WO1993016106A1

WO1993016106A1 - Novel megakaryocyte amplifier and production thereof

Info

Publication number: WO1993016106A1
Application number: PCT/JP1993/000155
Authority: WO
Inventors: Shuhei Kondo; Hiroki Shigematsu
Original assignee: Asahi Kasei Kogyo Kabushiki Kaisha
Priority date: 1992-02-07
Filing date: 1993-02-05
Publication date: 1993-08-19
Also published as: AU3463493A

Abstract

A substantially pure, novel megakaryocyte amplifier protein which has a molecular weight of 23,000 ± 8,000 as measured by gel filtration and an isoelectric point of 5.9 or less as measured by isoelectric electrophoresis, is differentiated immunologically from human erythropoietin, interleukin 1α, interleukin 1β, interleukin 6, interleukin 7 and interleukin 11, does not have a megakaryocyte colony stimulating activity, and has the activity of activating megakaryocyte amplification. The protein can be produced by utilizing cell culture techniques. It has the activities of promoting megakaryocyte amplification and increasing the content of platelets in the peripheral blood, thus being efficacious in preventing and treating thrombocytopenia and the like.

Description

Detail

Novel megakaryocyte amplification factor and method for producing the same

Technical field

The present invention relates to a novel megakaryocyte mitogen and a method for producing the same. More specifically, the present invention relates to a novel megakaryocyte amplification factor protein having an activity of promoting proliferation of megakaryocytes, which are platelet precursor cells, and having an action of promoting platelet production, and its production by cell culture. About the method. The present invention also relates to a pharmaceutical composition containing the novel protein as a megakaryocyte amplification factor useful for prevention and treatment of diseases such as thrombocytopenia.

Background art

Platelets play an important role in promoting the process of blood clot formation and blood clotting, which the body naturally stops bleeding caused by torn blood vessels. Thrombopoetin (TPO), a factor that specifically promotes the production of platelets, has been used by many researchers for more than 20 years with a passion for its acquisition. We have not yet succeeded.

When blood plasma from a thrombocytopenic animal is administered to healthy animals, platelet production increases, and conversely, when platelets are transfused, platelet production decreases. The existence of DPO, which has the effect of regulating its production, has been proposed for a long time. Subsequent studies show that platelets are released from the bone marrow stem cells into the blood from the megakaryocytes, which are differentiated and matured in the bone marrow from megakaryocyte progenitors. In addition, the results of invio showed that megakaryocytes differentiated at an early stage of the amplification process, and that megakaryocyte colony stimulating factor (Meg-CSF) exerted a force S action. It was revealed that TPO, which has the activity to promote megakaryocyte maturation, acts in the late stage. First, Meg-CSF acts, progenitor cells repeat cell division, and megakaryocyte components increase.Next, TPO acts, and each megakaryocyte progenitor performs endamisis. As the chromosome multiple is increased (up to 32 N), the cytoplasm matures and increases, and platelet production begins. TPO is also sometimes referred to as megakaryocyte potentiator (Meg-P0T).

The activity of Meg-CSF can be estimated by measuring the activity of megakaryocyte colony formation in soft agar culture of human or mouse bone marrow cells in vitro. Currently, the activity of Meg-CSF is measured in urine of patients with aplastic anemia and patients with idiopathic thrombocytopenic purpura, in blood of patients with myelomegaly aplastic thrombocytopenia, and in kidney bean lectin-stimulated cells. G. It is found in leukocyte culture supernatant, mouse leukemia cell line WEHI-3 culture supernatant, etc.

Among various site-induced proteins, interleukin 3 (IL-3) (interleukin is abbreviated as IL) acts non-specifically on many strains including megakaryocytes. It is becoming clear that it is ti-CSF. In addition, Meg-CSF in WEH 3 culture supernatant was completely consistent with IL-3, indicating that Meg-CSF activity in Many are attributed to IL-3. However, Meg-CSF, which has been shown to specifically act on the platelet system, is not yet known.

—The activity of TPO can be estimated by measuring the strong effect of the colony forming activity of Meg-CSF and the effect of promoting the maturation of or megakaryocytes. Attempts have been made to prepare some factors having TP〇-like activity. It is prepared from the culture supernatant of a human fetal kidney cell line and has the effect of promoting protein synthesis in megakaryocytic cells having a molecular weight of 15,000 on SDS-PAGE and an isoelectric point of 5.1. A Megakaryocyte Stimulatory Factor (MSF) and a method for its production have been reported (see US Pat. No. 4,894,440). Recently, it was discovered as a glycoprotein that induces B cell antibody production, and has been implicated in the immune system, acute-phase reaction system, and malignant tumors. IL-6 is also involved in the hematopoietic system and shows Meg-! ^ OT activity and megakaryocyte maturation promoting activity in vitro (Ishibashi, T. eta 1. rproc. Natl. Acad. Sc i. USAJ 86, 5953 (1989)), in

It has been confirmed that V i V 0 exhibits a platelet production promoting effect (Asano, S. eta 1. “Blo.d” 75, 1602 (1990)). Furthermore, 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 or not these are constitutive (healthy) hematopoietic factors inherent to living organisms. In order to find a novel megakaryocyte amplifying factor having the action of specifically and strongly promoting megakaryocyte amplification and the action of promoting platelet production in the above-mentioned technical background, the present inventors As a result of surprising studies, surprisingly, a completely new megakaryocyte amplification factor was found in the culture supernatant of human normal diploid cells, and by adding an appropriate production promoter to the culture medium, It has been found that a large amount of the factor is produced. Further, the factor was isolated and purified from the collected culture supernatant, and its properties were clarified, and its usefulness as a drug was demonstrated. The megakaryocyte amplification factor can also be expressed by applying genetic engineering technology. The present invention has been completed based on these findings.

