JPH0692997A - New megakaryocyte amplification factor and its production - Google Patents

Abstract

PURPOSE:To obtain a new megakaryocyte amplification factor having activity for promoting amplification of megakaryocyte, a precursor cell of blood platelet and promoting action on production of blood platelet and a medicine composition containing a new megakaryocyte amplification factor protein useful for preventing and treating diseases such as thrombocytopenia. CONSTITUTION:A megakaryocyte amplification factor protein has 23,000+ or -8,000 molecular weight measured by gel filtration, 3.5+ or -1 or 4.9+ or -1 isoelectric point measured by isoelectric point electrophoresis, is immunologically distinguished from human erythropoetin, interleukin 1alpha, interleukin 1beta, interleukin 6 interleukin 7 and interleukin 11, has no megakaryocyte colony stimulating activity and activity for activating megakaryocyte amplification and is substantially pure. The megakaryocyte amplification factor protein is produced by cell culture technology and its production can be extremely increased.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel megakaryocyte amplification factor and a method for producing the same. More specifically, the present invention provides
The present invention relates to a novel megakaryocyte amplification factor protein having an activity of promoting the amplification of megakaryocytes, which are progenitor cells of platelets, and an activity of promoting platelet production, and a method for producing the same by cell culture. The present invention also relates to a pharmaceutical composition containing the above novel protein as a megakaryocyte amplification factor useful for the prevention and treatment of diseases such as thrombocytopenia.

[0002]

2. Description of the Related Art Platelets play an important role in promoting bleeding caused by rupture of blood vessels in the process of thrombus formation and blood coagulation that the body naturally stops. Thrombopoietin (TPO), which is a factor that specifically promotes the production of platelets, has been enthusiastically acquired by many researchers over the past 20 years, but it has not yet succeeded. .

When plasma of a thrombocytopenic animal is administered to a healthy animal, platelet production is accelerated, and when platelets are transfused, on the contrary, platelet production is decreased. Therefore, the action of regulating the production of platelets depending on the increase / decrease of platelets is exerted. The existence of TPO has been advocated for a long time. Subsequent study results indicate that platelets are released into blood from megakaryocytes that have been differentiated and matured in bone marrow from bone marrow stem cells to megakaryocyte progenitor cells, and in vitro results indicate that megakaryocytes have differentiated.・ Megakaryocyte colony stimulating factor (Megakaryocyte Colo)
ny Stimulating Factor; Meg-CSF), and TPO having an activity of promoting the maturation of megakaryocytes has been revealed to act in the latter stage. That is, Meg-CSF acts first, progenitor cells repeat cell division to increase megakaryocyte compartments, and then TPO acts to cause each megakaryocyte progenitor cell to undergo endomitosis and increase its chromosome polyploidy. (-32N), the cytoplasm matures and increases, and platelet production begins. TPO is also a Megakaryocyte Potentiator (Meg-
Sometimes called POT).

The activity of Meg-CSF is estimated by measuring the activity of forming megakaryocyte colonies in human or mouse bone marrow cells in soft agar culture in vitro. Currently, Meg-CSF activity is shown in urine of patients with aplastic anemia and idiopathic thrombocytopenic purpura, in plasma of patients with myeloid megakaryocytic aplastic thrombocytopenia, kidney bean lectin-stimulated human leukocyte culture supernatant, It has been found in mouse leukemia cell line WEHI-3 culture supernatant and the like.

Among various cytokines, Interleukin 3 (IL-3) (hereinafter interleukin is abbreviated as IL) non-specifically acts on many lineages including megakaryocytes Multi-CS
It is becoming clear that it is F. In addition, Meg-CSF in WEHI-3 culture supernatant completely matches IL-3, and it is believed that most of Meg-CSF activity in conventional cell culture supernatant is due to IL-3. However, Meg-CSF, which has been shown to act specifically on the platelet system, is not yet known.

On the other hand, the activity of TPO is estimated by measuring the enhancing action of Meg-CSF colony forming activity and / or the promoting action of megakaryocyte maturation. Until now, preparation of several factors having TPO-like activity has been attempted.
SDS-PA prepared from culture supernatant of human embryonic kidney cell line
A megakaryocyte stimulatory factor (MSF) having a molecular weight of 15,000 and an isoelectric point of 5.1, which has an action of promoting protein synthesis in megakaryocyte cells, and a method for producing the same have been reported [US See Japanese Patent No. 4,894,440 (Japanese Patent Laid-Open No. 63-239298)]. Also recently
IL, a multifunctional cytokine found as a glycoprotein that induces B cell antibody production and has been shown to be involved in the immune system, acute phase reaction system and malignant tumors
-6 is also involved in the hematopoietic system, and Meg-
It shows POT activity and megakaryocyte maturation promoting activity (Ishibashi,
T. et al. "Proc. Natl. Acad. Sci. USA" 86,5953 (19
89)), and has been confirmed to exhibit a platelet production promoting action in vivo (Asano, S. et al. “Blood” 75,16).
02 (1990)). Further, it has been reported that IL-7, IL-11 and the like also have megakaryocyte amplification activity. However, the megakaryocyte-amplifying activity of these factors is weak, and it is unclear whether they are constitutive hematopoietic factors that the body originally has.

[0007]

SUMMARY OF THE INVENTION In view of the above technical background, the inventors of the present invention have developed a novel megakaryocyte having a specific and potent action of promoting the amplification of megakaryocytes and an action of promoting platelet production. As a result of intensive studies to find a sphere-amplifying factor, surprisingly, a completely new megakaryocyte-amplifying factor was found in the culture supernatant of normal human diploid cells, and an appropriate production promoter was added to the medium. Therefore, it was found that a large amount of the factor was produced. From the collected culture supernatant,
The factor was isolated and purified, its properties were clarified, and its usefulness as a drug was shown. In addition, genetic engineering techniques can be applied to express the megakaryocyte amplification factor. The present invention has been completed based on these findings.

