JP2850293B2 - γ-interferon sensitive disease agent - Google Patents

Description

Description: BACKGROUND OF THE INVENTION [0001] The present invention relates to a humanized culture.
Myelomonocytic cells (myelomonocyte)
-Cell) is effective in producing γ-interferon.
Γ-interferon sensitive disease prophylactic agent
Relates to therapeutic agents. 2. Description of the Related Art Interferons are described in
Tarferon ", published by Kodansha Co., Ltd. (1975),
D. A. J. Tyrrell, "Interferon
And It's Clinical Potential (Inte
rferon and it's Clinical Po
tenial) ", William Heinema
nn Medical Books Ltd. , Lon
don (1976), "Protein Nucleic Acid Enzyme", 2nd ed.
It is also described in Volume 1, Issue 4 (1976), etc.
Sea urchin, virus, bacteria, protozoa, rickettsas, nucleic acids, en
Produce interferon inducers such as dotoxins and polysaccharides
By acting on cells, the induced
Glycoproteins to be formed, and various viruses in the cells
Substances that have the function of non-specifically inhibiting the growth of
Name. Interferon has such a machine
Noh, interferon has been in use since the beginning of its discovery.
It has been expected as a prophylactic or therapeutic agent for lupus diseases. Ma
In recent years, if interferon was only a viral tumor,
And has antitumor properties against non-viral tumors
As a result, interferon as a drug is
It came to be. Interferon includes α-interferon
Feron (also known as leukocyte interferon, lymphoblast)
Cell-like interferon), β-interferon (separate
Name, fibroblast interferon) and γ-inter
Feron (also known as immune interferon, type II
Interferon), of which α-interfero
As for β-cells, from β-cells and lymphoblastoid cells, β-
Production of interferon from fibroblasts, etc.
Methods have been established, and recently pharmaceuticals utilizing these have become commercially available.
It was reached. On the other hand, γ-interferon
Although many manufacturing methods have been proposed,
It has not been implemented industrially. [0003] For example, Japanese Patent Application Laid-Open No. 57-58891,
JP-T-57-500961, JP-T-58-502
032, JP-A-59-82092, JP-A-59-82092
60-70099, JP-A-60-87300
Report, JP-A-60-139700, JP-A-60-1
From human peripheral blood proposed in Japanese Patent No. 49600
The method using leukocytes and T-lymphocytes in
It is difficult to supply large quantities in a stable manner, and
Production is also insufficient. Also, Japanese Patent Application Laid-Open No. 55-981
No. 18, the method proposed in Japanese Patent Application Publication No.
Transplantation of non-human-derived cells into warm-blooded animals
Or mounted inside or outside of a warm-blooded animal other than human
In the diffusion chamber, receive the supply of the body fluid of the warm-blooded animal.
And γ-
This is a method for producing interferon,
It is extremely excellent in that it can supply a large amount of incoming cells stably.
I have. [0004] However, this method involves culturing.
Depending on the type of human-derived cell line,
-It was found that there was a change in
Improved still to produce highly active γ-interferon
And still have to be implemented industrially
Absent. γ-interferon has a cytostatic effect,
Tumor action is α-interferon or β-interfero
Is stronger and stronger than
In combination with these, their antiviral action,
Is known to enhance cell growth suppression and antitumor effects
It is strongly desired to establish an industrial production method.
You. [0005] The present inventors have found that
For producing gamma-interferon that can be easily carried out on a large scale
For the purpose of establishing a method, various human cultures
Original cells, especially various lymphoid buds derived from human cultures
Comparative study on gamma-interferon producing ability of spheroid cells
Γ-interferon is converted to γ-
As a preventive and therapeutic agent for interferon-sensitive disease
We studied diligently whether it was useful or not. [0006] As a result, unexpectedly,
Cultured human bone marrow monocytic cells are
High gamma-interferon production unlike pablast-like cells
It has the viability and is suitable as a cell for γ-interferon production
Suitable for the myeloid cells,
Γ-interferon obtained from γ-interferon
That it is an excellent preventive and therapeutic agent for
Upon confirmation, the present invention has been completed. [0007] The human-derived bone cultured in the present invention.
Medullary monocyte cells are published by Iwanami Shoten, Tadazo Kishimoto, Takeshi Watanabe
Ed., “Iwasho Lecture on Immunology 3 Immunocompetent Cells”, 181
~ Page 204 (1986) and Mikio Shik
ita and Isao Yamane, "Mamari
Ann Cell Culture Technology (Mammal
ian Cell Culture Technology
gy) ", 141-162 (1985), Soft
Science Publications, Tok
yo, Japan, etc.
Cells that do not belong to the cells or B-cells,
More myelomonocytic antigens
cells that are identified by indicating the presence of
U. For example, HB newly established by the present inventors has
L-38 cells, HL- described in the above-cited references.
60 cells, KG-1 cells, ML-1 cells, ML-2 cells
Vesicles, ML-3 cells, THP-1 cells, U-937 cells,
Further, "Gann", Vol. 75, 660-6
CTV-1 cells reported on page 64 (1984)
And the like can be used as appropriate, but especially HBL-38 cells
Has a high ability to produce γ-interferon,
It can be used advantageously. [0009] The γ-interferon of these cells
Genes capable of producing, for example, polyethylene glycol
Cell fusion means using virus or Sendai virus
DNA ligase, restriction enzyme (nuclease), DNA
Known gene recombination utilizing enzymes such as limerase
A culture strain that can be more easily subcultured by means, etc.
To further increase the rate of proliferation by introducing into cells
Also further enhances its ability to produce γ-interferon.
Can also be advantageously implemented. [0010] The human origin of the culture used in the present invention
Method for expanding myeloid monocytic cells
Can be. For example, it has the ability to produce γ-interferon.