Disclosure of the invention

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

Another object of the present invention is to cultivate animal cells in a medium, produce megakaryocyte amplifying factor in the culture solution, collect a culture supernatant from the culture solution, and use a megakaryocyte from the collected culture supernatant. An object of the present invention is to provide a method for producing a megakaryocyte amplification factor, which comprises purifying a sphere amplification factor.

Still another object of the present invention is to increase the amount of the megakaryocyte amplification factor produced by adding the megakaryocyte amplification factor production promoter to the medium in the cell culture and culturing the cell. 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 amplifying factor as an active ingredient, and a therapeutic method using the same.

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

(a) Molecular weight: 230,000 ± 800,000 (measured by gel filtration) (b) Isoelectric point: p i ≤ 5.9 (measured by isoelectric focusing)

(C) human collar bets Ropoechin, interleukin I _alpha, I centers B A rk 1) 3, b Ntaro Lee Kin 6, using each antibody against Lee centers B A rk 7 and I interleukin 1 1 Activity is not substantially reduced in the megakaryocyte amplification activity neutralization test.

(d) It does not have megakaryocyte colony-stimulating factor activity.

Examples of the megakaryocyte amplification factor proteins of the present invention include the following two substantially pure megakaryocyte amplification factor proteins that have the activity of activating megakaryocyte expansion and have the following properties: Can be mentioned.

1-(a) Molecular weight: 230 000 ± 800 ◦ (measured by gel filtration)

(b) Isoelectric point: pi 3.5 ± 1 (measured by isoelectric focusing) (c) Human erythropoietin and interlocutin ΐ _α -indolokin I, interlocutin 6, interlocutin 7, and inter-pikin Activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against 11.

(d) It does not have megakaryocyte colony stimulating factor activity.

2. (a) Molecular weight: 230 000 ± 800 (measured by gel permeation)

(b) Isoelectric point: p i 4.9 ± 1 (measured by isoelectric focusing)

(c) Human erythropoietin, interlokin 1 interlokin 1 ^ 3, interlokin 6, interlokin 7 and interlokin 11. The activity is not substantially reduced in the megakaryocyte amplification activity neutralization test using each antibody against the above.

(d) It does not have megakaryocyte colony stimulating factor activity.

According to another aspect of the present invention, animal cells are cultured in a medium, megakaryocyte amplification factor is produced in the culture medium, a culture supernatant is recovered from the culture medium, and the culture supernatant is recovered from the recovered culture supernatant. There is provided a method for producing a megakaryocyte amplification factor, comprising separating and purifying a megakaryocyte expansion factor.

The animal cell used in the method of the present invention has an activity of activating megakaryocyte amplification and increases platelets in peripheral blood. Various cells having the ability to produce megakaryocyte expansion factor having the activity to be added 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 from lung, foreskin, and even more preferably cells from human fetal lung can be used.

The megakaryocyte amplification factor can be separated and purified from these tissue extracts, but preferably, these cells are cultured in a suitable growth medium, and Then, the megakaryocyte amplification factor is produced, the culture supernatant is recovered from the culture solution, and it can be separated and purified from the recovered tissue culture solution. It is desirable that these cells be propagated by a culture method used for normal cell culture, for example, the method described in “Tissue culture” (Junnosuke Nakai et al., Asakura Shoten, edited by Showa 51) and used in the present invention. The cells can produce megakaryocyte spreading factor by culturing them in a medium solution containing carbons, nitrogen source and, if necessary, inorganic salts and / or other additives. Further, according to the present invention, the megakaryocyte produced in a culture solution is obtained by adding a megakaryocyte amplification factor production promoter, preferably an animal meat enzyme-decomposing peptone, and culturing the cells. The amount of the sphere width factor can be greatly increased. The concentration of animal meat enzyme-degrading peptone can be (! -4 _W / V%, preferably 0.1-2 w / v%, based on the culture medium. , Which is commonly used for bacterial culture media Therefore, it is usually called proteose peptone, proteosebuton, or meat peptone.

The method for preparing this animal meat enzyme-degrading peptide is well-known, for example, according to the method described in "Bacterial Culture Studies Lecture Series 2" (Sakazaki Toshiichi, Naya Shoten, 1967). That is, as animal meat, meat or offal such as cows, pigs, nits, sheep, and whales are used, and beef is most commonly used. As enzymes for the degradation, trypsin, papine, pepsin, and no. There is creatine. These animal meats are crushed, mixed with water, and adjusted to a pH suitable for enzymatic degradation with sodium carbonate, concentrated hydrochloric acid and the like. The enzyme is added to this and the enzyme is digested at 20-40 ° C for 1-20 days, usually at 37 ° C for 2-3 days. After digestion, heat to 100 ° C or higher to inactivate degrading enzymes and heat coagulate undigested proteins, remove them by filtration, and then concentrate, dry, and finely powder. . Concentration and drying methods include boiling into powder and concentrating at a low temperature using a vacuum drying device, followed by fine powdering. Commercial products include Proteose Peptone No. 1 (Pr. Te.se Pep t. No. 1), Proteose Peptone No. 2 and Proteose Peptone manufactured by Difco Inc. of the United States. No. 3, Thiopepton, Proteorpeptin L46, Pokton PL46, manufactured by Oxide of the United Kingdom, Thioton (Thiot on), manufactured by BBL of the United Kingdom, Daigo There is a proteose plot etc. manufactured by Nutrition Chemistry.

