JPH02245185A - Human monoclonal antibody reactive with e 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 E 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 E serum-type Pseudomonas aeruginosa, the objective human-human hybridoma (FERM P-2272, 2273) is prepared.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to Pseudomonas aeruginosa of serotype E.
A human-human hybridoma cell line capable of stably supplying a large amount of human monoclonal antibodies against a), the human monoclonal antibodies it produces, and the prevention and treatment of Pseudomonas aeruginosa infections using the same as an active ingredient It relates to formulations for

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, the amount of antibodies that have affinity for Pseudomonas aeruginosa and are effective for treatment is variable, and because the content is small, these preparations cannot be used for prevention or treatment. Many people question the effectiveness of the 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 Pseudomonas aeruginosa study group classification (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.
n, Nature, 256.495-497 (19
75)) [for example, Hanco
ck 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.
We reported that mouse monoclonal antibodies against polysaccharide side chains have high protective activity against lethal infection by bacteria of the corresponding serotype in mouse infection experiments (Sa
Doff et al., Abstracts of the 198
2 Interscience Conference
onAntimicrobial Agents a
nd Chemotherapy, Nn253 (19
82) ). Subsequent reports also included the serotype specificity of Pseudomonas aeruginosa.
The efficacy of mouse or human monoclonal antibodies directed against antigens in in vivo and in vitro test systems has been demonstrated [e.g., Sawada et al., J. Infect, D.
is,, 150.570576 (1984) - Nakamura Keibo et al., Japanese Journal of Bacteriology, 39°337 (1984)
, Pennington, Infect, Immu
n,, 54°239-244 (1986), 5uz
uki et al., Microbiol, Immunol.

31, 959-966 (], 987), Zwerrn
ik et al., Infect.

■mmunity, 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 antibodies that commonly recognize multiple 〇-polysaccharide side chains have been disclosed in the patent application specification [International Publication No.
8104669] and several other patent application specifications [
Genetic Systems Corporate Simin, EP
163493 and BE 905890, Teijin Ltd.
WO36103754, Yukinaga Pharmaceutical Co., Ltd., JP-A-61
-091134, Merck End Company, Inc., EP 256713].

Problems to be Solved by the Invention The production of human monoclonal antibodies involves the introduction of the Epstein-Barr virus (Epstein Barr virus) into human B cells.
n-Barr virus (hereinafter abbreviated as EB virus)
EB virus-transformed cells can be obtained by infecting human antibody-producing cells such as B cells, or human-mouse heterohybridoma or human-human hybridoma can be obtained by fusing parent cells of human antibody-producing cells such as B cells with a parent cell line that has unlimited proliferation potential. It is done by doing.

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 media. 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 demonstrated that it is possible to produce human-human hybridomas that produce human monoclonal antibodies that are reactive with Pseudomonas aeruginosa serotype E, and that the human-human hybridomas can be used in a variety of ways. 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, we will use a cell line that has only human chromosomes, unlimited proliferation ability, and resistance to 8-azaguanine and ouabain as the parent cell line, and use it as a human antibody-producing EB virus-transformed cell that is reactive with E serotype Pseudomonas aeruginosa. 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) 1. 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 the American Type Culture Collection (ATCC), the Institute for Fermentation (IFO), and the Institute of Medical Science, the University of Tokyo.

2. Preparation of human-human hybridoma The human-human hybridoma according to the present invention, which produces a human monoclonal antibody reactive with serotype Pseudomonas aeruginosa, is derived from the parent cell line MP 4109 for hybridoma production or its passages. strains and human antibody-producing cells using known methods.
NOcLONAL ANTIBODIESJ p363
．． Plenum Press (1980) et al.], it can be produced by cell fusion. Human antibody-producing cells include
B cells obtained by known methods from peripheral blood, lymph nodes, tonsils, lungs, cord blood at delivery, etc. of healthy individuals who produce antibodies against Pseudomonas aeruginosa or patients with a history of Pseudomonas infection. However, after infecting B cells with EB virus, transforming them, and culturing them for a certain period of time, EB virus-transformed cells in which secretion of antibodies reactive with Pseudomonas aeruginosa is detected in the culture supernatant. It is preferable to use a colony or a single cell line selected from these EB virus-transformed cell colonies.

