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WO1992017500A1 - Nouveau facteur d'amplification de megacaryocytes et son procede de production - Google Patents

Nouveau facteur d'amplification de megacaryocytes et son procede de production Download PDF

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Publication number
WO1992017500A1
WO1992017500A1 PCT/JP1992/000372 JP9200372W WO9217500A1 WO 1992017500 A1 WO1992017500 A1 WO 1992017500A1 JP 9200372 W JP9200372 W JP 9200372W WO 9217500 A1 WO9217500 A1 WO 9217500A1
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Prior art keywords
megakaryocyte
cells
amplification factor
activity
interleukin
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PCT/JP1992/000372
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English (en)
Japanese (ja)
Inventor
Shuhei Kondo
Kohei Ogawa
Original Assignee
Asahi Kasei Kogyo Kabushiki Kaisha
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Publication of WO1992017500A1 publication Critical patent/WO1992017500A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel megakaryocyte amplification factor 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 the expansion of megakaryocytes which are platelet precursor cells and having an action of promoting platelet production, and a cell culture or genetic engineering technique. It relates to the manufacturing method. The present invention also relates to a pharmaceutical composition containing the above novel protein as a megakaryocyte potentiator useful for the prevention and treatment of diseases such as thrombocytopenia.
  • Thrombopoetin (TPO) a factor that specifically promotes platelet production, has been enthusiastically acquired by many researchers for more than 20 years, but has not yet been successful .
  • TPO which acts as a factor (Mega-karyocyt e Co- lony Stabilizing Factor; Meg-CSF) and exerts an activity to promote megakaryocyte maturation in the late stage, acts. That is, first, the action of Meg-CSF causes the progenitor cells to repeat cell division and increase the megakaryocyte component, and then, by the action of TPO, each megakaryocyte progenitor performs endomi tosis, and its chromosome multiples As the cell size increases ( ⁇ 32N), the cytoplasm matures and increases, resulting in the production of platelets. TPO is also sometimes called megakaryocyte potentiator (eg-POT).
  • Meg-POT megakaryocyte potentiator
  • Meg-CSF The activity of Meg-CSF is determined by measuring the activity to form megakaryocyte colonies in soft agar culture of human or mouse bone marrow cells in vitro.
  • the activity of Meg-CSF is measured in the urine of patients with aplastic anemia and idiopathic thrombocytopenic purpura, in the plasma of patients with myeloid megakaryocytic aplastic thrombocytopenia, and in kidney bean lectin-stimulated human leukocytes. Culture supernatant, found in mouse leukemia cell line WEHI-3 culture supernatant, etc.
  • interleukin 3 (1L-3) (interleukin is abbreviated as IL) acts non-specifically on many lines, including megakaryocytes. -It is clear that it is a CSF.
  • Meg-CSF in WEHI-3 culture supernatant was completely IL-3. Most of the Meg-CSF activity in conventional cell culture supernatants has been attributed to IL-3, including all matches. However, Meg-CSF, which has been shown to specifically act on the platelet system, is not yet known.
  • the activity of TPO can be determined by measuring the effect of enhancing the colony-forming activity of Meg-CSF and / or the effect of promoting megakaryocyte maturation. So far, attempts have been made to provide factors having TPO-like activity.
  • a megakaryocyte prepared from the culture supernatant of a human fetal kidney cell line, which has the molecular weight of 15000 on SDS-PAGE and has an isoelectric point of 5.1 and has the effect of promoting protein synthesis in megakaryocyte cells.
  • An enhancer Megakaryocyte Stimulatory Factor or SF
  • a method for producing the same have been reported (see US Pat. No. 4,894,440).
  • IL a multifunctional cytokine that has recently been discovered as a glycoprotein that induces B cell antibody production and has been shown to be involved in the immune system, the acute phase response system, and malignant tumors -6 is also involved in the hematopoietic system and exhibits Meg-POT activity and megakaryocyte maturation promoting activity in vitro (Ishibashi, T. eta 1. “Proc. Nat 1. Acad. Sc USA” 5953 (1989)), and has been confirmed to exhibit a platelet production promoting effect in vivo (Asano, S. et al., "B1ood", 1602 (1990)).