Next, an example of production of a megakaryocyte amplification factor by a cell culture method will be described. Use primary culture cells or commercially available cells that produce the factor directly removed from the tissue, and culture them by adherent culture or suspension culture. One example, a suitable cell density, favored properly in a density of 1 0 ⁵ C el I s / ml, with 0. 1~ l Om g / ml of cell culture bead carrier Implantation, serum-containing 15-45 ° C, preferably 25-40. In the temperature range of C,. 5 to 9 favored properly in culture p H range of 6-8, are cultured in air containing normal 5% CO _2. When using a megakaryocyte amplification factor production promoter, serum-free conditions should be used. (Production culture is performed at a concentration of 3 to 4%, preferably 0.1 to 2 o / o. The cells are allowed to grow sufficiently and are transferred to a production culture, preferably in a confluent state.The number of culture days for production is usually 1 to 60 days, but can be more than 60 days. The production rate of megakaryocytes is gradually slowed in the latter half of production, so the most efficient days are selected for industrial production. It is produced in solution from cells, and the amount of production is measured by the megakaryocyte amplification activity measurement method described in Reference Examples (a) and (b).

The megakaryocyte amplification factor of the present invention is obtained by expressing the megakaryocyte amplification factor in a suitable host cell by using a commonly used genetic technique, and collecting and purifying the megakaryocyte amplification factor. Can also be obtained.

That is, it produces a megakaryocyte amplification factor having an activity of activating megakaryocyte amplification and an activity of increasing platelets in peripheral blood. 'Cha,

106

Ten

A variety of cells, such as human kidney, intestine, lung, heart, urinary tract, skin, human foreskin, tongue, thyroid, placenta, uterus, or human fetus Total RNA is extracted from cells from the kidney, lung, foreskin, and more preferably from cells from fetal human lung, and Liboli A ⁺ RNA is further purified. Base click coater for appropriate expression, like properly is to prepare c DNA library over using vectors and Po Li A ^÷ RNA and linker one for eukaryotic expression, suitable using this line bra rie A host cell, for example, Escherichia coli is transformed, and lipase DNA is prepared from the culture solution. The plasmid is used to transfect a suitable host cell, preferably a cell derived from an animal, or more preferably, a monkey-derived c0s cell to obtain a megakaryocyte amplification factor gene. The megakaryocyte-amplifying factor can be produced by expressing, collecting and purifying it.

Next, a specific example will be described. To produce megakaryocytes. Ball amplification factor of the appropriate amount of cells, for example, human fetal lung cells favored properly is 1 0 ⁸ ce 11 s Yo Li, RNA eye Soviet Reshi ® Nki' door for example the United States, Lee Nbi door androgenic Co., Ltd. Using the product (catalog NO.K1592-01), total RNA is extracted by the guanidine isothiosinate method according to the attached manual, and poly A + RNA is obtained according to the conventional method. At this time, Oligotechs dT30 (manufactured by Nippon Synthetic Rubber Co., Japan) can be used. Usually, according to the above method, about 200 jig of total RNA and 1 to 2 / ig of poly A10 RNA are obtained. Next, a cDNA library is prepared according to the method of Okayama Ichiberg. For example, the eukaryotic expression vector 3'-oligo (dT) -tai led pcDV-1 (manufactured by Pharmacia, Sweden, No. 27-4955-01) and the poly A ⁺ RNA obtained above and 3'-o1 igo (<3G)-tai led pLl linker (No. 27-4957, manufactured by Pharmacia, Sweden) can be used. Alternatively, pcDL-SRα296 may be used. The resulting solution containing the cDNA library is divided into an appropriate number of pools, preferably 10 to 200, more preferably 50 to 100, and each of them is divided into E. coli MC1061 (ATCC533338). ). Incubate the transformed E. coli in the presence of ampicillin overnight. After collecting and lysing the cells, prepare plasmid DNA using Qiagen-tip-100 (manufactured by Qiagen, USA) according to the attached manual. The obtained recombinant DNA is, for example, According to the Norredextran method (CURRENT PROTOCOLS IN MOLECULAR BIO LOGY 9.2.1.-9,2.6), suitable host cells, preferably monkey kidney cells, C0S1 cells (ATCC, CRL165) After that, the gene is expressed almost in the same manner as in the method described in WO 88/05053, Example 2. That is, under appropriate culture conditions, for example, a D-MEM medium containing 10% fetal bovine serum (manufactured by Flora Laboratory, USA). (: After culturing under conditions of 5% CO ₂ for 40 hours, change to serum-free D-MEM medium, and collect the culture medium three times every two days.

Further, according to the present invention, the expression cells incorporating the megakaryocyte amplification factor gene can be screened using the activity of the megakaryocyte amplification factor as an index to clone the megakaryocyte amplification factor gene. You.

That is, after concentrating each collected medium, the megakaryocyte amplifying factor activity is measured by a method such as acetylcholinesterase activity measurement in liquid culture, and the pool containing the gene of this substance is used as an index. Can be narrowed down. Further, for the positive DNA, the E. coli is transformed again, and the obtained colonies (about 2000) are cultured as a group of about 10 cells, and the DNA is prepared in the same manner as above. In addition, introduction and expression into COS 1 cells and measurement of acetylcholinesterase activity can be performed to narrow down a secondary cDNA library. Usually, by repeating this method several times, Escherichia coli having a cDNA plasmid expressing the megakaryocyte amplifying factor activity is isolated. (Hayash i da, K. eta 1 "Hema topoiec Factor" 1, No. 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 megakaryocyte amplification factor and use it to clone the megakaryocyte expansion factor gene.

Furthermore, according to the present invention, using the cloned megakaryocyte amplification factor gene, for example, Escherichia coli, yeast, monkey kidney cells (COS cells), Chinese hamster ovary cells (CH〇 cells), mouse Transfect host cells such as C127 cells, human fetal kidney cell line, silkworm cell SF9, etc., and express the megakaryocyte amplifying factor more efficiently, collect and purify it. Thus, megakaryocyte amplification factor can be produced.