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 Hawkelyde mite-diene (PINM) to proliferate the B cells, and then subjected to cell fusion.

The method for transforming B cells with EB virus is a known method [
For example, 5teinitz et al., Nature, 269
．． 420422 (1977)). BO2-8 cells (marmoset leukocyte-derived cells that produce infectious EB virus) were cultured in RPMI 1640 medium (hereinafter sometimes abbreviated as culture medium) containing 20% fetal calf serum (hereinafter abbreviated as FC5), and centrifuged. The culture supernatant obtained by isolation 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, 399
401 (19'74)). B cells are centrifuged, the supernatant is removed by suction, a virus solution is added to the resulting pellet, dispersed, and then incubated at 37° C. in the presence of 5% carbon dioxide for 30 minutes to 1 hour. After culturing, centrifuge
After removing the supernatant by aspiration, the pellet has a cell density of I.
Add culture medium to disperse the cells at a concentration of 5 x 10'' cells/ml to 5 x 10'' cells/ml. 2 cell dispersions
The mixture is dispensed into each well of a 4-well culture plate or a 96-well culture plate, and cultured for 2 to 4 weeks at 37°C in the presence of 5% carbon dioxide. During this time, it is desirable to replace half of the culture solution with fresh culture solution every 3 to 4 days.

Detection of antibodies reactive with Pseudomonas aeruginosa can be carried out by methods such as general radioimmunoassay or enzyme-linked antibody assay (hereinafter abbreviated as ELISA) [Naritai "Monoclonal Antibody J p144,
Published by Kodansha (1983), 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. Dot immunobinding assay method (hereinafter abbreviated as DIBA method) (hereinafter abbreviated as DIBA method), which measures the presence or absence of production of the target antibody and the production amount based on the coloring rate of the substrate caused by the enzyme reaction.
nal.

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

The culture supernatant of each well in which proliferating colonies of EB virus-transformed cells were observed was analyzed by the above ELISA method.
After selecting wells containing the antibody of interest.

The cells in the wells were collected using the soft agar method [Naritai, Tissue Culture Applied Research Methods J p289, published by Soft Science Co., Ltd. (1985).
[etc.] or limiting dilution method [Altai “Monoclonal Antibodies” p. 7
3, published by Kodansha (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, mix MP 4109 and cells in wells that have been found to produce the desired antibody after transformation with EB virus, or antibody-producing cells isolated from peripheral blood, etc. at a ratio of 1:1 to 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 at 6 cells/ml. 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 hybridoma 15- 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 HAT medium) or a medium containing hypoxanthine and azaserine (hereinafter referred to as HAT medium). , HA medium) can be used. In addition, if the human antibody-producing cells are cells with unlimited proliferation ability such as EB virus-transformed cells, a medium containing HAT medium supplemented with ouabain (HA
T-0 medium) or l (A medium with ouabain added ()IA-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 104 cells/ml to 2 x 10' cells/ml and seeding into a suitable cell culture container.

It can be cultured at 37°C in the presence of 5% carbon dioxide. As an example of the culture solution, a basal medium such as RPMI 1640 or DMEM to which an appropriate amount of Fe2 is added is suitable. Also,
Various low-serum or serum-free media can also be used as appropriate. For example, NYSF 404 serum-free medium alone or NYSF 404 serum-free medium alone;
SF 404 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.

Cryopreservation of the human-human hybridoma of the present invention can be carried out by conventional techniques. For example, add cells to an appropriate cell cryopreservation solution at IX 10' cells/ml to 5 X 10' cells/ml.
The cells can be dispersed to a cell density of 200° C. and stored frozen in liquid nitrogen or liquid nitrogen gas, or in a freezer at -20°C to -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.

Restoration of frozen cells can be performed using standard techniques. For example, it is preferable to rapidly thaw a preservation solution containing frozen cells in warm water, wash the cells with a culture solution to wash out DMSO contained in the preservation solution, and then disperse the cells in the culture solution and culture.

Measurement of the amount of immunoglobulin in the culture supernatant.