Accordingly, one object of the present invention is to provide a substantially pure novel megakaryocyte amplification factor having a strong action.

Another object of the present invention is to culture animal cells in a medium, to produce megakaryocyte amplification factor in the culture medium, to collect the culture supernatant from the culture medium, and to collect the culture medium. It is intended to provide a method for producing a megakaryocyte amplification factor, which comprises purifying a megakaryocyte amplification factor from Qing.

Still another object of the present invention is to increase the amount of the megakaryocyte amplification factor produced by adding a megakaryocyte amplification factor production promoter to the medium in the above cell culture and carrying out the cell culture. Another object of the present invention is to provide a method for producing a megakaryocyte amplification factor.

Still another object of the present invention is to provide a pharmaceutical composition containing a therapeutically effective amount of a megakaryocyte amplification factor as an active ingredient, and a therapeutic method using the same.

[0012]

[MEANS FOR SOLVING THE PROBLEMS] According to the present invention, there is provided a megakaryocyte amplification factor having an activity of activating megakaryocyte amplification and an activity of increasing platelets in peripheral blood. More specifically, there is provided a substantially pure megakaryocyte amplification factor protein having an activity of activating megakaryocyte amplification and having the following properties.

(A) Molecular weight: 23000 ± 8000 (measured by gel filtration) (b) Isoelectric point: pI ≦ 5.9 (measured by isoelectric focusing) (c) Human erythropoietin, interleukin 1
The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against α, interleukin 1β, interleukin 6, interleukin 7, and interleukin 11. (D) It does not have megakaryocyte colony stimulating factor activity.

Examples of the megakaryocyte amplification factor protein of the present invention include the following two substantially pure megakaryocyte amplification factor proteins having the activity of activating megakaryocyte amplification and having the following properties. Can be mentioned. 1. (A) Molecular weight: 23000 ± 8000 (measured by gel filtration) (b) Isoelectric point: pI 3.5 ± 1 (measured by isoelectric focusing) (c) Human erythropoietin, interleukin 1
The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against α, interleukin 1β, interleukin 6, interleukin 7, and interleukin 11.

(D) Does not have megakaryocyte colony stimulating factor activity. 2. (A) Molecular weight: 23000 ± 8000 (measured by gel filtration) (b) Isoelectric point: pI 4.9 ± 1 (measured by isoelectric focusing) (c) Human erythropoietin, interleukin 1
The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against α, interleukin 1β, interleukin 6, interleukin 7, and interleukin 11.

(D) Does not have megakaryocyte colony stimulating factor activity.

According to another aspect of the present invention, animal cells are cultured in a medium, a megakaryocyte amplification factor is produced in the culture medium, a culture supernatant is collected from the culture medium, and the collected culture medium is collected. Provided is a method for producing a megakaryocyte amplification factor, which comprises separating and purifying the megakaryocyte amplification factor from a silkworm.

As animal cells used in the method of the present invention, various kinds of cells having an activity of activating megakaryocyte amplification and an ability of producing a megakaryocyte amplification factor having an activity of increasing platelets in peripheral blood Cells can be used. Normal diploid cells can be used advantageously, for example cells from human kidney, intestine, lung, heart, ureter, skin, foreskin, tongue, thyroid, placenta, uterus, preferably human fetal kidney,
Cells derived from lung or foreskin, and even more preferably cells derived from human fetal lung can be used.

The megakaryocyte amplification factor can be separated and purified from these tissue extracts, but more preferably, these cells are cultured in an appropriate growth medium and the megakaryocytes are added to the culture medium. It is possible to produce a sphere amplification factor, collect the culture supernatant from the culture medium, and separate and purify from the collected tissue culture medium. It is desirable that these cells are proliferated by the culture method used for culturing ordinary cells, for example, the method described in "Tissue culture" (edited by Junnosuke Nakai et al., 1975, published by Asakura Shoten) and used in the present invention. The cells can be cultured in a medium solution containing carbons, nitrogen sources, and, if necessary, inorganic salts and / or other additives to produce megakaryocyte amplification factor. Further, according to the present invention, by adding a megakaryocyte amplification factor production promoter, preferably animal meat enzyme-degrading peptone and culturing the cells, the amount of the megakaryocyte amplification factor produced in the culture leaps. Can be increased. The concentration of enzymatically digested animal meat peptone in the medium is 0 to 4 w / v%, preferably 0.1 to 2 w / v%.
Can be used. Animal meat enzyme-decomposed peptone is generally used for bacterial culture medium, and is usually called proteose peptone, proteose peptone, or animal meat peptone.

The method for preparing the enzyme-degraded animal meat peptone is publicly known, and for example, it may be carried out according to the method described in "Bacterial Media Science Lecture No. 2" (Riichi Sakazaki, Naya Shoten, 1967). That is, as animal meat, meat or internal organs such as beef, pig, chicken, sheep, whale, etc. are used, and of these, beef is most commonly used. Trypsin, an enzyme for decomposition,
Examples include papain, pepsin, and pancreatin. These animal meats are crushed, mixed with water, and adjusted to a pH suitable for enzymatic decomposition with sodium carbonate, concentrated hydrochloric acid or the like. Enzyme is added to this, and at 20-40 ° C for 1-20 days, usually at 37 ° C.
Enzymatic degradation for 2-3 days. After digestion, in order to inactivate the degrading enzyme and to heat-coagulate undigested protein, 10
After heating to 0 ° C. or higher and removing this by filtration, the mixture is concentrated, dried and pulverized. The methods of concentration and dryness include boiling down to powder and concentrating at a low temperature using a vacuum drying device, followed by pulverization. As a commercially available product, Proteose Pepton No. 1 (Proteose Pepton, manufactured by Difco, USA)
No. 1), Proteose Peptone No. 2, Proteose Peptone No. 3, Thiopepton, Proteose Peptone L46, Peptone PL4 manufactured by Oxoid, UK
6. There are Thioton manufactured by BBL, UK, and Proteose peptone manufactured by Daigo Nutrition Chemicals.