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, the culture supernatant is collected when the production of megakaryocyte expansion factor by the production cells reaches a desired production amount or days. I do. Examples of the method for separating and purifying the megakaryocyte amplification factor include methods usually used in protein chemistry, for example, adsorption using a carrier, salting out, electrophoresis, ion exchange, gel filtration, and the like. A variety of chromatographic methods, etc. that apply ligand affinity can be used alone or in combination. As a chromatographic method, preferably, a CM cell using Sepharose to which a carboxymethyl group is bonded is used. Q-Sepharose column chromatography using phenyl sucrose chromatography, quaternary aminoethyl groups bonded to quaternary aminoethyl groups, cross-linked dextra Use gel filtration column chromatography using particles such as gel, hydrophobic column chromatography, and antibody affinity chromatography that binds antibodies that specifically bind to the substance of the present invention. it can. In particular, the megakaryocyte amplification factor of the present invention is obtained from the fraction having the activity of the megakaryocyte amplification factor obtained during the purification using, for example, isoelectric focusing electrophoresis or ion exchange chromatography. The fraction that is considered to have an electric point can be isolated by separating and further purifying it.

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 amplifying factor is used as a reagent for studying the differentiation, proliferation and maturation of megakaryocytes from bone marrow stem cells or bone marrow megakaryocyte progenitor cells, or as a megakaryocyte amplifying factor alone or therapeutically. An effective amount of the megakaryocyte amplification factor is added to at least one selected from pharmaceutically acceptable carriers, diluents and excipients to form a suitable dosage form, and the resulting drug is Can also be used. As the carrier, diluent and excipient, those usually used in this field can be used. The megakaryocyte amplifying factor, IL-11, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, At least one factor selected from IL-11, GM-CSF, G-CSF, M-CSF, SCF, IFNs, L-IF, TNF and EPO, and a pharmaceutically acceptable carrier; At least one selected from diluents and excipients can be added to make a suitable dosage form and used as a pharmaceutical.

The megakaryocyte amplification factor of the present invention is useful for the treatment of certain thrombocytopenia, for example, thrombocytopenia after administration of anticancer drugs, thrombocytopenia after radiation therapy, thrombocytopenia due to megakaryocyte amplification factor deficiency, and aplastic anemia. It can be used to treat and prevent or prevent thrombocytopenia, thrombocytopenia after bone marrow transplantation, and thrombocytopenia in autoimmune diseases. It can also be used to treat leukemia. Further, it can be used as an alternative or adjuvant to platelet transfusion, or for growth culture of i.nvitro for bone marrow cells for transfusion.

The megakaryocyte amplification factor of the present invention can be used as an injection. In this case, sucrose, glycerin, methylcellulose, a thickener such as carboxymethylcellulose, a pH adjuster for various inorganic salts, and the like can be added as additives.

The dosage of the megakaryocyte amplification factor of the present invention per adult dose varies depending on the age, sex, weight, symptoms, etc., but is generally about 0.1 μg to 100 mg per day. It can be administered once or several times as needed.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to describe the present invention in more detail, reference examples and examples were used. Although described again, the scope of the present invention is not limited to these examples. .

Reference example Megakaryocyte amplification activity measurement method

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-P〇T activity assay by soft agar culture

Preparation of bone marrow cell suspension

The IMDM solution (Iscoes modei cation of Da 11 becco's med ium) used in the following method for preparing a bone marrow cell suspension was powdered IMDM (for 1 liter) (manufactured by Gibco, USA) 3 重 024 g of sodium bicarbonate, β-Menolecaptoethanol, 3.04 microliters, adjust ρ に to 7.1, then adjust to 1 liter, and further increase to 50 IU / It was prepared by adding Millirit Norrenicillin and 50 μg Z Milliliter Norest Streptomycin (both manufactured by Floraborate Lease Co., USA).

The femurs of 6- to 9-week-old C57BLZZ6 mice (male) were harvested, the upper part was cut, and 10 milliliters of 10 milliliters of IMD M solution was added. Using a plastic syringe (22 G needle), the bone marrow was pushed out vigorously into the 100 mm plastic dish from the knee joint side. After dispersing the cells by pipetting operation about 8 times (6 times with a 19 G needle and 2 times with a 22 G needle), transfer the cells to a 15-milliliter Falcon tube, Of the cells, wash them twice with 10 milliliters of IMDM, and finally The bone marrow cell suspension obtained by suspending and dispersing the mice in a 2.5-milliliter IMDM solution per mouse was further subjected to the following colony assay. The cell concentration was measured with a hemocytometer by staining with Trypan Blue (produced by Flora Bola Treasure Co., USA).

Colonia Tusei

The acetyl cholesterol esterase staining solution used in the next experiment was 1.73 mM acetyl thiocholin iodide, 0.5 mM potassium ferricyanide, 5 mM sodium citrate, and 3 mM copper sulfate (all in Japan). (Manufactured by Wako Pure Chemical Industries, Ltd.), and dissolved in 400 milliliters of a 75 mM phosphate buffer of pH 6.0.

10 ⁵ bone marrow cells were added to a 100-mm plastic dish with horse serum (manufactured by J.R. Scientific, USA) at 15 V / V ° / ₀ . 3 (manufactured by Genzym, USA) COS 1 cell culture supernatant containing 50 ng or IL-3 was added to a 500 microliter IMDM liquid culture medium added to 1 v / v%. The test solution was added, the mixture was made into a soft agar under the conditions of 0.3% Bata Toaga (manufactured by Difco, USA), and cultured at 37 ° C and 5% CO ₂ for 7 days. As a culture supernatant of COS 1 cells containing IL-3, COS 1 cells (ATCCCRK165) were prepared using a plasmid in which mouse IL-3 cDNA was linked to the SV-40 promoter. With the method described in WO 88/05053, Example 2. The culture supernatant containing IL-3 expressed in the same manner was used.