This can be carried out by a common ELISA method. For example, E
In the case of the LISA method, an anti-human immunoglobulin antibody is immobilized on a solid phase (the antibody used at this time is hereinafter abbreviated as immobilized antibody), and a portion of the culture supernatant is reacted with it. 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. Measurement of human IgM amount is
Anti-human IgM (Mu chain specific) antibody was used as the immobilized antibody, and peroxidase-labeled anti-human I as the enzyme-labeled antibody.
This can be done by using gM (Mu chain specific) 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 serotype E 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 serotype Pseudomonas aeruginosa E 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 of the present invention that is reactive with serotype Pseudomonas aeruginosa E or a preparation containing the same can be directly administered to humans for the prevention and treatment of Pseudomonas aeruginosa infections. 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 Hi1-monoclonal IgG-producing EB virus-transformed cell line MP 5038, which is cross-reactive with Pseudomonas aeruginosa serotypes E and F, has been deposited with the Microtech Institute since December 7, 1988, under number 15 of the Microtech Institute.
It has been deposited as No. 96. As the standard serotype of Pseudomonas aeruginosa classified by the Pseudomonas aeruginosa research group, the E serotype Pseudomonas is ATCC275.
81 (IID 1005) and AT for F serotype Pseudomonas aeruginosa.
CC27582 (IID 1006) was used.

Example 1. Preparation of anti-Pseudomonas aeruginosa antibody-producing hybridoma-1 (1) Cell fusion MP4], 09 and MP5038 were grown in RPMI 1640 medium containing 10% FC3 (hereinafter sometimes abbreviated as 10% FC5 culture medium). Collect each RPM
Washed with I 1640 medium. 3×10′ 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% PEG directly to the cell pellet.
(M, lit, 1500, Wako Tsumugi Pharmaceutical) and 10%
RPMI 1640 medium containing DMSO was gently added and rotated slowly to fuse the cells. 2 minutes later, 10m1
After adding RPMI 1640 medium and stirring gently, the mixture was centrifuged (175×g, 10 minutes). The supernatant was aspirated and the cell pellet was incubated with 20% FC5, 2 x 10-'M hypoxanthine (Sigma), 1 μg/ml azaserine (Sigma), 5 x 10-'M'77pine (Sigma).
Add RPMI 1640 medium (hereinafter sometimes abbreviated as HA-0 medium) and suspend at a density of I x 10 cells/ml, then seed 0.1 ml per well of a 96-well flat bottom culture plate. (576 wells in total).

Cells were statically cultured at 37°C in the presence of 5% carbon dioxide.

After 4 days, 0.1 ml of HA-0 medium was added, and then 3
Half of the HA-0 medium was replaced with fresh HA-0 medium every 5 days. After 4 to 5 weeks, IF5.3E2
．． Cell proliferation was observed in a total of 3 wells of 4A11.

5 x 106 pieces each (7) MP 4109 to MP 5
Cell fusion was performed using 038 in the same manner as described above. The cell pellet was treated with 2o% FC3, 2X 10-'M hypoxanthine, 0.66 μg/ml azaserine, 6 X 10-
'M I in RPMI 1640 medium containing ouabain.
After suspending the cells at a density of 10' cells/ml, 0.1 ml per well of a 96-well flat bottom culture plate was seeded (96 wells in total). The cells were statically cultured at 37°C in the presence of 5% carbon dioxide gas. 20%FC3.2X after 4 days
RPM containing 10-'M hypoxanthine, 0.33 p g/ml azaserine, 3 X 10-7M ouabain
I 1640 medium 0.1 resistant and then for 2 weeks, 3
Half of the selection medium was replaced with fresh selection medium every 5 days. In addition, after the second week, 20% FC8 and 2X10
-'M Replaced with RPMI 1640 medium containing hypoxanthine. After 4 to 5 weeks, 5A10.5C3,5
Cell proliferation was observed in a total of 4 wells of C8 and 5G5.

(2) Detection of anti-Pseudomonas aeruginosa antibodies The presence or absence of anti-E 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 E serotype Pseudomonas aeruginosa ATCC275 per dot.
Gridded nitrocellulose membrane filter fixed with 81 formalin-treated dried bacterial cells = (3.1 mm)
(corner) in a 96-well U-bottom microplate. The reaction was carried out for 2 hours at room temperature, and then inoculated with peroxidase-labeled rabbit anti-human immunoglobulin antibody (Dako).
After a time reaction, a color was developed using 4-chloro-1-naphthol as a substrate, and those whose color development was observed with the naked eye on the nitrocellulose membrane filter on which the antigen was immobilized were determined to be positive for antibody production.