Human bone marrow monocytic cells
Tissue culture performed in vitro, or γ-interface
Human-derived myeloid monocytic cells capable of producing
Transplanted into a non-human warm-blooded animal or a non-human
Diffusion chamber attached to the inside or outside of a warm-blooded animal
Implanted in a bar and grown while receiving its fluid supply
And in vivo. First, a case where the cells are grown in vitro is explained.
I will tell. The nutrient medium used at this time is human bone marrow
As long as it can be grown by inoculating spheroid cells, for example,
For example, RPMI 1640 medium, Eagle's minimal basic medium
And vitamins and minerals as needed
Supplements such as carbohydrates, carbohydrates, amino acids and mammalian serum
Can also be improved. Culture methods include monolayer culture methods.
Alternatively, a suspension culture method can be appropriately selected. The culture temperature is about 20
-40 ° C, preferably about 35-38 ° C,
About one week after seeding, you can see the maximum cell growth
Number of cells per ml of medium, preferably
About 10 ^Four -10 ⁷ Individual. The medium inoculated with the cells
Culture under the conditions for about 4 to 10 days.
Replace with fresh ones and supply enough nutrients,
Wash or dilute metabolites released into the medium for growth
It is desirable to make it. Next, a method for growing cells in a living body will be described.
Will be described. In this method, γ-interferon production
Non-human warm-blooded human bone marrow monocytic cells
Implanted in the animal or provided with the body fluid
Housed in an accessible chamber for normal breeding
Body fluid containing nutrients supplied from the body of a warm-blooded animal
Because the cells can be easily proliferated using
Includes expensive serum such as tissue culture in vitro
Large amounts of nutrient medium without or with significant savings
γ-interferon can be produced. That is, a warm-blooded animal other than a human is used.
The method should be easy to maintain during cell growth
Cell growth compared to in vitro culture
Is stable, and γ-interface
Increased production of lon, especially 2 to 10 times,
Is extremely advantageous because it is even higher. This way
Warm-blooded animals used for human
Whatever can be bred, for example, chicken, pigeon
Which birds, dogs, cats, monkeys, goats, pigs, cows, cormorants
Ma, rabbit, guinea pig, rat, hamster, ordinary ma
A mammal such as a mouse or a nude mouse can be used. This
It is preferable to transplant human-derived myeloid monocytic cells into these animals.
May cause an unwanted immune response.
To keep as little as possible, we can use animals
Young, i.e. eggs, embryos, fetuses, or neonatal, young
Are preferred. [0015] In addition, for example, about 200 to
Irradiate X-ray or gamma ray of about 600 rem
Or inject antisera or immunosuppressants
Pretreatment, such as to reduce the immune response
You may. If the animal used is a nude mouse,
Even if the prolonged one has a weak immune response,
Human-derived cultures without the need for treatment
Especially because bone marrow monocytic cells can be transplanted and rapidly proliferated
It is convenient. In addition, human-derived bone marrow monocytes cultured as a culture
For example, first, transplant the lineage cells into hamsters and allow them to proliferate.
After transplanting these cells into nude mice,
As described above, human-derived bone is transplanted between non-human warm-blooded animals.
Stabilize the proliferation of medullary monocytic cells, or
Increases the amount of γ-interferon produced by induction
You are free. In this case, of course,
That is, the same class, same class transplant may be used. Of human origin
The site in the animal body where the bone marrow monocyte cells are to be transplanted
Any site where cells can proliferate, for example, urine cavity,
Vein, peritoneal cavity, subcutaneous, etc. are freely selected. In addition, human bone marrow monocytes can be directly injected into an animal body.
Can prevent the passage of animal cells without transplanting lineage cells
Porous filtration membrane, for example, having a pore size of about 10 ^-7 -10 ^-Five m
Having a membrane filter, ultrafiltration membrane or hollow
Diffusion of various known shapes and sizes with fibers etc.
The chamber is embedded in the animal body, for example, in the abdominal cavity, and
While receiving a supply of body fluids containing nutrients from the object,
Human-derived bone marrow monocytes cultured as described above in Chamber
Any of the lineage cells can be grown. Also, if necessary,
Connect perfused solutions with nutrients to body fluids in the animal
Such a chamber, for example, attached to the body surface,
Proliferation status of human bone marrow monocytic cells in the chamber
Being able to see through, this chamber part
Allows only parts to be removed and replaced, without sacrificing animals
Proliferate full-life cells to increase cell production per animal
It can be even higher. Method using these diffusion chambers
Indicates that human-derived bone marrow monocytic cells come into direct contact with animal cells.
No, only human bone marrow monocytic cells can be easily collected
Worry not only of possible but also of unwanted immune response
Pretreatment to suppress the immune response is not necessary.
It has the characteristic that various warm-blooded animals can be used freely.
You. The maintenance of the transplanted animal is based on normal breeding of the animal.
No special handling is necessary even after transplantation
This is convenient. Human bone marrow monocytic cells
Is usually about 1 to 10 weeks.
Target can be achieved. The thus obtained human bone marrow alone
The number of spheroid cells is about 10 per animal ⁷ -10 ¹² Individual
Reach even more. In other words, non-human warm blood
-Derived bone marrow monocytes grown by the method using
The number of lineage cells is about 10 times the number of cells transplanted per animal. ^Two ~
10 ⁷ Different times or more, in vitro nutrient media
About 10 when inoculating and growing ¹ -10 ⁶ Double or
More than that, the production of γ-interferon
It is very convenient for Human-derived bone marrow grown in this way
Producing γ-interferon using live monocyte cells
The method is free. It remains in the animal body where it proliferated
It is also possible to cause a γ-interferon inducer to act.
Wear. For example, human
Tumor cells arising from or subcutaneously in native myeloid cells
Γ-interferon inducer is allowed to act directly on
Induced production of interferon and then its ascites
Or purifying and collecting γ-interferon from tumors
Good. In addition, human bone marrow monocytic cells
Removed from inside, ex vivo γ-interferon inducer
To induce and produce γ-interferon
Also included. For example, bone marrow isolated from human grown in ascites
Human bone marrow obtained by sorting spheroid cells or subcutaneously
Cells obtained by removing and dispersing tumors containing monocyte cells
In a nutrient medium maintained at about 20-40 ° C.