Next, an example of production of a megakaryocyte amplification factor by a cell culture method will be shown. Using primary culture cells or commercially available cells that produce the factor directly extracted from the tissue, they are cultured in adherent culture or suspension culture. As an example, a suitable cell density, preferably 10 ⁵ cells / ml, is implanted together with 0.1 to 10 mg / ml of cell culture bead carrier, and is then incubated in serum at 15 to 45 ° C., preferably 25 to 40 ° C.
Culturing is carried out in the temperature range of 5 ° C, preferably in the pH range of 5 to 9 and preferably in the range of 6 to 8, in the air containing 5% CO ₂ . When a megakaryocyte amplification factor production promoter is used, serum-free conditions are used, and production culture is performed by adding at a concentration of 0 to 4%, preferably 0.1 to 2%, but preferably, the cells are sufficiently proliferated and It is preferably transferred to production culture in a confluent state. The culture period for production is usually 1 to 60 days, but 60
It is possible to exceed the day. Since the production rate of the megakaryocyte amplification factor gradually decreases in the latter half of production, the most efficient days are selected for industrial production. Megakaryocyte amplification factors are produced in solution from cells under the conditions described above. The production amount is measured by the megakaryocyte amplification activity measurement method shown in Reference Examples (a) and (b).

The megakaryocyte amplification factor of the present invention can be obtained by expressing the megakaryocyte amplification factor in an appropriate host cell using a commonly used genetic engineering technique, recovering it, and further purifying it. You can also get it.

That is, various cells having an activity of activating megakaryocyte amplification and an ability of producing a megakaryocyte amplification factor having an activity of increasing platelets in peripheral blood, such as human kidney and intestine. , Lungs, heart, ureters, skin, foreskin,
Tongue, thyroid gland, placenta, cells from the uterus, preferably a human fetal kidney, lung, cells from foreskin, and even more preferably total RNA was extracted from human fetal lung-derived cells, further than this poly A ⁺ RNA To purify. A suitable expression vector,
Preferably, a cDNA library is prepared using a eukaryotic expression vector, poly A ⁺ RNA and a linker, and an appropriate host cell such as Escherichia coli is transformed with this library, and a plasmid DNA is obtained from the culture solution. To prepare. Using this plasmid, a suitable host cell, preferably an animal-derived cell, more preferably a monkey-derived COS cell, is transfected to express a megakaryocyte amplification factor gene, which is recovered and further purified. By doing so, a megakaryocyte amplification factor can be produced.

Next, a specific example thereof will be described.
From a cell producing an appropriate amount of megakaryocyte amplification factor, for example, human fetal lung cells, preferably 10 ⁸ cells, using an RNA isolation kit such as that manufactured by Invitrogen, USA (catalog No. K1592-01), Total RNA is extracted by the guanidine isothiocyanate method according to the attached manual, and poly A ⁺ RNA is obtained according to a conventional method. At this time, Oligotex-dT30 (manufactured by Japan Synthetic Rubber Co., Ltd.) can be used. Usually about 200 μg according to the above method
As a result, 1 to 2 μg of poly A ⁺ RNA is obtained. Next, a cDNA library is prepared according to the method of Okayama-Berg. For example, eukaryotic expression vector 3 '
-oligo (dT) -tailed pcDV-1 (No.27-4955-01 manufactured by Pharmacia, Sweden) and the poly A ⁺ RNA obtained above.
And 3'-oligo (dG) -tailed pL1 linker (No. 27-4957, Pharmacia, Sweden) can be used. Alternatively, pcDL-SRα296 may be used. The solution containing the obtained cDNA library was divided into an appropriate number of pools, preferably 10 to 200, more preferably 50 to 100, and E. coli MC1061 (A
TCC53338) is transformed. The transformed E. coli is cultured overnight in the presence of ampicillin. After collection and lysis, plasmid DNA is used, for example, using Qiagen-tip-100 (manufactured by Qiagen, USA) according to the attached manual.
To prepare. The obtained recombinant DNA is subjected to, for example, the diethylaminoethyl-dextran method (CURRENT PROTOCOLS
IN MOLECULAR BIOLOGY 9.2.1.-9.2.6), suitable host cells, preferably monkey kidney COS1 cells (AT
CC, CRL1650) and then WO88 / 05
The gene is expressed in substantially the same manner as in the method described in Example 2 in the specification of 053 (JP-A-64-6219). That is,
Appropriate culture conditions such as D-MEM containing 10% fetal calf serum
After culturing in a medium (manufactured by Flow Laboratories, USA) under the conditions of 37 ° C., 40 hours and 5% CO ₂ , the medium is replaced with a serum-free D-MEM medium, and the culture solution is collected every 2 days for 3 times.

According to the present invention, the megakaryocyte amplification factor gene can also be cloned by screening the expression cells into which the megakaryocyte amplification factor gene has been incorporated, using the activity of the megakaryocyte amplification factor as an index.

That is, after concentrating each recovered medium, the megakaryocyte amplification factor activity is measured by a liquid culture acetylcholinesterase activity measuring method and the like, and this is used as an index to narrow down the pool containing the gene of this substance. You can Colonies obtained by transforming E. coli again with the positive DNA (approximately 2000)
Approximately 10 cells were cultivated as a group, DNA was prepared, introduced into COS1 cells, expressed and acetylcholinesterase activity was measured in the same manner as above, and secondary cDNA
It is also possible to narrow down the A library. Usually, this method is repeated several times to isolate Escherichia coli having a cDNA plasmid expressing the megakaryocyte amplification factor activity. (Hayashida, K. et al. "Hematopoietic Factor" 1, N
o.2, 102-108 (1990)).