  • IL-7, I-I, and the like also have megakaryocyte amplification factor activity.
  • the megakaryocyte spreading factor activity of these factors is weak, and they are constitutive (c 0 nst It is unknown whether this is a hematopoietic factor.
  • one object of the present invention is to provide a substantially pure novel megakaryocyte amplifying 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 megakaryocyte spreading factor produced by adding a megakaryocyte amplification factor production promoter to a medium in the above-described cell culture and performing cell culture.
  • Let the giant An object of the present invention is to provide a method for producing a nuclear cell 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.
  • 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.
  • animal cells are cultured in a medium, megakaryocyte amplification factor is produced in the culture solution, a culture supernatant is collected from the culture solution, and the collected culture is
  • the present invention provides a method for producing a megakaryocyte amplification factor, comprising separating and purifying the megakaryocyte amplification factor from a bacterium.
  • the animal cell used in the method of the present invention has an activity of activating megakaryocyte amplification and an ability to produce a megakaryocyte amplification factor having an activity of adding platelets in peripheral blood.
  • Various cells can be used. Normal diploid cells can be advantageously used, for example, cells from human kidney, intestine, lung, heart, ureter, skin, foreskin, tongue, thyroid, placenta, offspring, preferably human fetal kidney Cells from lung, foreskin, and even more preferably cells from human fetal lung can be used.
  • the megakaryocyte amplifying factor can be separated and purified from these tissue extracts, but more preferably, these cells are cultured in an appropriate growth medium, and the resulting culture is used as a culture medium.
  • a megakaryocyte amplification factor can be produced, the culture supernatant can be recovered from the culture, and can be separated and purified from the recovered tissue culture.
  • These cells are It is preferable that the cells be propagated by the method described in the culture method used for cell culturing, for example, “tissue culture” (edited by Junnosuke Nakai et al., Asakura Shoten, Showa 51) and used in the present invention.
  • Cells can produce megakaryocyte amplifying factors by culturing them in a medium solution containing carbons, a nitrogen source and, if necessary, inorganic salts and / or other additives.
  • a megakaryocyte amplifying factor production promoter preferably an animal meat enzyme-degrading peptide is added to a medium, and the cells are cultured, whereby the cells are produced in a culture solution.
  • the amount of the megakaryocyte amplification factor can be dramatically increased.
  • the concentration of the enzymatically degraded peptide for animals may be from 0.0 to 4 w / v%, preferably from 0.1 to 2 w / v%, based on the culture medium.
  • the animal meat enzyme-degrading peptide is generally used in a bacterial culture medium, and is usually called a proteospeptone, a proteosepeptone, or a meat peptone.
  • a method for preparing this animal meat enzyme-decomposed peptone is well-known, and may be, for example, the method described in “Bacterial Culture Studies Vol. 2” (written by Toshikazu Sakazaki, 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.
  • Examples of enzymes for decomposition include trypsin, papine, pepsin, and noncreatine. 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.
  • Proteose Peptone No.1 Proteose Peptone No.1
  • Proteose Peptone No.2 Proteose Peptone No.3, and Chopepeptone (manufactured by Difco Inc., USA).
  • Thiopepton Proteo-peptone L46
  • Peptone PL46 manufactured by Oxide, UK, Thioton (Thioton), manufactured by BBL, UK, Proteose-peptone, manufactured by Daigo Nutrition Chemistry of Japan, etc.
  • Thiopepton Proteo-peptone L46
  • Peptone PL46 manufactured by Oxide
  • BBL Thioton
  • BBL Thioton
  • a suitable cell density a density of favored properly is 10 5 ce lls / m ⁇ , 0.1 ⁇ 10 mg / m £ implant with the beads carrier for cell culture, chromatic serum under 15 to 45 ° C, preferred properly in a temperature range of 25 to 40 ° C,. 5 to 9 favored properly in culture pH range of 6-8, is cultured usually 5% C0 2 in including the air.
  • a megakaryocyte amplification factor production promoter serum-free conditions are used, and production culture is performed at a concentration of 0 to 4% or 0.1 to 2%, but preferably cells are sufficiently grown.