0 ^Five -10 ⁸ Cells / ml, and γ-a
By acting an interferon inducer, γ-I
Interferon is induced to be produced, purified and collected.
I just need. Further, human bone marrow monocytic cells are
When growing in a chamber, the grown cells should be
Leave in chamber or remove from chamber
Γ-interferon can be induced to produce
You. In addition, for the induced production of γ-interferon,
If necessary, for example, an animal with high species specificity
Priming using Interferon
Public induction such as the super induction method using
By adopting the method of knowledge, the generated γ-inter
-You can freely increase the amount of feron. In addition, for example, expanded human bone marrow
First, the γ-interfering cells were added to monocyte cells in the animal body.
After induced production of the same animal,
Human bone marrow monocytic cells collected from the site or whole
To induce the production of γ-interferon outside the animal body
Method, and once used to induce the production of γ-interferon.
The cells used were further induced at least twice for γ-interferon.
The method used for conduction generation, or burying or
Or the chamber to which it is connected
Animal individuals to be used by methods such as increasing
Γ-interferon production per unit
Be free. As the γ-interferon inducer, there are
Usually, for example, phytohemagglutinin, concanavalin A,
Pork weed mitogen, lipopolysaccharide, lipi
Mitogens such as de-A, endotoxins, polysaccharides and bacteria
Is preferred. Also, for sensitized cells,
Gen is also a gamma-interferon inducer. These γ-a
When an interferon inducer is used, usually about 0.0
Used at a concentration of 01 μg to 10 mg / ml. necessary
For example, viruses, nucleic acids, polynucleotides, etc.
Γ-interferon inducer
-Further increase in ferron production can also be
-Feron and γ-interferon simultaneously
You are free to do it. The γ-interface thus induced and generated
-Feron can be purified and separated by known methods such as salting out,
Analysis, filtration, centrifugation, concentration, freeze-drying, etc.
Therefore, it can be easily purified, separated, and collected. Change
In cases where a high degree of purification is required, for example,
Adsorption / elution to exchanger, gel filtration, affinity chromatography
Chromatography, isoelectric focusing, high-performance liquid chromatography
Well-known methods such as electrophoresis
Chromatography using monoclonal antibodies
The highest purity γ-interfero
Can also be collected. The thus obtained γ-interface
Is a prophylactic and / or therapeutic agent for gamma-interferon sensitive disease.
It can be used advantageously as a remedy. γ-interfero
-Sensitive disease is prevented by γ-interferon
Or a disease to be treated, which is a viral disease
Patients, for example, epidemic hepatitis, herpes keratitis,
Ruenza, rubella, serum hepatitis, AIDS, etc.
Non-viral diseases such as colon cancer, lung cancer, liver cancer,
Malignant tumors such as osteosarcoma, as well as atopic allergies
ー, myasthenia gravis, collagen disease, pernicious anemia, rheumatoid arthritis,
Even for immune diseases such as systemic lupus erythematosus
Good. [0029] In addition, γ-interferon sensitive disease
The shape of the inhibitor or therapeutic agent depends on its purpose.
You can choose freely. For example, sprays, eye drops
Agents, gargles, liquids such as injections, ointment-like pace
And solid preparations such as tablets, powders, granules and tablets. this
Γ-interferon is used for prophylactic and therapeutic agents.
Usually contains about 1 to 10,000,000 units per gram
Γ-interferon if necessary
Together with other lymphokines such as α-interfering
Ron, β-interferon, Tsumore necrosis
Actor, lymphotoxin, interleukin 2, B fine
Lymphokases other than γ-interferon such as vesicle differentiation factor
And other natural or synthetic chemotherapeutic agents.
More than one kind or two or more to prevent and treat
The increase can also be carried out advantageously. If necessary, auxiliaries, extenders, stabilizers
It is optional to use one or more of these in combination.
You. The γ-interface of the present invention thus produced
For example, prophylactic and therapeutic agents for
As anti-tumor agents and antitumor chemotherapeutic agents
Anti-tumor effect enhancer, malignant tumor metastasis prevention, recurrence prevention agent,
It can be used advantageously as an immunomodulator, therapeutic agent for immune diseases, etc.
You. Interferon highly species-specific to humans
The activity is described in "Protein Nucleic Acid Enzymes", Vol. 20, No. 6,
Humans reported on pages 616-643 (1975)
A well-known plaque half-life method using FL cells derived from amniotic membrane
It was measured. Note that the activity of γ-interferon is similar to that of anti-α
-Interferon antibody and anti-β-interferon anti
Coexist with α-interferon and β-interferon
After neutralization of -feron, the measurement was performed. The hemagglutination value is determined according to J. E. FIG. By Salk,
"The Journal of Immunology (The Jo
urnal of Immunology) ", No. 49
Vol., Page 87 (1944). DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a bone newly established according to the present invention will be described.
HBL-38 cells, which are medullary monocytic cells, will be described.
You. Acute myeloid leukemia patient (male 55 years old)
Culture of these leukocytes in an in vitro nutrient medium
As a result, cell proliferation was observed 21 days later. It
Subculture and ensure that one of them is grown stably.
This was named HBL-38 cells. <(1) Proliferation ability> Fetal bovine serum 10 v / v%
Growth ability in RPMI 1640 medium supplemented with
However, the doubling time was about 30 hours. <(2) Form> The bottom of the flask during growth
Had the property of adhering to
Released. In addition, the formation of cell clumps was observed during proliferation,
It is not strong and can be easily separated into single cells when touched lightly.