(3) IF5 among the culture supernatants of 7 wells in which cloning cell proliferation was observed
．． Anti-E serotype P. pyogenes antibody production was observed in the culture supernatants of 5 wells of 3E2.4A11.5C3 and 5G5.

Cells in 5 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 in advance, 0.1 ml of the cell suspension was added to each well. sow seeds,
Stationary culture was performed at 379C in the presence of 5% carbon dioxide gas. One feeder plate was used for each cell. After 4 days, add 0.1 ml of HA-0 medium, then from 3 to 5 days.
Half of the HA-0 medium was replaced with fresh HA-0 medium every day. After 2 to 4 weeks, the presence or absence of anti-E 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 E Pseudomonas aeruginosa were re-cloned as described above. Hybridomas MP 5133, HP 5135, MP 5137, and M that produce human monoclonal antibodies that cross-react with serotypes E and F of Pseudomonas aeruginosa classified by the Pseudomonas aeruginosa research group through two rounds of cloning.
Five strains, P5138 and MP5139, were obtained.

The fully grown hybridomas were gradually expanded in 96-well flat bottom culture plates. cells at 75% FC3,
The cells were suspended in a cell preservation solution consisting of 10% DMSO, 1 x 5% RPMI 1640 medium at a density of I x 10'' cells/ml, and then dispensed into 2 ml freezing tubes. -20
After cooling to 1°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 8 culture medium at a density of I x 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. 2
After 4 hours, the culture supernatant was separated by centrifugation (250 x g, 10 minutes), and the amount of human IgM in the supernatant was determined by ELISA.
Quantitated by method. Hybrid 1 (-Ma MP 5133, M
P 5135, MP 5137, MP 5138, MP
5139, 106 cells secreted 18 μg, 30 μg, 44 μg, 7 μg, and 45 μg of human IgM into the culture supernatant over 24 hours, respectively.

(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 placed in 2 lines = 28 independent rows at 5 x 104 cells/ml in 10% FC3 culture medium.
After suspending to a density, 4
The cells were seeded in ml portions and cultured stationary at 37° C. in the presence of 5% carbon dioxide. Collect cells every 3 to 4 days and store in fresh 10% F.
After resuspending the cells in C3 culture medium to the same density, the cells were cultured stationary for 3 consecutive months.

The growth curve was measured as follows. Cells in the logarithmic growth phase independently cultured in two lines were collected, suspended in 10% FC8 culture medium at a density of 5X and 104 cells/ml, and then
1 ml was inoculated into each well of a well culture plate into a total of 6 wells, and cultured stationary at 37° C. in the presence of 5% carbon dioxide gas. Every day, the culture in one well was harvested, the density of live cells and dead cells was measured, and the culture supernatant was treated with IgM.
The amount was measured by ELISA method.

MP 5139 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 26 hours. In addition, 10' cells secrete Ig in 24 hours, but at the start of culture, 4
5 μg, 40 μg after 1 month from the start of culture, 38 μg after 2 months, and 36 μg after 3 months, and there was no major change in antibody production during subculturing for 3 months.

MP 5139 was deposited with the Institute of Fine Technology as Article No. 2272.