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

(b) Measurement of acetylcholinesterase activity by liquid culture

The mouse bone marrow cell suspension prepared in the same manner as in the measurement in (a) above was added to diisopropylfluorophosphate (DFP) (Sigma, USA) to a final concentration of 0.4 mM. Was added and left at room temperature for 20 minutes with occasional agitation to eliminate the intrinsic acetylcholinesterase activity of the bone marrow cells. The cell number was counted by a hemocytometer as described above.

Eliminate endogenous acetylcholinesterase activity with DFP so that the cell density of the bone marrow cell suspension is 2.5-5 × 10 ⁵ ce 11 s / milliliter (J.Cel. .122 159 (1985)) was suspended in IMDM solution containing 15 V / v%, and placed in a 96-well culture dish (Sumitomo Bakelite, Japan). , 2 milliliters were dispensed. 20 microliters of the sample solution was added, and the cells were cultured at 37 ° C and 5% CO ₂ for 7 days. After culturing, the cells are spun down by centrifugation, and the supernatant is removed. In each well, 20 microliters of 5.6 mM acetylacetylcholine iodide (Sigma, USA) and 180 microliters Was added to a 50 mM epes buffer (pH 7.5) containing 0.12 M saline, 1 mM ethylenediaminetetraacetic acid, and 0.2% triton X-100, and reacted at room temperature for 3 to 4 hours. . Transfer 20 microliters of the reaction mixture to a 96-well plate for fluorescence measurement (Gleiner, Germany), and add 160 microliters of 1 mM ethylenediaminetetraacetic acid, 0.2% PH 5.0 acetate buffer and 20 microliters of 0.4 mM CPM (7-diethylamino-3- (4-malimidylphenyl) -4-methylcoumar in) (US, Acetonitrile solution (Molecular Probes) was added and reacted at room temperature for 1 hour. The acetylcholinesterase activity was measured by using PANDEXFCA (manufactured by Baxter, USA) by measuring the fluorescence at 450 nm with excitation light at 365 nm.

Example 1

Production of megakaryocyte amplifying factor by human cell culture method

Commercially available human fetal lung normal diploid cells (the United States, Furorapora door made rie, Inc.) to the Garasubo torr 1 0 0 liters capacity, at a density of 1 0 ⁵ eel Is / ml 2.5 mgZ of ml concentration Size Lee Todekkusu I (bead carrier for cell culture, Sweden, Pharmacia Co.) with implantation in 3 7 ° C, 5% C_〇. air containing _2, a growth medium Containing 10% fetal calf serum 60 liters of PEM MME medium was added, and suspension culture was performed with stirring at a rotation speed of 60 rpm. After culturing for 8 days and allowing the cells to grow sufficiently, wash the bead carrier to which the cells are adhered with a physiological saline solution, and contain or contain 0.75% serum-free proteose peptone No. 3 The medium was replaced with 60 liters of medium 199 medium, and cultured with stirring at a rotation speed of 60 rpm. While exchanging this medium every third day, a culture medium containing the substance of the present invention was collected. The culture solution was concentrated 10-fold, and the megakaryocyte amplifying factor activity contained therein was evaluated by the Meg-POT activity measurement method using the soft agar culture method shown in Reference Example (a). Table 1 shows the results. By reference, several other cell cultures were also evaluated. It was shown that human fetal lung cells have the ability to produce megakaryocyte amplifying factors, and that when cultured in the presence of proteose peptides, the production of megakaryocyte amplifying activity was significantly increased.

Two

Table 1 Sample of culture solution Knee number 1 0 ⁵ cel Is Human fetal lung cell culture solution First recovery solution 3 3

(+ P P) Twice a day recovery solution 2 8

Fifth recovery liquid 3 1

1 0th recovery liquid 3 2

Human fetal lung cell culture solution First recovery solution 5

(1 P P) 5th recovery liquid 4

TH P—1 (leukemia cells) 2

Hep G 2 (liver cancer cells) 3

T 24 (bladder cancer cells) 2

MC F 7 (breast cancer cells) 0

[Notes] 1) The number of times indicates the number of times every third day described above.

2) P P; Proteose peptone ''

3) The number of colonies in each sample was calculated by subtracting 3 from the number of colonies generated when only L-13 was added to the Atsushi strain.

Example 2

Purification of megakaryocyte amplification factor

Approximately 1.2 liters of acetic acid was added to 190 liters of the culture supernatant of normal diploid human fetal lung cells obtained in Example 1 to adjust the pH to 4, and then filtered. Thus, cell fragments and generated insolubles were removed. Carboxymethyl sepha-mouth, which had been sufficiently equilibrated with a 20 mM acetic acid buffer solution of pH 4.0 containing 0.2 M salt in advance

(CM Sepharose; manufactured by Vanoremasia, Sweden) Adsorbed on a column (9 cm in diameter x 23.5 cm in height), 13.5 liters and 0.4 ml of the same equilibration buffer After washing with 6 liters of 2OmM acetate buffer at pH 4.0 containing M salt, 2M at pH 4.0 containing 0.75M salt and 1OmM lysine hydrochloride The adsorbed protein was eluted with 12 liters of mM acetate buffer. By this operation, approximately 5.5 liters of eluate was obtained as crude purified solution I containing megakaryocyte amplification factor activity. Proteopeptide components contained in the culture supernatant in large amounts decreased to i% or less. Since the eluate usually contains a large amount of tissue plasminogen activator (tPA), it was specifically removed. That is, after adding a 5 M sodium hydroxide solution and adjusting the pH to 7.0, a 20 mM Tris-HCl buffer solution (pH 7.5, containing 0.5 M salt) is sufficient in advance. Monoclonal antibody against tPA was equilibrated to Sepharose and bound to Sepharose (3 mg / milliliter gel), antibody column (diameter 9 cm x height 29 cm) Was passed through. By this operation, about 6 liters of the crude solution II was obtained as crude solution II. By this column chromatography, megakaryocyte amplification factor activity was almost quantitatively recovered. Further, the tPA activity contained in the crude purified solution I in a large amount was removed.