Was scattered. Observation of these cells with a phase contrast microscope
As shown in FIG. The morphology of the cells is approximately 15 μm
It was round. Nuclei are circular as a result of Giemsa staining
In addition to those that show irregular cutting and lobing tendency,
Admitted. <(3) Number of chromosomes>
Cells in several growth phases were used. Table showing the frequency distribution of chromosome numbers
1 is shown. As a result of observing 150 cells,
The number of chromosomes is in the low diploid region, and its frequency distribution is 45 lines.
The largest number was 53. In addition, 44 cells are 42 cells
Admitted. [Table 1] <(4) Karyotype Analysis> The results of the karyotype analysis are shown in FIG.
2 is shown. The sex chromosome of the cell is XY,
And matched. Chromosome No. No. 17 and No. 17 18
Was all missing. No. No. 5 short arm (p) and No. 5
Chromosome insertion was observed in 12 long arms (q). Also,
One unidentifiable marker monochromosome and one chromatid
Admitted. <(5) Cell Surface Traits> Various Cell Surface Antibodies
Table 2 shows the results of identification of HBL-38 cells using
It was shown to. Sheep red blood cells (E), antibody-sensitized bovine red blood cells
(EA), using human complement-sensitized bovine erythrocytes (EAC)
Analysis showed that EA had 10% rosette formation,
No other was found. Using goat anti-human antibody
As a result of detecting cell surface immunoglobulin,
All species were negative. Also, use monoclonal antibodies
As a result of the search for the surface marker, 3A1, MCS-2,
B3 / 25, MY-9 show high positive rate, NU-T
2, Leu-5, Leu-4, A-50, BA-2, O
KT-1, NU-N1, B2, MO-1, MO-2 are:
All were negative. [Table 2] <(6) EB virus-specific nuclear antigen (EBN)
A) Search> For EBNA, early after cell line establishment
Searches were performed several more times and were always negative. <(7) Colony formation in soft agar medium>
In 0.3% agar medium containing colony forming factor (CSF)
Colony formation was tested on an inverted microscope on day 14 of culture.
Results in the formation of myeloid-like colonies
Cells were observed. Their frequency is 1-2%
Was. If no colony forming factor is added, no
won. From the above results, it was found that HBL-38 cells
It was found to belong to the medullary monocytic cells. Next, γ-in
Experiment I for the production of terferon is described. [Experiment I] <γ of various lymphoblastoid cells derived from human cultures
-Comparison of interferon-producing ability [Experiment I-1] <Production of interferon by cells grown in vitro>
Raw> RPMI 16 supplemented with fetal calf serum at 20 v / v%
40 medium (pH 7.2)
Various cells are inoculated, respectively, and cultured at 37 ° C according to a conventional method.
And then RPMI 1640 medium without serum
(PH 7.2), and washed with the same medium at a concentration of 1 × 10 ⁶ Individual
/ Ml. The various human-derived cell suspensions thus obtained
In each of the suspensions, about 10 ml of lipopolysaccharide was added per ml.
μg and keep at 37 ° C for 2 days
And centrifuged.
Interferon activity and γ-interferon activity
Was measured. Table 3 summarizes the results. [Table 3] As is clear from the results in Table 3, the culture
Of various human lymphoblastoid cells
Comparison of feron production ability shows that all
Unexpected production from myeloid monocytic cells
However, it was also found that the production amount was large. Bird
That is, HBL-38 cells produce γ-interferon.
The performance is remarkably high and can be advantageously used in the present invention. [Experiment I-2] <Production of interferon by cells grown in vivo
Raw> A newborn hamster is known from rabbits.
Immunization of hamsters
And then subcutaneously cultured human-derived bone marrow
Each monocyte cell is transplanted, and then
They were raised for 3 weeks. Subcutaneous tumor was removed and minced
After that, suspend cells in trypsin-containing saline to disperse cells.
And took it out. Each of the obtained cells was subjected to Experiment I-1.
A suspension was prepared in the same manner as described above, and the activity was measured in the same manner. as a result
Are summarized in Table 4. [Table 4] As apparent from the results in Tables 3 and 4,
In addition, human bone marrow monocytic cells
In addition, HBL-38 cells are better than cells grown in vitro.
Also, cells grown in vivo have significantly higher γ-
It was found to show interferon production. Hereinafter, a production example of γ-interferon will be described.
This is shown as Example A. Example A-1 HBL-38 cells were calfed with 10 v of calf serum.
/ V% supplemented RPMI 1640 medium (pH 7.2)
5 × 10 cells ^Five The cells were inoculated so as to obtain the number of cells / ml.
Then, regularly replace with fresh medium according to the usual method.
While culturing at 37 ° C, then washing with fresh same medium
And 2 × 10 ⁶ Suspended to be pieces / ml
did. Add about 10 μl of lipopolysaccharide per ml to this
And keep at 37 ° C for 2 days to induce interferon
Led. This was centrifuged, and about
5,100 units of γ-interferon were obtained. Example A-2: Newborn hamsters were released from rabbits
Inject the antiserum prepared by known methods in advance and
After weakening the immune response, HBL-38 cells were transferred subcutaneously.
After planting, they were reared for 4 weeks in the usual manner. Subcutaneously
After removing about 20 g of the resulting tumor, collagenase containing
The cells were suspended in physiological saline, dispersed, and sorted. this
After washing the cells with Eagle's minimal basal medium,
The cell concentration was about 2 × 10 ⁶ Individual
/ Ml of phytohema per ml.
200 μg of glutinin and 5 μg of lipid A were added, and 3
Interferon was induced at 7 ° C. for 2 days.
This is centrifuged and about 93,000 units per ml of supernatant
γ-interferon was obtained. Hamster-about 1 per animal
83,000,000 units of γ-interferon were obtained.
Was done. Example A-3 Intravenous injection of KG-1 into newborn rats
After transplanting the cells, they were bred for 4 weeks in the usual manner. leather
After removing about 20 g of the underlying tumor, Example A-2
Was dispersed in the same manner as described above to obtain a cell suspension. To this, ml
About 100 hemagglutination titers and
Add about 5 μl of polysaccharide and keep at 37 ° C for 2 days.