Furthermore, according to the present invention, it is also possible to prepare a gene probe encoding a part of the primary structure of the protein of the megakaryocyte amplification factor, and use this to clone the megakaryocyte amplification factor gene.

Furthermore, according to the present invention, the cloned megakaryocyte amplification factor gene is used to transform, for example, E. coli,
Yeast, monkey kidney cells (COS cells), Chinese hamster ovary cells (CHO cells), mouse C127 cells, human embryonic kidney cell lines, silkworm cells SF9, and other host cells are transfected, and the megakaryocytes are more efficiently transfected. A megakaryocyte amplification factor can be produced by expressing the amplification factor, recovering it, and further purifying it.

In the method for producing a megakaryocyte amplification factor protein according to the present invention, for example, in the case of production by cell culture, when the production of megakaryocyte amplification factor by the producing cells reaches a desired production amount or number of days, the culture supernatant is to recover. The method for separating and purifying the megakaryocyte amplification factor is a method usually used in protein chemistry, for example, adsorption method using a carrier, salting out method, electrophoresis method, ion exchange, gel filtration, affinity to an appropriate ligand. The various chromatographic methods and the like applying can be used alone or in combination. As the chromatographic method, preferably, particles of CM sepharose column chromatography using sepharose having a carboxymethyl group bound thereto, Q sepharose column chromatography using sepharose having a quaternary aminoethyl group bound thereto, particles of crosslinked dextran gel and the like. Gel filtration column chromatography, hydrophobic column chromatography, and antibody affinity column chromatography in which an antibody that specifically binds to the substance of the present invention is bound can be used. In particular, the megakaryocyte-amplifying factor of the present invention is considered to have an acidic isoelectric point from the obtained fraction having a megakaryocyte-amplifying factor activity in the course of purification using, for example, isoelectric focusing or ion exchange chromatography. It can be isolated by separating the fractions and further purifying.

The novel megakaryocyte amplification factor thus obtained has an activity of activating megakaryocyte amplification and an activity of increasing platelets in peripheral blood. The megakaryocyte amplification factor is used as a reagent for studying differentiation, proliferation and megakaryocyte maturation of megakaryocytes from bone marrow stem cells or bone marrow megakaryocyte precursor cells, and the megakaryocyte amplification factor alone or in a therapeutically effective amount. At least one selected from pharmaceutically acceptable carriers, diluents and excipients may be added to the megakaryocyte amplification factor to prepare a suitable dosage form for use as a pharmaceutical product. As the carrier, diluent and excipient, those usually used in this field can be used. In addition, the megakaryocyte amplification factor, IL-1, IL-2, IL-3, I
L-4, IL-5, IL-6, IL-7, IL-8, I
L-9, IL-11, GM-CSF, G-CSF, M-
CSF, SCF, IFNs, LIF, TNF and EPO
At least one factor selected from the above and at least one selected from pharmaceutically acceptable carriers, diluents and excipients can be added to obtain a suitable dosage form, which can be used as a medicine.

The megakaryocyte-amplifying factor of the present invention is used in certain thrombocytopenia such as thrombocytopenia after administration of anti-cancer agent, thrombocytopenia after radiotherapy, thrombocytopenia due to megakaryocyte-amplifying factor deficiency, and aplastic anemia. Can be used for the treatment and / or prevention of thrombocytopenia, thrombocytopenia after bone marrow transplantation, and thrombocytopenia of autoimmune disease. It can also be used to treat leukemia. Furthermore, as an alternative or an adjunct to platelet transfusion, or in vitro of bone marrow cells for transfusion
It can also be used for growth culture at 0 ° C.

The megakaryocyte amplification factor of the present invention can be used as an injection. In this case, a thickening agent such as sucrose, glycerin, methyl cellulose, carboxymethyl cellulose, a pH adjusting agent for various inorganic salts and the like can be added as additives.

The dose of the megakaryocytic amplification factor of the present invention per adult is generally about 0.1 μg to 100 mg, although it varies depending on age, sex, body weight, symptoms, etc., once a day or as needed. Can be administered several times.

In order to describe the present invention in more detail, reference examples and examples will be described, but the scope of the present invention is not limited to these examples.

[0035]

[Reference example]

(Method for measuring megakaryocyte amplification activity) The megakaryocyte amplification factor activity of the novel protein obtained by the present invention was measured by the following two methods (a) and (b).

(A) Meg-POT by soft agar culture method
Activity measurement method Preparation of bone marrow cell suspension IMDM solution (Is
cove's modeificationof Dallbecco's medium) is powder IMDM (for 1 liter) (manufactured by Gibco, USA) and baking soda.
After adding 3.024 g and 3.04 microliters of β-mercaptoethanol to adjust the pH to 7.1, the volume was adjusted to 1 liter, and 50 IU / ml penicillin and 50
It was prepared by adding μg / milliliter streptomycin (all are manufactured by Flow Laboratories, Inc., USA).

6 to 9-week-old C57BL / 6 mice (male)
The femur of the mouse is collected, the upper part is cut off, and the bone marrow is vigorously pushed into the 100 mm plastic dish from the knee joint side using a 10 ml plastic syringe (22 G needle) containing 10 ml of IMDM solution. It was
The cells were dispersed by pipetting operation about 8 times (6 times for 19G needle, 2 times for 22G needle) and then transferred to a 15 ml Falcon tube to collect non-precipitating cells, which were then added to 10 ml of IMDM. The resulting bone marrow cell suspension was washed twice with 2 times, and finally suspended in 2.5 ml of IMDM solution per mouse, further dispersed well, and used for the next colony assay experiment. The cell concentration was measured with a hemocytometer by Trypan Blue (Flora Laboratories, USA) staining.