  • the culture days for production are usually 1-60 days, but 60 days It is also possible to exceed. Since the production rate of 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 was measured by the megakaryocyte amplification factor activity measurement method shown in Reference Examples 1 (a) and (b), and the degree of maturation of the megakaryocyte was determined by the megakaryocyte DNA amount measurement method shown in Reference Example 2. Can be confirmed by law.
  • the megakaryocyte-amplifying factor is expressed in a suitable host cell by using a commonly used genetic technique, and is collected and further purified.
  • a method for producing a megakaryocyte amplification factor is provided.
  • RNA is extracted from the cells of origin, and poly A + RNA is further purified.
  • a cDNA library is prepared using an appropriate expression vector, preferably a eukaryotic expression vector, and polyA + RNA and a linker, and the appropriate library is used using the library.
  • a host cell for example, Escherichia coli is transformed, and liposome DNA is prepared from the culture.
  • a suitable host cell is Transfects animal-derived cells, more preferably monkey-derived COS cells, to express the megakaryocyte spreading factor gene, collects it, and purifies it further.
  • Sphere amplification factor can be produced.
  • Cells that produce an appropriate amount of megakaryocyte amplifying factor such as human fetal lung cells, preferably 10 8 ce 11 s, RNA isolation kits such as those manufactured by Invitrogen, USA Using mouth No. K1592-01), extract the total RNA by the guanidine isothiosinate method according to the attached manual, and obtain poly A + RNA according to the conventional method.
  • Oligodex-S d T30 manufactured by Nippon Synthetic Rubber Co., Ltd. in Japan
  • about 2 total RNAs and 1 to 2 g of poly A + RNA are obtained.
  • a cDNA library is prepared according to the method of Okayama Ichiberg.
  • the eukaryotic expression vector 3′-oligo (dT) -tailed pcDV-1 (Pharmacia, Sweden, No. 27-4955-01) and the poly A + RNA and 3 obtained above '-ol igo (dG) -tai led p L1 linker (No. 27-4957, manufactured by Pharmacia, Sweden) can be used.
  • Certain players may use pcDL-SR 296.
  • the resulting solution containing the cDNA library is divided into an appropriate number of pools, preferably 10 to 200, and more preferably 50 to 100, and E. coli MC 106 1 (ATCC 5 33 38) ).
  • plasmid DNA is prepared, for example, using Qiagen-tip-100 (manufactured by Qiagen, USA) according to the attached manual.
  • the obtained recombinant DNA is transformed into a suitable host cell, preferably monkey kidney cell C, by, for example, the getylaminoethyl monodextran method (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 9.2.1-9.2.6).
  • CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 9.2.1-9.2.6 (4) After introduction into S1 cells (ATC CRL 1650), the gene is expressed in substantially the same manner as described in Example 2 of W088 / 05053.
  • the megakaryocyte amplification factor activity is measured by a method such as acetylcholinesterase activity measurement in liquid culture, and this is used as an index to determine the pool of the pool containing the gene for this substance. You can refine your search. Further, the positive DNA is again transformed into Escherichia coli, and the obtained colonies (about 2,000 values) are cultured as a group of about 10 cells. Introduce and express COS 1 cells and measure acetylcholinesterase activity. It is also possible to narrow down the cDNA library. Usually, by repeating this method several times, Escherichia coli having a cDNA plasmid expressing the megakaryocyte amplifying factor activity is isolated. Riayashi da, K. et a 1. tic Factor ”l, No. 2, 102-108 (1990)) o
  • megakaryocyte amplification factor gene for example, Escherichia coli, yeast, monkey kidney cells (COS cells), Chinese hamster ovary cells (CHO cells), mice Transfecting host cells such as C127 cells, human fetal kidney cell lines, silkworm cells SF9, etc., expresses the megakaryocyte amplification factor more efficiently, collects it, and further recovers it. By purifying, megakaryocyte amplification factor can be produced.
  • the culture supernatant is used. to recover.