To induce interferon. Centrifuge this
And about 49,000 units of γ-inter
I got Feron. About 97,000,000,00 per rat
0 units of γ-interferon were obtained. Example A-4 Membrane having a pore size of about 0.5 micron
A plastic circle with a filter capacity of about 10 ml
In a cylindrical chamber, float CTV-1 cells with saline.
The chamber is implanted in the abdominal cavity of a grown rat.
Established. The rats were bred for 4 weeks in the usual way
Later, the chamber was taken out. This cell was used in the Example
The same treatment as in A-1 was conducted to induce interferon.
Was. This was centrifuged, and about 41,000
Units of γ-interferon were obtained. Per rat,
About 78,000,000 units of γ-interferon
Obtained. Example A-5 Receiving of chicken kept at 37 ° C. for 5 days
After transplanting HBL-38 cells into sperm eggs,
Kept for a week. After breaking this egg, collect the proliferating cells,
The cells were treated in the same manner as in Example A-2, and
Ron was induced. This was centrifuged and per ml of supernatant
About 36,000 units of γ-interferon were obtained. Receiving
About 60,000,000 units of γ-in per 10 sperm eggs
Terferon was obtained. Next, anti-γ-interferon monochrome
Method for producing null antibody and high-purity γ-in using the same
A production example of terferon is shown as Example B. Example B-1 <(1) Preparation of partially purified γ-interferon>
Contains γ-interferon prepared by the method of Example A-2
The solution is pH 8.5, 0.01 M Tris hydrochloride buffer
Dialysis for 20 hours, and the filtrate obtained by microfiltration
α-interferon antibody and anti-β-interfero
Flow through the antibody column with immobilized antibody
And then apply it to the chromatofocusing method.
The antivirally active fraction was collected, concentrated, and lyophilized.
Γ-interferon containing powder,
Obtained at about 30%. The specific activity of this product is about 10 ⁶ Unit / mg
It was a protein. <(2) Anti-γ-interferon monochrome
Preparation of internal antibody> Part obtained by the method of Example B-1 (1)
Purified purified γ-interferon in physiological saline
Dissolve so that the concentration becomes about 0.05 w / v%.
And Freund's complete adjuvant emulsion are mixed in equal amounts
0.2 ml of this mixture was injected subcutaneously into mice,
One day later, the mice were immunized with the same injection. The antibody
Induce anti-γ-interferon antibody to cells capable of producing
The spleen is excised from this mouse and dissociated
Spleen cells and mouse myeloma cells P _Three -X ₆₃ −
Ag8 (Flow Laboratories) and
Was prepared with 50 w of Eagle's minimal basal medium without serum.
/ V% polyethylene glycol-1000 solution (pH
7.2, temperature 37 ° C) ^Four Pcs / ml
And keep it for 5 minutes.
Dilute 0-fold and then Davison etc.
, Somatik Cell Genetics (Somat
ic Cell Genetics), Volume 2, 175
~ 176 (1976)
And hypoxanthine-aminopterin-thymidine culture solution
A fused cell that can grow in
-Select fusion cells with the ability to produce interferon antibodies
did. The obtained fused cells were intraperitoneally injected into the mouse in an amount of about
10 ⁶ After transplanting and rearing for 2 weeks, this was killed
Collect body fluids such as ascites and blood, centrifuge and collect the supernatant.
Ammonium sulfate salting out, collecting a precipitate fraction having a saturation degree of 30 to 50%,
Then, the solution was dialyzed, and the solution was further subjected to Example B-1 (1).
Γ-interferon obtained by the method of
Immobilized γ obtained by reaction with immobilized Sepharose at room temperature
-Affinity chromatography using interferon gel
Performed chromatography, anti-γ-interferon antibody fraction
, Dialyzed, concentrated, freeze-dried and powdered γ-
Interferon monoclonal antibodies were collected. The product is a human γ-a derived from bone marrow monocytic cells.
Neutralizing activity immunologically specific for interferon activity
Showed sex. Stability of this monoclonal antibody in aqueous solution
As a result of examining the neutralization activity by measuring the neutralization activity, at pH 7.2
80% or more activity at 60 ° C under the condition of holding for 30 minutes
Remained, and at 70 ° C., 90% or more of the activity was lost. Further, under the condition of holding at 4 ° C. for 16 hours, p
H is stable in the range of 4.0 to 11.0 and at pH 2.0
Lost more than 90% of its activity. Furthermore, this monochrome
As a result of examining the properties of the internal antibody, 2-mercaptoethanol
Unstable, anti-mouse immunoglobulin M antibody
And a specific antigen-antibody reaction. Therefore, this monoclonal antibody is
It is an antibody classified into the noglobulin M class. <(3) Highly Purified γ-Interface
Preparation of Heron> Part prepared by the method of Example B-1 (1)
The partially purified γ-interferon was prepared according to Example B-1 (2).
Gel immobilized with monoclonal antibody prepared by the method
Column chromatography using
Collect the active fraction of feron, dialyze, concentrate and freeze-dry
Γ-interferon solid with an activity yield of about 80%
Obtained. This product is highly purified γ-interfero
And its generality is about 1.5 × 10 ⁷ Unit / mg
It was a protein. Example B-2 <(1) Preparation of partially purified γ-interferon>
Contains γ-interferon prepared by the method of Example A-3
The solution was partially purified according to the method of Example B-1 (1).
And a specific activity of about 10 ⁶ Γ-interface of unit / mg protein
Was obtained in a yield of about 20%. <(2) Anti-γ-interferon monochrome
Preparation of internal antibody> Part obtained by the method of Example B-2 (1)
Except that purified γ-interferon was used as the antigen.