Colony Assay As the acetylcholinesterase staining solution used in the next experiment, 1.73 mM acetylthiocholine iodide,
40 including 5 mM potassium ferricyanide, 5 mM sodium citrate, 3 mM copper sulfate (all manufactured by Wako Pure Chemical Industries, Ltd.)
A solution dissolved in 0 ml of 75 mM phosphate buffer having a pH of 6.0 was used.

Horse serum (manufactured by JR Scientific Co., USA) was added to 10 ⁵ bone marrow cells in a 100 mm plastic dish at 15 v / v%, and further added.
IL-3 (manufactured by Genzyme, USA) 50 ng or IL-3 containing COS1 cell culture supernatant was added at 1 v / v% 500
Add the test solution to microliters of IMDM medium,
Soft agar was formed under the condition of 0.3% Bacto agar (manufactured by Difco, USA), and cultured at 37 ° C. under 5% CO ₂ for 7 days. As a culture supernatant of IL-3 containing COS1 cells, a plasmid in which mouse IL-3 cDNA was ligated to a promoter of SV-40 was used to prepare COS1 cells (ATCC CR
K1650) and transformed into WO 88/05053 (JP-A 64-62).
No. 19), the IL-3 containing culture supernatant expressed in the same manner as in the method described in Example 2 was used.

After the culture was completed, the agar disc was transferred onto a slide glass, dried, and then fixed with 2% glutaraldehyde. After fixation, the cells were washed with phosphate buffered saline (PBS), and megakaryocytes were specifically stained with an acetylcholinesterase staining solution. The number of colonies was counted using an Olympus AHB type microscope as one colony consisting of 6 or more positive cells.

(B) Method for measuring acetylcholinesterase activity by liquid culture A mouse bone marrow cell suspension prepared in the same manner as in the above measurement (a) was diluted to a final concentration of 0.4 mM with diisopropylfluorophosphate (DFP). (Manufactured by Sigma, USA) was added, and the mixture was left for 20 minutes at room temperature with occasional stirring to eliminate the endogenous acetylcholinesterase activity of bone marrow cells. The number of cells was counted by a hemocytometer as described above.

The cell density of the bone marrow cell suspension was 2.5 to 5 × 10 5.
Eliminate the endogenous acetylcholinesterase activity with DFP to ⁵ cells / ml [J.Cell.Physio
l.122159 (1985)] Horse serum was suspended in IMDM solution containing 15 v / v%, and 0.2 ml per well was dispensed into a 96-well culture dish (Sumitomo Bakelite Co., Ltd.). 20 microliters of the sample solution was added, and the mixture was cultured at 37 ° C. and 5% CO ₂ for 7 days.

After culturing, the cells were submerged by centrifugation and the supernatant was removed. Then, 20 μl of 5.6 mM acetylthiocholine iodide (manufactured by Sigma, USA) and 180 μl of 0.12 M sodium chloride, 1 were added to each well. 50 mM of pH 7.5 containing mM ethylenediaminetetraacetic acid, 0.2% Triton X-100
Hepes buffer was added, and the mixture was allowed to react at room temperature for 3 to 4 hours to give 11111. Transfer 20 microliters of the reaction solution to a 96-well fluorescence measurement plate (Greiner, Germany),
0 microliter of 1 mM ethylenediaminetetraacetic acid,
Acetate buffer pH 5.0 containing 0.2% Triton X-100 and 20 microliters of 0.4 mM CPM (7-diethyla
mino-3- (4'-maleimidylphenyl) -4-methylcoumarin)
An acetonitrile solution (manufactured by Molecular Probes, Inc., USA) was added and reacted at room temperature for 1 hour. Acetylcholinesterase activity is 45 by excitation light of 365 nm.
0 nm fluorescence was measured using PANDEX FCA (manufactured by Baxter, USA).

[0044]

【Example】

(Example 1) Production of megakaryocyte amplification factor by human cell culture method Commercially available human fetal lung normal diploid cells (manufactured by Flow Laboratories, USA) were placed in a 100-liter glass bottle.
Cytodex I (bead carrier for cell culture, at a concentration of 2.5 mg / ml at a density of 10 ⁵ cells / ml,
Implanted with Sweden, Pharmacia),
As a growth medium at 37 ° C in the air containing 5% CO ₂ , 1
60 liters of a medium MEM medium containing 0% fetal bovine serum was added, and suspension culture was performed while stirring at a rotation speed of 60 rpm. After culturing for 8 days and allowing the cells to proliferate sufficiently, the bead carrier to which the cells were adhered was washed with a physiological saline solution to obtain serum-free 0.75% Proteose Peptone No. The medium was replaced with 60 liters of the medium 199 medium containing or not containing 3, and the cells were cultured with stirring at a rotation speed of 60 rpm. A conditioned medium containing the substance of the present invention was collected while exchanging the medium every 3rd day.
The culture solution was concentrated 10-fold, and the megakaryocyte-amplifying factor activity contained therein was measured by the soft agar culture method M shown in Reference Example (a).
It was evaluated by the egg-POT activity measuring method. The results are shown in Table 1. For reference, this was also evaluated for some other cell cultures. It was shown that human fetal lung cells have the ability to produce a megakaryocyte-amplifying factor, and that when cultured in the presence of proteose peptone, the amount of megakaryocyte-amplifying activity is significantly increased.

[0045]

[Table 1] [Note] 1) The number of times indicates the number of times every 3rd day described above.

2) PP; proteose peptone 3) The number of colonies of each sample is the IL added to the assay system.
It was calculated by subtracting 3 from the number of colonies to be twisted when only -3.