  • the method for separating and purifying the megakaryocyte amplification factor include methods generally used in protein chemistry, for example, adsorption using a carrier, salting out, electrophoresis, ion exchange, gel filtration, and the like. Each application of affinity to a natural ligand Various chromatographic methods can be used alone or in combination.
  • CM sepharose column chromatography using sepharose bonded with carboxymethyl group preferably, CM sepharose column chromatography using sepharose bonded with carboxymethyl group, gel filtration column chromatography using particles such as cross-linked dextran gel, etc. 1.
  • Dye adsorption column chromatography, antibody affinity column chromatography to which an antibody that specifically binds to the substance of the present invention is bound can be used.
  • the novel megakaryocyte spreading 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.
  • the carriers, diluents and excipients those usually used in this field can be used.
  • 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, 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 bone marrow cells for transfusion at in Vr0.
  • the megakaryocyte amplification factor of the present invention can also be used as an injection.
  • thickeners such as sucrose, glycerin, methylcellulose, carboxymethylcellulose, etc., and pH adjusters of various inorganic salts, etc. can be added as additives.
  • the dosage of the megakaryocyte amplifying factor of the present invention per adult per dose varies depending on the age, sex, weight, symptoms, etc., but is generally 0.1 g to 100 mg per day, and is preferably once per day. Or it can be administered several times as needed.
  • the megakaryocyte amplification factor activity of the novel protein obtained by the present invention was measured by the following two methods (a) and (b).
  • IMDM solution (Is 0 c 0 Ves modification of Dal 1 beccos medium) used in the following method for preparing bone marrow cell suspension (., Powder ⁇ ) IDM (for 1 ⁇ ) (Gibco, USA ) was added to baking soda 3.G 24 g, iS — melcaptoethanol 3.04 ⁇ , and ⁇ 7.1 was adjusted to 7.1, and then messed up to 1 ⁇ , followed by 50 IU / m ⁇ ⁇ It was prepared by adding 50 ⁇ g / m ⁇ -strept mycin (all manufactured by Floraborate Lease).
  • the femurs of 6 to 9-week-old C57BL6 mice male were collected, the upper part was cut off, and a lOm fi plastic syringe (22 G needle) containing ⁇ : 1 MDM was added. ) was used to push the bone marrow vigorously into the 10-mm plastic dish from the knee joint side. After dispersing the cells by pipetting 8 times (6 times with a 19 G needle and 2 times with a 22 G needle), transfer the cells to a 15 ml £ phenolic con- verter and collect non-precipitating cells.
  • Acetylcholinesterase staining for use in subsequent experiments The liquids were 1 ⁇ 73 mM acetylthiocholine iodide, 0.5 mM potassium ferricyanide, 5 mM sodium citrate, 3 mM copper sulfate (all in Japan, Wako Pure Chemical Industries, Ltd.) was dissolved in 400 mM ⁇ , 75 mM phosphate buffer at pH 6.0 containing the same.
  • COS 1 cells As the culture supernatant of COS 1 cells containing IL-13, COS 1 cells (ATCC CRK 1650) were prepared using plasmid DNA obtained by linking mouse IL-3 cDNA to the SV-4 ° promoter. ) was used, and IL-13-containing culture supernatant expressed in the same manner as described in Example 2 of WO 88/05053, Example 2 was used.
  • the cells were washed with phosphate buffered saline (PBS), and megakaryocytes were specifically stained with cetylcholinesterase staining solution.
  • PBS phosphate buffered saline
  • the number of colonies was counted using an AHB-type microscope manufactured by Olympus Corporation in Japan as a colony consisting of 6 or more positive cells.
  • a mouse bone marrow cell suspension prepared in the same manner as in the above method (a) was added to diisopropylpropylfluorophosphate (DFP) (Sigma, USA) at a final concentration of 0.4 mM.
  • DFP diisopropylpropylfluorophosphate
  • the cell number was counted by a hemocytometer in the same manner as described above.
  • the megakaryocyte maturation promoting activity of the megakaryocyte amplification factor according to the present invention obtained by culturing human-derived cells was directly confirmed by the following method.