Mice were immunized as in Example B-1 (2) and spleen cells were
Obtained. This spleen cell and mouse myeloma cell P3-NS-1
/ 1-Ag4-1 (Dainippon Pharmaceutical Co., Ltd.) and 14
0 mM NaCl, 54 mM KCl, 1 mM NaH
_Two P0 _Four , 2 mM CaCl _Two To a salt solution containing
10 each ^Four Float so that the number of cells / ml,
Before containing Sendai virus previously inactivated by ultraviolet light
The salt solution was mixed under ice-cooling, and the mixed solution was added 3 minutes after 5 minutes.
Dilute approximately 20-fold with RPMI medium at 7 ° C, then run
Anti-γ-interferon anti-antibody in the same manner as in Example B-1 (2)
Fusion cells with somatic productivity were selected. The obtained fused cells were immunized by a known method.
Per animal in the abdominal cavity of 7-day-old hamsters
About 10 ⁷ An individual was transplanted, and a module was transplanted in the same manner as in Example B-1 (2).
Noclonal antibodies were collected. This product was obtained from the model prepared in Example B-1 (2).
Γ-interferon activity
Showed immunologically specific neutralizing activity. This monoc
The stability of a nal antibody in aqueous solution is determined by measuring its neutralizing activity.
As a result of the examination by the measurement, the condition maintained at pH 7.2 for 30 minutes
In this case, 80% or more of the activity remains at 60 ° C.
Over 90% of the activity was lost. 16 hours at 4 ° C
Under the condition of keeping, stable in the range of pH 2.0 to 11.0
Met. Further, the properties of this monoclonal antibody were examined.
As a result, it is stable to 2-mercaptoethanol,
Mouse immunoglobulin G antibody and specific antigen-antibody reaction
It turned out to show. Therefore, this monoclonal antibody
Is an antibody classified into the immunoglobulin G class.
You. <(3) Highly Purified γ-Interface
Preparation of Heron> Part prepared by the method of Example B-2 (1)
The partially purified γ-interferon was prepared according to Example B-2 (2).
Gel immobilized with monoclonal antibody prepared by the method
Column chromatography using
-Collect the active fraction of feron, dialyze, concentrate and
A γ-interferon solution was obtained with a yield of about 85%. This product
Is highly purified γ-interferon.
And its specific activity is about 1.5 × 10 ⁷ Unit / mg protein
Met. Example B-3 The γ-a obtained by the method of Example A-1
The supernatant containing interferon was purified to pH 7.2, 0.0
Permeate for 15 hours with saline containing 1M phosphate buffer
The filtrate obtained by precipitation and further microfiltration was subjected to Example B-1.
Purify using an antibody column according to the method of (3) and concentrate
And freeze-dried to give a gamma-
A solid was obtained. This product is highly purified γ-in
Terferon, whose specific activity is about 1.5 × 10
⁷ Unit / mg protein. Example B-4 The γ-a obtained by the method of Example A-4
Interferon-containing supernatant was purified by the method of Example B-3.
The filtrate obtained by dialysis and microfiltration according to
Purification using an antibody column according to the method of B-2 (3)
Γ-interfero
Solution was obtained. This product is highly purified γ-internase.
-Feron having a specific activity of about 1.5 × 10 ⁷ single
Position / mg protein. Example B-5 The γ-a obtained by the method of Example A-5
Interferon-containing supernatant was purified by the method of Example B-3.
The filtrate obtained by dialysis and microfiltration according to Example B was used.
-1 Purification using an antibody column according to the method of (3),
Concentrate and freeze-dry to obtain a γ-interactant with an activity yield of about 70%.
-Feron solid was obtained. This product is highly purified γ
-An interferon, whose specificity is about 1.5
× 10 ⁷ Unit / mg protein. Next, γ-a by γ-interferon
Experiment I on prevention and treatment of interferon-sensitive disease I
I will be described. [Experiment II] <γ-interferon feeling by γ-interferon
Test for Prevention and Treatment of Receptive Disease> [Experiment II-1] <In Vitro Inhibition of Virus Proliferation> Diameter 6
primary culture of human fetal lung cultured in a single-layer culture dish
Γ-internatants prepared by the method of Example B-1 (3)
Add 0.1, 1.0 or 10.0 units of ferron
And in a 5% carbon dioxide gas incubator at 37 ° C for 20:00
After maintaining for a while, add
A total of about 100 plaque-forming varisas
Zostar virus (varicella-zoster virus), or
Human cytomegalovirus (virus that causes stillbirth and premature birth)
The number of plaques generated by adding was counted. The inhibitory effect on virus growth was determined by the γ
-The rate of plaque number reduction (%) by interferon
It was judged in degrees. ## EQU1 ## Table 5 shows the counting results. [Table 5] As is clear from the results in Table 5, in the present invention,
The γ-interferon used causes viral diseases.
It can be seen that the virus
Call [Experiment II-2] <Treatment of malignant tumor with γ-interferon> [Experiment II-2 (1)] <Evil in vitro due to γ-interferon
Suppressive action of primary tumor cells> Containing 15 v / v% fetal bovine serum
Example B-1 (3) in the RPMI 1640 medium having
The final concentration of γ-interferon prepared by the method of
Add it to 5, 50, 500 units / ml and add
In addition, 5 × 10 ^Five Individual/
5% charcoal inoculated to a concentration of 37 ml
The cells were cultured in an acid gas incubator for 3 days. As a control
Is the heat-inactivated γ-internatant kept at 100 ° C for 30 minutes.
-Add the ferrons in equal amounts, and
Was cultured. After the culture is completed,
Orology (Applied Microbio1og)
y) ", Vol. 22, No. 4, pp. 671-677 (197)
1 year) according to the method described in
Live cells are stained with Lalred and the stain is subsequently applied.
Elute with sidoethanol and elute at 540 nm
The amount of living cells was measured from the absorbance. Cell growth inhibition rate (%)
Was calculated by Equation 2. ## EQU2 ## Table 6 shows the measurement results. [Table 6] As is clear from the results in Table 6, the present invention
The γ-interferon to be used is KB cells, HEp-
2 cells, KATO-III cells, P-4788 cells, etc.