(Example 2) Purification of megakaryocyte amplification factor Culture supernatant 1 of human fetal lung normal diploid cells obtained in Example 1
To 90 liters, add about 1.2 liters of acetic acid,
After adjusting to 4, the cell fragments and the generated insoluble matter were removed by filtration. Carboxymethyl Sepharose (CM Sepharose; manufactured by Pharmacia, Sweden) column (9 cm in diameter x 2 in height) that has been sufficiently equilibrated with 20 mM acetate buffer having a pH of 4.0 containing 0.2 M sodium chloride in advance.
(3.5 cm) and washed with 13.5 liters of the same equilibration buffer and 6 liters of 20 mM acetate buffer of pH 4.0 containing 0.4 M sodium chloride, then 0.75 M sodium chloride and 10 m
20 mM acetate buffer 1 containing M lysine hydrochloride at pH 4.0
The adsorbed protein was eluted with 2 liters. By this operation, about 5.5 liters of an eluate was obtained as a crude purified liquid I containing megakaryocyte amplification factor activity. The proteose peptone component contained in a large amount in the culture supernatant was reduced to 1% or less.

Since the eluate usually contains a large amount of tissue plasminogen activator (tPA), it was specifically removed. That is, 5M sodium hydroxide solution was added to adjust the pH to 7.0, and the monoclonal antibody against tPA, which had been preliminarily equilibrated with 20 mM Tris-hydrochloric acid buffer containing pH 7.5 containing 0.5 M sodium chloride, was previously added to Sepharose. The bound (3 mg / ml gel) was passed through an antibody column (9 cm diameter x 29 cm height). By this operation, about 6 liters of the pass-through liquid was obtained as the crude purified liquid II. By this column chromatography, the megakaryocyte amplification factor activity was recovered almost quantitatively. Further, the tPA activity contained in the crude purified solution I in a large amount was removed.

6.2 liters of the crude purified liquid II was concentrated to 300 ml with an ultrafiltration module SIP-1013 (manufactured by Asahi Kasei Corp.) and 10 times volume of 20 m of pH 7.0.
M sodium phosphate buffer was added, the solution was concentrated again to 300 ml, and the buffer was exchanged. 50 mM in advance
A CM sepharose column (diameter 5 cm × height 15 cm) sufficiently equilibrated with 20 mM sodium phosphate buffer having a pH of 7.0 and containing sodium chloride was passed through the crude purified liquid III.
As a result, about 300 ml of the pass-through liquid was obtained.

FIG. 1 shows an example of the result of the CM sepharose column chromatography of the first purification step.

To the crude purified solution III, an equal volume of purified water and 3 ml of 0.5 M Tris were added, and the pH was adjusted to 8.0 with hydrochloric acid. 20m containing 50mM sodium chloride having a pH of 8.0 in advance
Adsorbed on a Q Sepharose (Pharmacia, Sweden) column (diameter 2.5 cm × height 10 cm) sufficiently equilibrated with M Tris-HCl buffer, washed with about 100 ml of the same buffer, 120 ml of the same buffer (E-1) containing 0.1 M sodium chloride, about 20
0 ml of the same buffer containing 0.25M sodium chloride (E
-2), the same buffer (E- containing 0.5 M sodium chloride)
Elution was performed in 3). The flow rate was 200 ml / hour. Megakaryocyte amplification factor activity is E-1, E-
Although observed in 2, E-3 and all fractions, the megakaryocyte amplification factor having an acidic isoelectric point was mainly recovered in the E-3 fraction. That is, about 50 ml of the E-3 fraction was recovered as the crude purified liquid IV at an activity recovery rate of about 10%. Figure 2
Shows an example of the result of Q sepharose column chromatography.

48 ml of the crude purified solution IV was concentrated to 3 ml using the above ultrafiltration hollow fiber, and this was sufficiently equilibrated with a 50 mM sodium phosphate buffer of pH 7.3 to obtain Sephacryl S-200 (Sweden). Gel filtration was performed on a column (manufactured by Pharmacia) (diameter 1.6 cm × height 90 cm). The megakaryocyte amplification activity is a fraction having an elution molecular weight of about 23 kd, that is, about 25
As the milliliter of the crude purified liquid V, the activity recovery rate is 30 to 4
Recovered at 0%. FIG. 3 shows an example of the result of Sephacryl S-200 column chromatography. After adding an equal volume of purified water to 25 ml of the crude purified solution V, concentrated and desalted, and added Ampholine (3.5-10) (Pharmacia, Sweden) to a final concentration of 2%, Carrier-free isoelectric focusing was performed at a constant voltage of 12 watts for 5 hours using a rotophor (manufactured by Bio-Rad, USA). An example of the results of isoelectric focusing is shown in FIG. The megakaryocyte amplification activity had peaks at pH 3.5 and 4.9. Active fractions around pH 3.5 and around 4.9 were respectively collected and designated as purified sample 1 and purified sample 2 of megakaryocyte amplification factor. The total protein amounts of the purified sample 1 and the purified sample 2 were 0.9 mg and 0.15 mg, respectively.

Table 2 shows the degree of purification in each purification step.

[0054]

[Table 2] [Note] The protein concentration of each sample was calculated on the assumption that the ultraviolet absorption coefficient at 280 nm was A280.1% = 10. The purified sample 2 was concentrated 10 times and the absorbance was measured.

Example 3 Physical Properties of Megakaryocyte Amplification Factor (Purified Sample 1) (a) Molecular Weight Sephacryl S-200HR Equilibrated with PBS in advance
Using a column (made by Pharmacia, Sweden) with a diameter of 1.6 cm and a height of 90 cm, the purified preparation 1 obtained in Example 2 was developed with PBS (flow rate 20 ml / hour), and 2 ml of this was used. Fractionation was performed and the megakaryocyte amplification factor activity of each fraction was measured. The molecular weight of this substance was measured by comparing the elution positions of the fraction having megakaryocyte amplification factor activity and the low molecular weight marker protein kit for gel filtration (Pharmacia, Sweden). This substance was eluted with a peak at a molecular weight of around 23,000.