Example 2. Cell culture and antibody purification - IMP 51
39 cryopreserved cells were recovered and expanded using 10% FC3 culture medium, and after further expansion culture in NYSF, 404 serum-free medium [Noritsugu Yabe, Tissue Culture, 11.458 (1985)], 1 The cells were collected, suspended in 500 ml of NYSF 4.04 serum-free medium to a density of 5 x 104 cells/ml, and placed in 10 flasks (bottom area: 175 CII?
) was sown. 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 carried out using a hydroxyapatite-packed column for high-performance liquid chromatography at a flow rate of 1 ml/min. HCA-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 (pi(7,0) (hereinafter abbreviated as solution A) containing 0.15M sodium chloride, and 2 ml of crude Added IgM fraction. Added solution A for 10 minutes.
Furthermore, 0 at a ratio of 25 volumes (25%) to 75 volumes of liquid A.
．． The column was washed for 15 minutes with a solution containing 25M sodium phosphate buffer (pH 7.5) (hereinafter abbreviated as solution B). 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(-). 10.6+ng of Ig from 480ml of culture supernatant
A M(Nl-5) 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
X]O'' cells/ml, suspended in RPMI 1640 medium containing 20% FC5 (hereinafter abbreviated as 20% FC3 culture medium), and in the presence of 5% carbon dioxide.
It was statically cultured at 37°C. The culture supernatant on day 7, which was close to the stationary phase, was collected by centrifugation (800 x g, 10 minutes) and filtered through a membrane filter (Millipore) with a pore size of 0.45 microns 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 E Pseudomonas aeruginosa was positive up to a 1000-fold dilution using the DIBA method. An equal volume of RPMI 1640 medium was added to the mixture to dilute it 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 (400 x g, 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 20% FC3 culture solution,
The centrifugation operation was repeated to obtain a pellet of =31 human lymphocytes (cell number: 5XIQ').

(3) Transformation with EB virus 50 ml of the virus solution prepared in (1) was added to 5×10' human antibody-producing cells and incubated at 37° C. for 1 hour. After incubation, centrifugation (
Cells were collected at 250×g for 10 minutes). Cells were dispersed in 20% FC8 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 bottom culture plate and cultured stationary at 37°C in the presence of 5% carbon dioxide gas. . After 4 days, 0.1+nl of 20% FC5 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-E serotype P. pyogenes human antibodies in the culture supernatant was determined using DIB, an enzyme-linked immunosorbent assay.
Measured using method A. 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 E 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 cells/ml was prepared, and 0.1 ml of this cell suspension was mixed with 0.3% agarose (G-Plaque agarose, FMC Corporation)
was added to 30 ml of culture solution containing the following ingredients and mixed. Next, 3 ml of a 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 a 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 C3 culture solution was transferred to each well of a 96-well flat bottom culture plate and cultured. 20% FC5 culture solution 0 after 2 days
．． After 2 days, the presence or absence of an anti-Pseudomonas aeruginosa monoclonal antibody in the culture supernatant 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 in which the anti-Aeruginosa 1 antibody activity in the culture medium was high were sequentially expanded and cultured to obtain an anti-E serotype P. aeruginosa human IgM-producing EB virus-transformed cell colony 6Z26AN6.

(5) Cell fusion of 2.5 x 107 anti-E serotype P. pyogenes human Ig each
M-producing EB virus transformed cell colony 86Z26A
Example 1 using N6 and MP4109. Cell fusion was performed in the same manner. The fused cells were added to 1 (lXl0 in A-0 medium)
A cell suspension with a density of 0.1 cells/ml was seeded in 0.1 ml per well of a 96-well flat bottom culture plate (480 wells in total). Cells were grown in the presence of 5% carbon dioxide for 3
It was statically cultured at 7°C. After 4 days, add 0.1 ml of HA-0 culture solution, then add half of HA-0 every 4 to 5 days.
The medium was replaced with fresh HA-0 medium. For 21 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. IF2.3A6.3H11,
Antibody activity was observed in the culture supernatant of 4 wells of 4B4. This was cloned and the hybridoma MP5 was created, which produces a human monoclonal antibody that reacts only with E serotype Pseudomonas aeruginosa.
Three strains, 140, MP 5141, and MP 5143, 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 5140, MP 514], MP
5143, 10'' cells secreted 54 μg, 53 μg, and 13 μg of human cells into the culture supernatant in 24 hours, respectively.

(7) Passage stability of cell lines Except for using MP 5140: Example 1. (5
) The proliferation of the cell line and the stability of the antibody production amount were measured in the same manner as described in (1).

HP 5140 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 24.5 hours. Also, 106
The IgM secreted by each cell in 24 hours was 54 μg at the start of culture, 45 μg after 1 month, 40 μg after 2 months, and 38 μg after 3 months, and during subculture for 3 months. There were no significant changes in antibody production.

HP 5140 was deposited with the Institute of Fine Technology as Article No. 2273. Example 4. Cell culture and antibody purification-2MP 51
40 cryopreserved cells were raised and expanded using 10% FC5 culture medium, and after further expansion culture in NYSF 404 serum-free medium, 50 ml of NYSF 404-free cells were grown to a cell density of 5 x 104 cells/ml. Suspended in serum medium.