Significantly suppresses the growth of malignant tumor cells
It can be seen that the concentration is effective at 5-500 units / ml.
You. [Experiment II-2 (2)] <Others by γ-interferon in vitro
Of lymphokine enhances the inhibitory effect on growth of malignant tumors> The lymphokines used were γ-in
Taferon 5 units / ml, α-interferon
50 units / ml and Tsumore necrosis factor
Was used at 10 units / ml. These lymphokines
Are all natural types derived from lymphoblastoid cells.
Used. The experimental method is the same as that of Experiment II-2 (1).
The cell growth inhibition rate (%) was determined according to the procedure. result
Is shown in Table 7. [Table 7] As is clear from the results in Table 7, γ-in
Terferon is a malignant tumor growth that other lymphokines have
It significantly enhances the inhibitory effect, and the effect is
It shows the synergistic and inhibitory effects of heron. [Experiment II-2 (3)] <In vitro conversion with γ-interferon
Enhancing Effect of Chemotherapeutic Agent on Inhibition of Growth of Malignant Tumor> Prepared according to the method of Experiment II-2 (1)
10 ml of human malignant tumor cells in 1 ml of nutrient medium ⁶ Individual
After culturing for 1 day, the mixture was added to Example B-1.
The final concentration of γ-interferon prepared by the method (3)
Saline containing 50 units per degree and / or chemotherapeutic agent
0.1 ml of water was added, and the cells were cultured at 37 ° C. for 2 days. Contrast
Includes γ-interferon and chemotherapeutic agents
No saline was used. After completion of the culture, experiment II-2
According to the method of (1), the cell growth inhibition rate (%) is determined.
Was. The concentration of the chemotherapeutic agent should be
Nimustine hydrochloride (ACNU) 1.0 × 10 ^-6 g
Luouracil (5-FU) 1.5 × 10 ^-8 g, doxo
Rubicin (ADM) 1.0 × 10 ^-Ten g, mitomaishi
C (MMC) 2.5 × 10 ^-9 g and vincris sulfate
Chin (VCR) 1.5 × 10 ^-Ten g. The result is a table
FIG. [Table 8] As is clear from the results in Table 8, γ-in
Terferon suppresses malignant tumor growth of various chemotherapeutic agents.
Production is significantly enhanced, and its action is
Demonstrates its inhibitory and additive or synergistic effects
You. [Experiment II-2 (4)] <In vivo inhibitory effect on malignant tumor growth> BALB
/ C-derived nude mouse subcutaneously with human breast cancer tissue fragments
Implanted, the tumor volume is about 200mm ^Three When did you become
Et al., The γ-interferon prepared by the method of Example B-1 (3).
Feron alone or with this γ-interferon
Other lymphokines and chemotherapy from lymphoblastoid cells
Daily in a state of being dissolved in physiological saline in combination with
Intravenous injection was performed once for 20 days. Thereafter, the nude mice were sacrificed and the tumor weight was
Was measured. As a control, saline was used.
Was. The results are shown in Table 9. [Table 9] As is clear from the results in Table 9, the in vivo
(In vivo) test also revealed that
Heron significantly suppresses the growth of malignant tumors. Also,
Antiproliferative effect of other lymphokines and chemotherapeutics
Significantly enhanced by combination, showing high antitumor effect
You. [Experiment II-2 (5)] <Acute toxicity test> The test was performed using a 20-day-old mouse.
Γ-interferon obtained by the method of Example B-1 (3)
Was tested for acute toxicity. Very low toxicity, intraperitoneal
LD when injected into ₅₀ Is 10 ⁹ Unit / kg mouse or more
Turned out to be. Hereinafter, the γ-interferon of the present invention has
Examples of the production of a drug contained as an active ingredient were described in Example C.
Shown. Example C-1 <Solution> A solution was prepared in physiological saline by the method of Example B-1 (3).
Prepared γ-interferon at a rate of 500 units per ml.
A liquid preparation was produced by containing them together. This product is used for epidemic conjunctiva
Prevention and cure of viral diseases such as inflammation and influenza
Suitable as a remedy for spraying, eye drops, nasal drops, and gargle
It is. Example C-2 <Injection> In a physiological saline, the method of Example B-2 (3) was used.
The prepared γ-interferon was added to 100,00 per ml.
0 units, and this was aseptically filtered.
Take 2 ml each of the filtrate obtained into a sterilized glass container and freeze
The mixture was dried and sealed to obtain a dry injection. This product is an example
As in the case of C-1, a prophylactic or therapeutic agent for a viral disease
It can be used advantageously. Also, breast cancer, lung cancer, liver cancer, leukemia
As a preventive or therapeutic agent for malignant tumors such as
Allergic, pernicious anemia, rheumatoid arthritis, systemic
As a preventive or therapeutic agent for immune diseases such as rhetmatosus
Are also suitable. Furthermore, melphalan, methotrexate,
Anti-tumor effect enhancer of chemotherapeutic agents such as doxorubicin
It is also convenient to use it. Example C-3 <Injection> Prepared in a physiological saline by the method of Example B-3.
Γ-interferon, natural from lymphoblastoid cells
Type α-interferon and lymphoblastoid cells
Add natural Tsumore Necrosis Factor per ml
10,000 units, 100,000 units and
100,000 units in a ratio of aseptic
And freeze-dried in the same manner as in Example C-2, followed by dry injection.
Agent was obtained. This product is a prophylactic and therapeutic agent for various viral diseases
It is suitable as an agent. In addition, breast cancer, lung cancer, liver cancer, stomach cancer, leukemia, etc.
As a preventive or therapeutic agent for any malignant tumor,
Allergic disease, collagen disease, rheumatoid arthritis, systemic erythema
As a preventive or therapeutic agent for immune diseases such as tematodes
It is suitable. Furthermore, tegafur, mitomycin C, sulfuric acid
Antitumor effect enhancer of chemotherapeutic agents such as vincristine acid
It is also convenient to use it as. Example C-4 <Ointment> γ-a prepared by the method of Example B-1 (3)
Interferon and native α from lymphoblastoid cells
-Use a small amount of liquid paraffin
Γ-interferon per gram of product
50,000 and α-interferon each
Petroleum jelly so as to obtain 500,000 units.