(B) Isoelectric point The purified sample 1 obtained in Example 2 was subjected to an isoelectric focusing column (110 ml) (manufactured by Kato Shoichi Shoten Co., Ltd.).
Glycerol density gradient isoelectric focusing was performed for 40 hours at a constant power of 3 watts. As the amphoteric carrier, 1% ampholine (manufactured by Pharmacia, Sweden) was used.
This substance had an isoelectric point in the pH range of 2.5 to 4.5.

(C) Antigenicity Whether or not this substance immunologically reacts with a known cytokine was examined using the purified preparation 1 obtained in Example 2.
Anti-human erythropoietin antibody, anti-human IL-1α antibody,
Anti-human IL-1β antibody, anti-human IL-6 antibody, anti-human I
Using L-7 antibody (all manufactured by Genzyme, USA) and anti-human IL-11 antibody, a megakaryocyte amplification factor activity of this substance was neutralized. As a result, it was shown that the substance did not decrease in activity even when treated with these antibodies and did not react with these antibodies. In addition, this substance did not exhibit adsorptivity to anti-human IL-1 antibody column and anti-human IL-6 antibody column (manufactured by Endogen, USA). This indicates that the megakaryocyte amplification factor of the present invention is immunologically distinguished from these known cytokines.

(D) Megakaryocyte-amplifying factor activity The activity of the megakaryocyte-amplifying factor of this substance was examined by a soft agar culture method. The results are shown in Table 3. The Meg-POT activity of IL-6 (manufactured by Genzyme) was only 2. when IL-3 alone was used, even when a high concentration of 200 ng / mL was used.
It was 5 times. In contrast, the purified preparation of this substance is IL-
The activity was 14 times higher than that of 3 alone. In addition, a large number of cells showing a strong acetylcholinesterase activity per colony was observed in the purified preparation of this substance as compared with the case of using only IL-3. In addition, this substance alone did not show megakaryocyte amplification factor activity.

Furthermore, the megakaryocyte amplification factor activity of this substance was evaluated by the acetylcholinesterase activity assay method by liquid culture. The results are shown in Fig. 5. This method also showed the megakaryocyte-amplifying factor activity of this substance.

(E) Thrombopoietin action The purified preparation 1 obtained in Example 2 was used as a mouse (C57BL male, 7).
It was intraperitoneally administered for 5 days continuously at 5 weeks of age (5 animals per group), and blood was collected 3 hours after the final administration to measure the number of platelets and the number of red blood cells. As shown in Table 4, it was revealed that this substance significantly increased the platelet count at a risk rate (P) of 1% or less and exhibited a thrombopoietin action. The number of red blood cells did not increase at this time. In Table 4, Group 1 was administered with 1 μg of the purified substance dissolved in PBS containing 150 μg / ml bovine serum albumin (BSA) per administration, and Group 3 was administered with BSA alone as a control. .

(Purified sample 2) (a) Molecular weight Sephacryl S-200HR previously equilibrated with PBS
The purified sample 2 obtained in Example 2 was developed with PBS (flow rate 20 ml / hour) using a column (made by Pharmacia, Sweden) with a diameter of 1.6 cm and a height of 90 cm, and 2 ml of each was added. Fractionation was performed and the megakaryocyte amplification factor activity of each fraction was measured. The molecular weight of this substance was measured by comparing the elution positions of the fraction having megakaryocyte amplification factor activity and the low molecular weight marker protein kit for gel filtration (Pharmacia, Sweden). This substance was eluted with a peak at a molecular weight of around 23,000.

(B) Isoelectric point Using the purified sample 2 obtained in Example 2 with an isoelectric focusing column (110 ml) (manufactured by Kato Shoichi Shoten),
Glycerol density gradient isoelectric focusing was performed for 40 hours at a constant power of 3 watts. As the amphoteric carrier, 1% ampholine (manufactured by Pharmacia, Sweden) was used.
This substance had an isoelectric point in the range of pH 3.9 to 5.9.

(C) Antigenicity Whether or not this substance immunologically reacts with a known cytokine was examined using the purified preparation 2 obtained in Example 2.
Anti-human erythropoietin antibody, anti-human IL-1α antibody,
Anti-human IL-1β antibody, anti-human IL-6 antibody, anti-human I
Using L-7 antibody (all manufactured by Genzyme, USA) and anti-human IL-11 antibody, a megakaryocyte amplification factor activity of this substance was neutralized. As a result, it was shown that the substance did not decrease in activity even when treated with these antibodies and did not react with these antibodies. In addition, this substance did not exhibit adsorptivity to anti-human IL-1 antibody column and anti-human IL-6 antibody column (manufactured by Endogen, USA). This indicates that the megakaryocyte amplification factor of the present invention is immunologically distinguished from these known cytokines.

(D) Megakaryocyte amplification factor activity The megakaryocyte amplification activity of this substance was examined by a soft agar culture method. The results are shown in Table 3. IL-6 (manufactured by Genzyme) M
The egg-POT activity was only 2.5 times higher than with IL-3 alone, even with the high concentration of 200 ng / ml. On the other hand, the purified preparation of this substance exhibited 6 times the activity of IL-3 alone. In addition, a large number of cells showing a strong acetylcholinesterase activity per colony was observed in the purified preparation of this substance as compared with the case of using only IL-3. In addition, this substance alone did not show megakaryocyte amplification factor activity.

Furthermore, the megakaryocyte amplification factor activity of this substance was evaluated by the acetylcholinesterase activity assay method by liquid culture. The results are shown in Fig. 2. This method also showed the megakaryocyte-amplifying factor activity of this substance.

(E) Thrombopoietin action The purified preparation 2 obtained in Example 2 was used as a mouse (C57BL male, 7).
It was intraperitoneally administered for 5 days continuously at 5 weeks of age (5 animals per group), and blood was collected 3 hours after the final administration to measure the number of platelets and the number of red blood cells. As shown in Table 4, it was revealed that this substance significantly increased the platelet count at a risk rate (P) of 1% or less and exhibited a thrombopoietin action. The number of red blood cells did not increase at this time. In Table 4, Group 2 was administered with 2 μg of the purified substance dissolved in PBS containing 150 μg / ml bovine serum albumin (BSA) per administration, and Group 3 was administered with BSA alone as a control. .

