is determined by measuring the antibody titer against G serotype Pseudomonas aeruginosa LPS.
It was determined by measuring by LISA method.

Antibody titer was measured as follows.

G serotype Pseudomonas aeruginosa LPS was added to 0.1M citrate buffer (pH
4,0) dissolved in L, 0.0 per well of a 96-well plate for EIA (Greiner).
It was dispensed into 5 ml portions. Leave at 37°C for 16 hours, LPS
was adsorbed onto the plate. The solution dilution series was allowed to react in wells for 2 hours at room temperature. Next, after reacting with peroxidase-conjugated goat anti-human IgM antibody (Tabo) for 2 hours,
Color was developed using '-azinobis(3-ethylbenzthiazoline sulfonic acid) (Sigma) as a substrate, and the absorbance at a wavelength of 414 nm was measured. The dilution factor at which the absorbance was 0.1 was calculated using the least squares method, and this was taken as the antibody titer.

N7-2 and N stored at 4℃ or 37℃ for 1 month
The antibody titer of the liquid preparation No. 8-2 was no different from the antibody titer of the control liquid preparation stored at 80° C., and the antibody activity was maintained.

Example 7. Preparation of lyophilized formulation N7-2 obtained in Example 2 and N8 obtained in Example 4
-2 and 0.2% (w/v) human serum albumin (Calbio) were prepared in PBS (-) and removed using a membrane filter with a pore size of 0.22 microns. Bacteria filtered. Each antibody was separately aseptically dispensed at 1 ml per vial and lyophilized to prepare a lyophilized preparation. When redissolved with distilled water and measured the antibody titer against G serotype Pseudomonas aeruginosa LPS in the same manner as in Example 6, the antibody titer of the redissolved solution of the freeze-dried preparations of N7-2 and N8-2 was as follows. There was no difference in antibody titer from that of the control lyophilized untreated solution, and the antibody activity was maintained.

Effects of the Invention By culturing the human-human hybridoma according to the present invention using an appropriate production medium and using human monoclonal antibodies purified from the culture alone, in combination of two or more, or in combination with other human antibodies. , excellent preventive and therapeutic effects against Pseudomonas aeruginosa infection are achieved.

The human-human hybridoma of the present invention producing a human monoclonal antibody reactive with serotype G Pseudomonas aeruginosa is
It can be stably subcultured and expanded for a long period of time in a medium supplemented with fetal bovine serum for animal cell culture.

Furthermore, when purifying human monoclonal antibodies from culture, it has the ability to produce antibodies in large quantities even in serum-free medium without the risk of contamination with unknown impurities derived from the medium.
It is ideal for obtaining human monoclonal antibodies that can be used as raw materials for preparing compositions for preventing and treating Pseudomonas aeruginosa infections. That is, the novel human-human hybridoma of the present invention has a high antibody secretion ability, and can shorten the culture period and reduce production costs when culturing in large quantities to produce human monoclonal antibodies.

Furthermore, the human-human hybridoma of the present invention can be used not only as a cell line for producing human monoclonal antibodies, but also as a material cell for preparing human antibody genes when introducing and expressing globulin genes into other host cells or microorganisms. It can also be used for

Agent Patent Attorney 1) Parent Male

Claims (1)

[Scope of Claims] 1. A human-human hybridoma that produces a human monoclonal antibody that is reactive with at least G serotype Pseudomonas aeruginosa and a cell line derived therefrom. 2. The first hybridoma is a hybridoma of a cell line with only human chromosomes and unlimited proliferation ability and human antibody-producing cells.
Human-human hybridomas and cell lines derived therefrom as described in Section 1. 3. The human-human hybridoma and cell lines derived therefrom according to item 2, wherein the cell line has only human chromosomes and has the ability to proliferate indefinitely, and is a cell line that has selection characteristics for hybridomas. 4. The human-human hybridoma and cell line derived therefrom according to item 3, wherein the cell line having the selective characteristics of a hybridoma is Kaikoken Jokyo No. 2129 or a cell line derived therefrom. 5. The human-human hybridoma and cell line derived therefrom according to item 2, wherein the human antibody-producing cells are EB virus-transformed cells. 6. EB virus transformed cell
6. The human-human hybridoma according to item 5, which is a cell line derived therefrom or a cell line derived therefrom. 7. The human-human hybridoma and cell line derived therefrom according to items 1 to 6, wherein the human monoclonal antibody produced is protective against infection with the corresponding serotype Pseudomonas aeruginosa. 8. Human-human hybridoma of FAIKEN Jyoyori No. 2269 or Jyoyori No. 2274 and cell lines derived therefrom. 9. A method for producing a human-human hybridoma according to items 1 to 8. 10. Reactive to G serotype Pseudomonas aeruginosa, which is obtained by culturing at least one of the human-human hybridomas and cell lines derived therefrom as described in paragraphs 1 to 8, and purifying the cultured product. Method for preparing human monoclonal antibodies. 11. Human monoclonal antibodies produced by the human-human hybridomas and cell lines derived therefrom according to items 1 to 8. 12. A human monoclonal antibody prepared by the preparation method described in Section 10. 13. A preparation for preventing or treating Pseudomonas aeruginosa infection, which contains at least one human monoclonal antibody according to items 10 to 12. 14. A preparation for preventing or treating Pseudomonas aeruginosa infection, wherein the preparation according to item 13 is a liquid preparation. 15. A preparation for preventing or treating Pseudomonas aeruginosa infection, wherein the preparation according to item 13 is a freeze-dried preparation. 16. A method for preventing or treating Pseudomonas aeruginosa infection, which comprises administering the preventive or therapeutic preparation according to items 13 to 15.