The crude solution II 6.2 liters were concentrated to 300 milliliters with an ultrafiltration module SIP 1103 (made by Asahi Kasei Corporation, Japan), and the volume of PH 7 was increased to 10 times. 2.0 of 2OmM sodium phosphate buffer was added, the mixture was again concentrated to 300 milliliters, and the buffer was exchanged. A CM Sepharose column (5 cm in diameter x 15 cm in height), which had been well equilibrated with 20 mM sodium phosphate buffer at pH 7.0 containing 5 OmM salt, was prepared. The mixture was passed through to give a roughly purified solution III of about 300 milliliters. Figure 1 — (a) shows an example of the results of the CM Sepharose column chromatography on the first purification stage. An equal volume of purified water and 3 milliliters of 0.5 M tris were added to the crude purified solution III, and the pH was adjusted to 8.0 with hydrochloric acid. Q Sepharose (manufactured by Pharmacia, Sweden) column (2.5 mm in diameter), which had been sufficiently equilibrated with 2 OmM Tris-HCl buffer containing 5 OmM salt at pH 8.0. After being adsorbed to a height of 10 and 111) and washed with about 100 milliliters of the same buffer, about 120 milliliters of the same buffer containing 0.1 M sodium chloride was added. Buffer (E-1), about 200 milliliters of the same buffer containing 0.25 M salt (E-2), and the same buffer containing 0.5 M salt (E-3) To elute each. The flow rate was 200 milliliters / hour. Megakaryocyte amplification factor activity was observed in E-1, E-2, E-3 and all fractions, but megakaryocyte amplification factor with acidic isoelectric point was mainly in E-3 fraction. Recovered. That is, about 50 milliliters of the E-3 fraction was recovered as the roughly purified liquid IV at an activity recovery rate of about 10%. Figure 1-(b) shows an example of the results of Q Sepharose column chromatography.

The crude solution IV 48 milliliters was concentrated to 3 milliliters using the above ultrafiltration hollow fiber, and this was previously concentrated to 50 mM sodium phosphate with a pH of 7.3. Gel filtration was performed on a column of Sephacryl S—200 (manufactured by Pharmacia, Sweden) which had been sufficiently equilibrated with a buffer solution (diameter: 1.6; height: 900; 111). The megakaryocyte amplification activity was recovered as a fraction having a peak at an eluted molecular weight of about 23 kd, i.e., a crudely purified solution V of about 25 milliliters at an activity recovery rate of 30 to 40%. Was. Figure 1 — (c) shows an example of the results of Sephacryl S-200 column chromatography. Equivalent volume of purified water was added to crude solution V25 milliliter. Then, concentrated and desalted, and added with ampholine (3.5-10) (Pharmacia, Sweden) to a final concentration of 2%, followed by rotophoresis (Biorad, U.S.A.). Was carried out at a constant voltage of 12 bits for 5 hours. An example of the results of isoelectric focusing is shown in Figure 1-(d). Megakaryocyte spreading activity had peaks at PH 3.5 and 4.9. ϋ H 3. Active fractions near 5 and 4.9 were collected, respectively, to give purified samples 1 and 2 of megakaryocyte amplification factor. The total protein of purified sample 1 and purified sample 2 was 0.9 mg and 0.9 mg, respectively.

It was 15 mg.

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

Sample name Volume Protein recovery Activity recovery Percentage of purification (Little) (%) (%) (times) On culture 淸 190000 100 100 1 Crude solution I 5500 0.42 60 143 Crude solution II 6200 0.32 54 168 Liquid III 300 0.12 21 175 Crude liquid IV 48 0.01 2.2 220 Crude liquid V 25 2.0X10— ⁴ 0.77 3900 Purified sample 1 2.5 0.08 13000 Purified sample 2 2.5 1.0X10— ⁵ 0.07 70000

[Note] The protein concentration of each sample was calculated assuming that the ultraviolet extinction coefficient at 28 O nm Α280, 1% = 10. The purified sample 2 was concentrated 10-fold and the absorbance was measured.

Example 3

Physical properties of megakaryocyte amplification factor

(Refined product 1)

(a) molecular weight

Purification obtained in Example 2 using a Sephacryl S—200 HR (manufactured by Pharmacia, Sweden) column (diameter 1.6 cm × height 90 cm) previously equilibrated with PBS. Sample 1 was developed with PBS (flow rate: 20 milliliter time), fractionated by 2 milliliters, and the megakaryocyte amplification factor activity of each fraction was measured. The molecular weight of the substance was determined 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 near the molecular weight of 23,000.

(b) Isoelectric point

The purified sample 1 obtained in Example 2 was applied to an isoelectric focusing column (1

Glycerol density gradient isoelectric focusing was performed using a 10-milliliter bottle (manufactured by Sho Kato, Japan) with a constant power of 3 bits for 40 hours. As an amphoteric carrier, 1% ampholine (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 reacts immunologically with known cytokines was examined using the purified preparation 1 obtained in Example 2. Anti-human Erythropoietin antibody, anti-human IL-1 antibody, anti-human IL-1 / 3 antibody, anti-human IL-16 antibody, anti-human IL-17 antibody (all manufactured by Genzym, USA) and anti-human IL-17 antibody Using the human IL-11 antibody, a neutralization experiment was performed on the megakaryocyte amplification factor activity of this substance. As a result, it was shown that the activity of this substance did not decrease even when treated with these antibodies, and it did not react with these antibodies. This substance is used for anti-human IL-11 antibody column and anti-human IL-6 antibody column.