  • the acetylcholinesterase stained microscope specimen prepared as described above was immersed in a DAPI (4'-6-diamidino-2-phenylindole) staining solution for about 10 hours to perform double staining. Approximately 200 megakaryocytes forming colonies were randomly extracted and analyzed using a BH2 epi-illumination microscope manufactured by Olympus Japan and an OSP-1 cell-DNA fluorescence microphotometer. Distribution of chromosome doubling numbers of those cells was measured.
  • the DAPI staining solution was prepared by mixing the following preservation solutions 1, 2, and 3 at a ratio of 0.5: 98.5: 1.0 m fi to obtain ⁇ .
  • Stock solution 1 DAPI lOmg dissolved in lOOOOmfi distilled water.
  • T H P—1 leukemia cells
  • CM Sepharose Canoleboxime tylsepharose
  • Column fully equilibrated with 20 mM acetate buffer (pH 4.0) containing 0.2 M salt beforehand (diameter 9 cm x height 23.5 cm) ) And washed with 20 mM acetate buffer 6 ⁇ of 4.0, containing the same equilibration buffer 13.5 ⁇ and 0.4 M salt, and then with G.
  • Fig. 1 shows an example of the results of C-separator chromatography on the first stage of the purification process.
  • tissue plasminogen activator tissue plasminogen activator
  • the crude purified solution ⁇ 6.2 ⁇ obtained above is concentrated to 300m ⁇ by ultrafiltration module SI ⁇ -101 (made by Asahi Kasei Corporation, Japan), and contains 0.5 ⁇ salt in advance.
  • Sephacrynolate S—200 (manufactured by Pharmacia, Sweden) fully equilibrated with 7.4 mM 20 mM sodium phosphate buffer, column 7.4 (cm ⁇ 90 cm height).
  • a fraction 290 ⁇ having a molecular weight of about 25 and having c-megakaryocytic amplifying factor activity at a flow rate of 800 m ⁇ h was fractionated as a crudely purified solution. Activity recovery was 10-20%.
  • Figure 2 shows an example of the results of the third step of the purification process, Cepharil S-200 column chromatography.
  • the crude purified solution ⁇ 29 £ obtained above was concentrated to 30m ⁇ with an ultrafiltration module SIP-1013 (made by Asahi Kasei Corporation, Japan), and 10 times the volume of m ⁇ 7.4 20mM Tris-HCl buffer was added and buffer exchange was performed. Adsorbed on a CM Sepharose column (diameter 26 cm x height 7 cm), which had been equilibrated sufficiently with 20 mM Tris-HCl buffer (pH 7.4) containing 50 mM sodium chloride beforehand.
  • FIG. 4 shows an example of the results of phenyls-per-lower force chromatography at the fifth stage of the purification process.
  • the purified sample obtained in Example 2 was purified using a Sephacryl S — 200 HR (manufactured by Pharmacia, Sweden) column (diameter 2.6 cm ⁇ height 94 cm) which had been sufficiently equilibrated with PBS in advance.
  • the plate was developed with PBS (flow rate: 27.6m ⁇ / h) and fractionated by 4.6m ⁇ .
  • the megakaryocyte amplifying factor activity of each fraction was determined in advance using a low molecular weight marker protein kit for gel filtration (Pharmacia, Sweden; Persera albumin (BSA); 67 kd; ovalbumin; 43 kd , Chymotrypsinogen; 25 kd, ribonuclease A; 14 kd), and blue dextran 2000, and the molecular weight was determined by comparing the elution positions with these. This substance eluted with a peak around 8 kd on a 25 kd soil.
  • BSA Persera albumin
  • Example 2 Using a 15% to 25% polyacrylamide gradient gel (manufactured by Daiichi Kagaku, Japan), the molecular weight of the purified sample obtained in Example 2 was measured. Load about 0.1 g of the purified sample for SDS-PAGE with 10 ⁇ onto the gel, and use electrophoresis buffer of PH 8.4 containing 0.025 M Tris, 0.192 M Glycine and 0.1% SDS. And electrophoresed at 30 mA for 1.5 hours.