And mixed to obtain an ointment. This product is herpes, skin
Prevention and treatment of various skin diseases such as cancer and atopic dermatitis
It is suitable as a therapeutic agent. Example C-5 <Enteric coated tablet> A starch and maltose were prepared in accordance with a conventional method.
In producing a tablet as a material, the method of Example B-5 was used.
Prepared γ-interferon and lymphoid cells
One tablet of the natural type Tsumore Necrosis Factor (2
Tablets containing 10,000 units per 100mg)
And methylcellose phthalate
To give an enteric coated tablet. This product is for small intestine, large intestine, etc.
Useful as a preventive and therapeutic agent for viral diseases
Wear. In addition, prevention of colon cancer, colon cancer, liver cancer, etc.
Agent, therapeutic agent, atopic allergy, severe
Myasthenia, rheumatoid arthritis, systemic lupus erythematosus, etc.
It can also be used advantageously as a therapeutic or prophylactic agent for immune diseases.
You. Furthermore, doxorubicin, fluorouracil
And anti-tumor effects of chemotherapeutic agents such as mitomycin C
It is also convenient to use as a strong agent. As described above, the conventional γ-interface
Is produced in an insufficient amount and must be manufactured industrially.
Was extremely difficult. On the other hand, the present invention
The established human-derived bone marrow monocytic cells are
Feron production was found to be extremely high.
Γ-interferon has high virus growth inhibitory action and
And has an inhibitory effect on malignant tumor growth, and furthermore, toxicity
Is extremely low, and this γ-interfero
Γ-interferon feeling comprising
This is a completed drug for receptive disease. Γ-Interface of the present invention
-Feron-sensitive disease drug is γ-interferon sensitive
Can be used as a prophylactic or therapeutic agent for
Such as viral diseases, malignant tumors, and immune diseases
It is extremely effective as a prophylactic and therapeutic agent. Thus, the present invention
Is of great industrial significance.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a phase contrast micrograph of HBL-38 cells. FIG. 2 is a micrograph showing the results of chromosome observation in karyotype analysis of HBL-38 cells.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07H 21/04 C12P 21/08 C07K 14/57 A61K 37/66 AAR 16/24 ABB C12N 5/10 ABC 15/02 ADY C12P 21 / 02 C12N 5/00 B 21/08 15/00 C

Claims (1)

(57) [Claims] H, which is a human bone marrow monocytic cell line
Can be obtained from BL-38 cells or KG-1 cells
Comprising γ-interferon as an active ingredient
A prophylactic or therapeutic agent for an interferon-sensitive disease. 2. H, which is a human bone marrow monocytic cell line
Can be obtained from BL-38 cells or KG-1 cells
Γ-interferon has a specific activity of about 1.5 × 10 ⁷
2. The preventive or therapeutic agent for gamma-interferon-sensitive diseases according to claim 1, wherein the unit is mg / mg protein. 3. The γ- according to claim 1 or 2, as an antiviral agent, an antitumor agent, an antitumor effect enhancer, and a therapeutic agent for an immune disease.
An interferon-sensitive disease preventive or therapeutic agent. 4. H, which is a human bone marrow monocytic cell line
BL-38 cells or KG-1 cells, either by transplantation into warm-blooded animal body other than a human, or implanted in the installed diffuser chamber to a warm-blooded animal body or outside of the non-human of the warm-blooded animal body fluids 4. The preventive or therapeutic agent for a y-interferon-sensitive disease according to claim 1, 2 or 3, which is a cell obtained by proliferating while receiving the supply. 5. An active ingredient γ- interferon, cultures of
HBL-38 cells, human bone marrow monocytic cells
The resulting isosamples contacting the inducing agent to the cells or KG-1 cells
The prophylactic or therapeutic agent for γ-interferon-sensitive disease according to claim 1, 2, 3 or 4, which is capable of being used. 6. Γ-interferon as an active ingredient is
HBL-38 cells, human bone marrow monocytic cells
Γ-interns obtainable from vesicles or KG-1 cells
-Feron, which has been purified and collected by column chromatography using an anti-γ-interferon monoclonal antibody,
6. The preventive or therapeutic agent for γ-interferon-sensitive disease according to 2, 3, 4 or 5 . 7. Anti-γ- interferon monoclonal antibodies, culture
HBL-, an established human-derived bone marrow monocytic cell
Γ -cells obtainable from 38 cells or KG-1 cells
A fused cell having anti-γ-interferon antibody-producing ability from a fused cell obtained by immunizing a warm-blooded animal other than human with interferon as an antigen, collecting antibody-producing cells from the animal, and fusing the cell with myeloma cells 7. A preventive or therapeutic agent for a γ-interferon-sensitive disease according to claim 6 , which is a monoclonal antibody having specificity for γ-interferon obtained by selecting and then growing the selected cells. 8. The preventive or therapeutic agent for a γ-interferon-sensitive disease according to claim 7 , wherein the monoclonal antibody is an immunoglobulin G class or an immunoglobulin M class. 9. H, which is a human bone marrow monocytic cell line
Can be obtained from BL-38 cells or KG-1 cells
With that γ- interferon, claim 1, characterized in that it contains other lymphokines as an active ingredient,
The agent for preventing or treating γ-interferon-sensitive disease according to 2, 3, 4, 5, 6, 7, or 8 . 10. Other lymphokines are α-interferon, β
-Prevention of gamma-interferon sensitive disease according to claim 9 , characterized in that it is one or two or more lymphokines selected from interferon, Tumore necrosis factor, lymphotoxin, interleukin 2, and B cell differentiation factor. Agent or therapeutic agent.