(Endogen, USA) showed no adsorptive properties. This indicates that the megakaryocyte spreading factor of the present invention is immunologically distinct from these known cytokines.

(d) Megakaryocyte amplification factor activity

The megakaryocyte amplification factor activity of this substance was examined by the soft agar culture method. Table 3 shows the results. The Meg-POT activity of IL-16 (Genzam) was only 2.5 times higher than that of IL-13 alone, even at a high concentration of 200 ng / milliliter. In contrast, the purified preparation of this substance showed 14 times the activity of IL-13 alone. Compared to the case of only IL-13, the purified sample of this substance showed a greater number of cells per colony that strongly showed acetylcholinesterase activity. The substance alone did not show megakaryocyte amplification factor activity.

Furthermore, the megakaryocyte amplifying factor activity of this substance was evaluated by a method of measuring acetylcholinesterase activity in liquid culture. Figure 2 shows the results. In this method, the megakaryocyte amplification factor of this substance Child activity was demonstrated.

(e) Thrombopoetin action

The purified sample 1 obtained in Example 2 was intraperitoneally administered to mice (C57BL male, 7 weeks old, 5 mice per group) for 5 consecutive days, blood was collected 3 hours after the final administration, and the platelet count and red blood cells were collected. The number was measured. As shown in Table 4, it was found that this substance significantly increased the platelet count at a risk factor (P) of 1% or less and exhibited a thrombopoietin effect. At this time, the number of red blood cells did not increase. In Table 4, group 1 administered 1 μg of this purified substance in PBS containing 150 μg / ml of serum serum albumin (BSA) per dose. 3 received BSA only as a control.

(Purified sample 2)

(a) molecular weight

Using a Sephaacryl S-200 HR (manufactured by Pharmacia, Sweden) column (diameter 1.6 cm x height 90 cm) previously equilibrated with PBS, the purified sample 2 obtained in Example 2 was used. Was developed in PBS (flow rate: 20 milliliter-torno time), fractionated by 2 milliliters, and the megakaryocyte amplification factor activity of each fraction was measured. The molecular weight of the 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 near the molecular weight of 23,000. (b) Isoelectric point

Purified sample 2 was obtained using isoelectric focusing column (110 milliliters) (manufactured by Shoichi Kato Shoten, Japan) at 3 Watts of constant power. Glycerol density gradient isoelectric focusing was performed for 40 hours. As an amphoteric carrier, 1% ampholine (Pharmacia, Sweden) was used. This substance had an isoelectric point in the pH range of 3.9 to 5.9.

(c) antigenicity

Whether the substance reacted immunologically with known cytokines was examined using the purified preparation 2 obtained in Example 2. Anti-human erythropoietin antibody, anti-human IL-11α antibody, anti-human IL-11 ^ 3 antibody, anti-human IL-16 antibody, anti-human IL-17 antibody (all in the US and Jenzym And anti-human IL-11 antibody were used to neutralize the megakaryocyte amplifying factor activity of this substance, and as a result, the activity of this substance decreased even when treated with these antibodies. The substance was not reacted with these antibodies, and this substance was used as an anti-human IL-11 antibody column and an anti-human IL-16 antibody column.

(Endogen, USA) showed no adsorptivity. This indicates that the megakaryocyte amplifying factor of the present invention is immunologically distinct from these known cytokins. (D) Megakaryocyte amplifying factor activity

The megakaryocyte amplification activity of this substance was examined by the soft agar culture method. Table 3 shows the results. Meg—of IL-6 (manufactured by Genzym) POT activity was only 2.5 times that of IL-13 alone, even at the high concentration of 200 ng / milliliter. In contrast, the purified preparation of this substance showed 6 times the activity of IL-13 alone. Compared with the case of IL-3 alone, the purified sample of this substance showed a greater number of cells showing strongly acetylacetylcholinesterase activity per colony. This substance alone did not show megakaryocyte amplification factor activity.

Furthermore, the megakaryocyte amplifying factor activity of this substance was evaluated by an acetylcholinesterase activity measurement method using liquid culture. Figure 2 shows the results. This method also showed megakaryocyte amplification factor activity of this substance.

(e) Thrombopoetin action

The purified sample 2 obtained in Example 2 was intraperitoneally administered to mice (C57BL male, 7 weeks old, 5 animals per group) for 5 consecutive days, blood was collected 3 hours after the final administration, and the platelet count and The red blood cell count was measured. As shown in Table 4, it was found that this substance significantly increased the platelet count at a risk factor (P) of 1% or less and exhibited a thrombopoietin effect. At this time, the number of red blood cells did not increase. In Table 4, for Group 2, 2 μg per dose of this purified substance was added to 150 μg

Group B was administered as a control dissolved in PBS containing 1 ml / ml serum serum albumin (BSA). Group 3 received BSA alone as a control.

Table 3 Sample name Mouth-One

I L—3 only

(15 My microliter)

I L 3 + purified sample 1

(30 My crottle / Milliliter) 1 6 (150 Mycroliter / Milliliter) 2 8

I L 3 + purified sample 2

(30 micro liters / "mill liter") 1 2 (150 micro liters liter) 1 2 Refined sample 1 only

(30 micro liters / mill liter) ■ 0 (150 micro liters liter) 0 Purified product 2 only

(30 My microliter / Milliliter) 0 (150 My microliter / Milliliter) 0

I L-3 + I L-6

(200 n gZ milliliter)

Table 4 Platelet (1 ◦ '/ mu 1) red blood cells (1 0 ^4/1) croup 1 1 2 6 ± 1 0 0 7 5 ± 4 4 ( purified sample 1, 1 mu g)

Ρ <-0 1

Group 2 pi 2 9 ± 1 3 1 0 2 ± 9 5

(Purified sample 2, 2 μg) P. 0 1 group 3 9 8 ± 1 2 0 9 5 ± 7 5

(Note) BSA: albumin albumin

Example 4

Hereinafter, the formulation examples of the pharmaceutical composition containing the megakaryocyte amplification factor of the present invention as an active ingredient and the method of preparing the pharmaceutical composition are shown, but the present invention is not limited to these formulation examples.