After seeding in one flask (bottom area: 175a#), the seeds were statically cultured at 37° C. for 5 days in the presence of 5% carbon dioxide gas. Collect the cells and divide them into cells at a density of 5 x 10' cells/50 cells.
The cells were suspended in 0 ml of NYSF404 serum-free medium and seeded in one Starling culture flask (Techne). 5%
Culture was carried out at 37° C. for 5 days with stirring at 20 rpm in the presence of carbon dioxide gas. Centrifuge (400X
g for 20 minutes) to obtain a 480 ml supernatant, which was filtered through a membrane filter with a pore size of 0.22 microns.

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

13.2 mg of IgM (Ni) was extracted from 480 ml of culture supernatant.
l-1) A solution was obtained.

Example 5. Test of protective activity of human monoclonal antibodies against Pseudomonas aeruginosa infection The human monoclonal antibodies N1-5 and Ni1-1 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 were treated with 50 ng of human monoclonal antibody per mouse, 5
0.2ml of a solution containing 00ng, 5μg, 50μ& was administered intraperitoneally, and 2 hours later, Pseudomonas aeruginosa E serotype (PA 103
) was challenged intraperitoneally. In the control group, only physiological saline was administered instead of human monoclonal antibody. PA 103 was seeded on heart infusion agar plates and cultured overnight at 37°C.

The grown bacterial colonies were scraped off, diluted with physiological saline, and 5% mucin was added to prepare a challenge bacterial mass 13.5 times the 50% lethal dose (LDsa value) per mouse. It was made into a bacterial solution. The 50% effective dose (
ED5o value) was determined. N1-5, ED of Ni1-1,
, o values were 0.135 μg and 0.39 μg, respectively. Each human monoclonal antibody had high protective activity against infection with Pseudomonas aeruginosa of serotype E.

Example 6. Preparation of liquid N1-5 obtained in Example 2 and Ni obtained in Example 4
1-1 at 1 mg/ml and human serum albumin (Calbio) at 0.2% (w/v) were prepared in PBS (-) and filtered using a membrane filter with a pore size of 0.22 microns. Sterilized and filtered. 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 E serotype Pseudomonas aeruginosa LPS.
It was determined by measuring by LISA method.

Antibody titer was measured as follows.

E serotype Pseudomonas aeruginosa LPS was added to 0.1M citrate buffer (pH
4°0) (2 μg/ml), 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,
``Adinobis (3-ethylbenzthiazoline sulfonic acid) (Sigma) was used as a substrate to develop color, and the absorbance at a wavelength of 414 nm was measured.The dilution ratio at which the absorbance was 0.1 was calculated using the least squares method. This was defined as the antibody titer.

N1-5 and N left at 4℃ or 37℃ for 1 month
The antibody titer of the solution of i1-1 was no different from that of the control solution stored at 80° C., and the antibody activity was maintained.

Example 7. Preparation of freeze-dried preparation N1-5 obtained in Example 2 and Ni obtained in Example 4
1-1 at 1 mg/ml and human serum albumin (Calbio) at 0.2% (en/v) in PBS (-) and filtered using a Membrane filter with a pore size of 0.22 microns. Sterilized and filtered. Each antibody was separately aseptically dispensed at 1 ml per vial and lyophilized to prepare a lyophilized preparation. The antibody titers against E serotype Pseudomonas aeruginosa LPS were measured in the same manner as in Example 6. There was no difference in antibody titer from that of the 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 human monoclonal antibodies reactive with serotype Pseudomonas aeruginosa E 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 and a cell line derived therefrom that produce a human monoclonal antibody that is reactive with at least E serotype Pseudomonas aeruginosa. 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 JYOYO NO. 2272 or JYOYO NO. 2273 and cell lines derived therefrom. 9. Method for producing human-human hybridomas according to paragraphs 1 to 8; 10. Cultivating at least one of the human-human hybridomas described in paragraphs 1 to 8 and cell lines derived therefrom; A method for preparing a human monoclonal antibody reactive with E serotype Pseudomonas aeruginosa, which comprises purifying it from a culture thereof. 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.