  • SDS-PAGE molecular weight marker protein kit (Pharmacia, Sweden, Phosphorilla) B) 94 kd, BSA 67 kd, ovalbumin 43 kd, canolebonic anhydrase 30 kd, soybean trypsin inhibitor 20.1 kd, ⁇ -lactalbumin 14.4 kd) were migrated. The molecular weight was measured. The substance migrated at 28 kd ⁇ 2 kd.
  • the purified preparation obtained in Example 2 was treated in PBS at 100 ° C. for 10 minutes, and the stability was evaluated based on the activity of the remaining megakaryocyte amplification factor.
  • the activity of this substance was unstable to less than 5%.
  • the substance was also highly sensitive to treatment with 0.125 mg / m ⁇ of trypsin at 37 ° C for 1 hour. Antigenicity.
  • the megakaryocyte amplification factor activity of this substance was examined using the purified sample obtained in Example 2 by the soft agar culture method described in Reference Example 1 (a). The activity was compared with that of IL-16 and IL-11.
  • IL-16 As the rhoL-6 derived from CHO cells, a commercially available product (manufactured by Genzym, USA) was used. HIL-11 was prepared from C ⁇ S 1 cells and from CH ⁇ cells as described below.
  • RNA was extracted according to the modified NAT method.
  • a total RNA isolation kit manufactured by Invitrogen, USA
  • RNA added with lipoA was isolated from the total RNA by rioligotex.
  • c DNA synthesis was performed according to the Okayama-ichi Berg method. That is, using the poly-A-added RNA, a 3 'oligo dT tail-added pCDV-1 (Pharmacia, Sweden) was used as a vector primer and a cDNA synthesis kit (Base, Germany). CDNA was synthesized according to the attached manual.
  • phenol was extracted and ethanol was precipitated, and then a dC tail was added using a tailing kit (Boehringer, Germany). Further, after phenol extraction and ethanol precipitation, the mixture was digested with a restriction enzyme HindIII, and after completion of the reaction, phenol black-mouthed form extraction was performed to precipitate ethanol.
  • an oligonucleotide having the following sequence:
  • 5′-I CCGAGGGTCTCTGGGGAAATCTC-3 ′ was resynthesized by a conventional method using a DNA synthesizer (Applied Biosystems, USA, DNA synthesizer, Model 980-A).
  • a DNA synthesizer Applied Biosystems, USA, DNA synthesizer, Model 980-A.
  • the 5 ′ end of the above oligonucleotide was phosphorylated with T4 polynucleotide kinase, and then used as an IL-11 synthetic DNA probe.
  • a clone of the cDNA library prepared above was cloned into an experimental book (Manias et al., Molecular Cloning 2nd. Edit ion 1.85, 1989, Cold Spring Harbor Laboratory). It was implemented according to.
  • plasmid DNA (referred to as pcDIL—11—12) was prepared in accordance with the experiment manual.
  • COS-1 cells generated by the plasmid pc DIL-11-1-12 into COS-1 cells were carried out as follows according to a conventional method.
  • COS-1 cells (ATCC CRL 165) were added to a tissue culture dish and supplemented with 10% (v / v) of fetal calf serum (hereinafter referred to as FCS; Gibco, USA).
  • FCS fetal calf serum
  • FCS fetal calf serum
  • FCS fetal calf serum
  • the CHO-dhfr-cell line was obtained from the University of Colombia, Dr.L.Chasi II, Dr.GU Chas in. First, 5 ⁇ 10 5 CHO-dhfr cells were placed in a tissue culture dish and grown in a growth medium (Ham's F-12 medium supplemented with 10% FCS (produced by Floraborate Inc., USA)). At 37 ° C for 1 day.
  • the obtained plasmid PcDIL-11-1-12 and the plasmid pSV2-dhfr were obtained by the method of FL Graham et al. (FLGraham “Virology” 52, 456 (1973) ))
  • the calcium method was introduced into CHO-dhfr-cell lines. After culturing in a growth medium for 3 days, 150 g / m ⁇ of proline and dialysed fetal serum (manufactured by Gibco, USA) in selective medium (DMEM) (Flow Laboratories, USA) ) To 10%). Thereafter, the medium was changed about every three days, and transformed cell colonies appeared about two weeks later.