(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 155.4 mg The above components are distilled for injection. Dissolve in 2 ml water and sterile vial Put in one, three and five. Primary drying at C for 0.75 Torr for 35 hours, followed by secondary drying at 3.0 ° C and a vacuum of 0.03 T0 rr for 5 hours. Manufactured. The obtained composition is dissolved in physiological saline or 500 ml of injection of glucose immediately before administration and used for intravenous drip infusion.

(Formulation Example 2)

Purified megakaryocyte amplifying factor of the present invention 1 μg arubin 5 mg mannitol 25 mg sodium chloride 1.95 mg sodium phosphate 3.85 mg A vial for injection was produced in substantially the same manner as in Formulation Example 1.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 11 (a) to 1-(d) are diagrams showing chromatograms and results of electrophoresis in each purification step of Example 2. Fig. 11 (a) is the first-stage purified CM Sepharose chromatographic graph, Fig. 11 (b) is the fourth-stage Q Sepharose chromatographic graph, Fig. 11 (c) shows the results of the gel filtration chromatography at the fifth stage, and Fig. 11 (d) shows the results of the isoelectric focusing at the sixth stage.

FIG. 2 shows that the megakaryocyte amplifying factors (purified samples 1 and 2) and the IL-6 megakaryocyte amplifying activity according to the present invention were compared with the acetylethylcholinesterase activity (AchEactivi) by liquid culture. ty) The results of evaluation by the measurement method are shown.

Industrial applicability

The megakaryocyte amplification factor protein of the present invention has an activity of promoting megakaryocyte amplification and increasing platelets in peripheral blood, and its activity is stronger than that of known factors having similar activities. It is. 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. .

Claims

The scope of the claims

1. A substantially pure megakaryocyte amplification factor protein having an activity of activating megakaryocyte amplification and having the following properties.

(a) Molecular weight: 230,000 ± 800,000 (measured by gel filtration) (b) Isoelectric point: pi ≤ 5.9 (measured by isoelectric focusing) (c) Elimination of human DOO Ropoechin, Lee Ntaroi Kin i _alpha, i Ntaroi Kin 1/3, y Ntaroi Kin 6, activity is substantially in megakaryocyte potentiating activity neutralization test using the antibody against Lee pointer one Roiki down 7 and Lee Ntaroi Kin 1 1 Does not decrease.

(d) No megakaryocyte colony-stimulating factor activity.

2. The megakaryocyte amplification factor protein according to claim 1, wherein the isoelectric point (p i) is 3.5 ± 1 (measured by isoelectric focusing).

3. The megakaryocyte amplification factor protein according to claim 1, wherein the isoelectric point (p i) is 4.9 ± 1 (measured by isoelectric focusing).

4. The megakaryocyte amplification factor protein according to any one of claims 1 to 3, which is derived from a human cell.

5. The megakaryocyte amplification factor protein according to claim 4, wherein the human cells are normal diploid cells.

6. The megakaryocyte amplification factor protein according to claim 5, wherein the normal diploid cells are derived from the lung.

7. Cultivate animal cells, produce megakaryocyte amplifying factor protein in the culture solution, recover culture supernatant from the culture solution, and recover the recovered culture medium. A method for producing a substantially pure megakaryocyte amplification factor protein, comprising separating and purifying a megakaryocyte amplification factor protein having an activity of activating megakaryocyte amplification and having the following properties from a culture supernatant.

(a) Molecular weight: 230,000 ± 800,000 (measured by gel filtration) (b) Isoelectric point: pi ≤ 5.9 (measured by isoelectric focusing) (c) Elimination of human collected by filtration Poechin, Lee Ntaroi Kin i _alpha, i Ntaroi Kin 1/3, y Ntaroi Kin 6, Lee Ntaroi key sheet 7 and activity substantially in megakaryocyte potentiating activity neutralization test using the antibody to interleukin-1 1 Does not decrease.

(d) No megakaryocyte colony-stimulating factor activity.

8. The method according to claim 7, wherein the megakaryocyte amplification factor protein has an isoelectric point (pi) of 3.5 soil 1 (measured by isoelectric focusing).

9. The method according to claim 7, wherein the megakaryocyte amplification factor protein has an isoelectric point (pi) of 4.9 soil 1 (measured by isoelectric focusing).

10. The method according to any one of claims 7 to 9, wherein the animal cell is a human cell.

11. The method according to claim 10, wherein the human cells are normal diploid cells.

1 2. The method according to claim 11, wherein the normal diploid cells are lung-derived cells.

13. The method according to any one of claims 7 to 9, 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 broadening factor protein production promoter is a meat peptone.

15. The method according to claim 14, wherein the animal cell is a cell derived from human lung.

16. It contains the megakaryocyte amplification factor protein according to any one of claims 1 to 3, which is therapeutically effective, and at least one of pharmaceutically acceptable carriers, diluents and excipients. Pharmaceutical composition.

1 7. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-1, IL-9, IL-11, GM-CSF, G- 17. The pharmaceutical composition according to claim 16, further comprising at least one factor selected from the group consisting of CSF, M—CSF, SCF, IFNs, LIF, TNF and EPO.