[0067]

[Table 3]

[0068]

[Table 4] (Note) BSA: Bovine Serum Albumin (Example 4) The following is an example of a pharmaceutical composition containing the megakaryocyte-amplifying factor of the present invention as an active ingredient and a method for preparing the pharmaceutical composition. It is not limited to the example.

(Formulation Example 1) Purified megakaryocyte amplification factor of the present invention 1 mg Purified gelatin 20 mg Mannitol 100 mg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg The above ingredients were dissolved in distilled water for injection 2 ml and put in a sterile vial. , Primary drying at −35 ° C. and vacuum degree of 0.075 Torr for 35 hours, then 30 ° C., vacuum degree of 0.03 Torr
The vial for injection was manufactured by secondary drying for 5 hours. The obtained composition is dissolved in 500 ml of physiological saline or glucose injection immediately before administration and used for intravenous infusion.

(Formulation Example 2) Purified megakaryocyte amplification factor of the present invention 10 μg Albumin 5 mg Mannitol 25 mg Sodium chloride 1.95 mg Sodium phosphate 3.85 mg Injection was carried out in substantially the same manner as in Formulation Example 1 with the above components. Vials were manufactured.

[0071]

INDUSTRIAL APPLICABILITY The megakaryocyte-amplifying factor protein of the present invention has an activity of promoting the amplification of megakaryocytes and increasing the number of platelets in peripheral blood, and the activity is a known factor having similar activities. Is more powerful than. Therefore, the megakaryocyte amplification factor protein of the present invention can be effectively used alone or in the form of a pharmaceutical composition containing it as an active ingredient for the prevention and treatment of thrombocytopenia and the like.

[Brief description of drawings]

FIG. 1 C of the first purification step in the purification process of Example 2
The result of M sepharose column chromatography is shown.

FIG. 2 is the Q of the fourth stage of purification in the purification process of Example 2.
The result of sepharose column chromatography is shown.

FIG. 3 shows the results of gel filtration chromatography at the fifth purification step in the purification step of Example 2.

FIG. 4 shows the result of isoelectric focusing at the sixth purification step in the purification step of Example 2.

FIG. 5 shows the results of evaluating the megakaryocyte-amplifying factors (purified preparations 1 and 2) and the megakaryocyte-amplifying factor activity of IL-6 according to the present invention by a liquid culture acetylcholinesterase activity (AchE activity) assay method.

Claims (17)

[Claims] 1. A substantially pure megakaryocyte amplification factor protein having an activity of activating megakaryocyte amplification and having the following properties. (A) Molecular weight: 23000 ± 8000 (measured by gel filtration) (b) Isoelectric point: pI ≤ 5.9 (measured by isoelectric focusing) (c) Human erythropoietin, interleukin 1
The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against α, interleukin 1β, interleukin 6, interleukin 7, and interleukin 11. (D) It does not have megakaryocyte colony stimulating factor activity. 2. The megakaryocyte amplification factor protein according to claim 1, which has an isoelectric point (pI) of 3.5 ± 1 (measured by isoelectric focusing). 3. The megakaryocyte amplification factor protein according to claim 1, which has an isoelectric point (pI) of 4.9 ± 1 (measured by isoelectric focusing). 4. The megakaryocyte amplification factor protein according to claim 1, which is derived from human cells. 5. The megakaryocyte amplification factor protein according to claim 4, wherein the human cell is a normal diploid cell. 6. The normal diploid cell is derived from lung.
The megakaryocyte amplification factor protein described in 1. 7. A method for culturing animal cells, producing a megakaryocyte amplification factor protein in the culture solution, collecting a culture supernatant from the culture solution, and activating the megakaryocyte amplification activity from the collected culture supernatant. A method for producing a substantially pure megakaryocyte amplification factor protein, which comprises isolating and purifying a megakaryocyte amplification factor protein having the following properties. (A) Molecular weight: 23000 ± 8000 (measured by gel filtration) (b) Isoelectric point: pI ≤ 5.9 (measured by isoelectric focusing) (c) Human erythropoietin, interleukin 1
The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against α, interleukin 1β, interleukin 6, interleukin 7, and interleukin 11. (D) It does not have megakaryocyte colony stimulating factor activity. 8. The isoelectric point (p of the megakaryocyte amplification factor protein)
The method according to claim 7, wherein I) is 3.5 ± 1 (measured by isoelectric focusing). 9. The isoelectric point (p of the megakaryocyte amplification factor protein)
The method according to claim 7, wherein I) is 4.9 ± 1 (measured by isoelectric focusing). 10. The animal cell according to claim 7, which is a human cell.
9. The method according to any of 9. 11. The method according to claim 10, wherein the human cell is a normal diploid cell. 12. The method according to claim 11, wherein the normal diploid cells are lung-derived cells. 13. The method according to claim 7, wherein the cells are cultured by adding a megakaryocyte amplification factor protein production promoter to the medium. 14. The method according to claim 13, wherein the megakaryocyte amplification factor protein production promoter is animal meat peptone. 15. The method according to claim 14, wherein the animal cell is a cell derived from human lung. 16. A medicament containing the therapeutically effective megakaryocyte amplification factor protein according to any one of claims 1 to 3 and at least one kind of a pharmaceutically acceptable carrier, diluent and excipient. Composition. 17. IL-1, IL-2, IL-3, IL
-4, IL-5, IL-6, IL-7, IL-8, IL
-9, IL-11, GM-CSF, G-CSF, MC
The pharmaceutical composition according to claim 16, further comprising at least one factor selected from the group consisting of SF, SCF, IFNs, LIF, TNF and EPO.