  • the megakaryocyte amplifying factor activity of the supernatant was measured according to Reference Example l ( a ), and the activity was detected in several clones.
  • the clones whose activity was detected were further cultured for about 2 weeks on a selective medium containing 20 ⁇ of meso-rexate ( ⁇ ⁇ X) (manufactured by Wako Pure Chemical Industries, Japan). ⁇ Obtained X resistant colony.
  • the megakaryocyte amplifying factor activity of the supernatant was measured according to Reference Example 1 (a). Those with higher activity were detected.
  • ⁇ ⁇ X concentration of ⁇ ⁇ X was increased to 200 ⁇ ⁇ , and a strain resistant strain was obtained by the same procedure.
  • a strain resistant strain was obtained by the same procedure.
  • One of them was used as an IL-11 producing strain in the following experiments. This strain was cultured in a selective medium until it became confluent. Then, after culturing in a selective medium from which serum had been removed, a supernatant was recovered. About 23 kd of IL-11 was confirmed by SDS-PAGE in the culture supernatant.
  • the megakaryocyte amplification factor activity was examined by the soft agar culture method. Table of results Figure 3 shows.
  • the Meg-P ⁇ T activity of IL-6 (Genzam, USA) cannot be detected at lng / m ⁇ or 50ng / mjS, and a high concentration of 200ng / m ⁇ was about three times that of IL-13 alone.
  • the purified product of this substance already showed about 5 times the activity at 1 ng / m ⁇ compared to IL-13 alone, and at 10 ng / m ⁇ , it gave more than about 40 colonies and was saturated.
  • the activity of IL-l1 was examined using COS1 and expression culture supernatants from CHO cells.
  • GCSF (10 ng / m ⁇ ) 0
  • the megakaryocyte amplifying factor activity of this substance was evaluated by a method of measuring acetylcholinesterase activity (Ac hE activity) by liquid culture.
  • IL-16 was also evaluated.In this measurement method, IL-3 was used as the Meg-CSF, and the control at the time when 2 ⁇ was added was that when only IL-3 was added. is there.
  • the results are shown in Figure 7- (a) and Figure 7- (b).
  • the bar graphs on the left side of FIGS. 7 _ (a) and 7-(b) are data when IL-3 is not added at all.
  • Relative ⁇ 1 uorescence is shown as a relative value with 1 when nothing is added to the test sample (control).
  • Figure 7-(a) shows the culture results under serum-free conditions
  • Figure 7-(b) shows the culture results under conditions containing 15% DFP-treated horse serum (HS). This assay also showed strong megakaryocyte amplification factor activity of this substance.
  • the purified substance was intraperitoneally administered to mice (C57BL male, 7 weeks old, 5 animals per group) intraperitoneally for 5 days, and blood was collected 3 hours after the final administration to measure the platelet count and erythrocyte count. As shown in Table 5, it was found that this substance significantly increased the platelet count at a risk factor (P) of 1% or less, and showed a topo-mbopoetin effect. At this time, the number of red blood cells did not increase.
  • P risk factor
  • 1 ⁇ g and 0.2 g of this purified substance were administered in PBS containing 150 ⁇ g / m ⁇ bovine serum albumin (BSA) per administration Group 3 received BSA alone as a control. (Table 4)
  • 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.
  • Purified megakaryocyte amplifying factor of the present invention 1 mg Purified gelatin 20 mg Mannitol sodium lOOmg Sodium chloride 7.8 mg Sodium phosphate 15.4 mg The above components were dissolved in distilled water for injection 2m ⁇ , and sterile vials were prepared. The mixture was first dried at -35 ° C at a vacuum of 0.075 TGrr for 35 hours, and then secondarily dried at 30 ° C and a vacuum of 0.03 Torr for 5 hours to produce a vial for injection. The obtained composition is used for intravenous drip infusion in a physiological saline solution or 50 ⁇ m ⁇ -sugar injection solution immediately before administration. (Formulation Example 2)
  • Purified megakaryocyte amplification factor of the present invention 10 ⁇ g
  • FIGS. 1 and 2 show chromatograms in the first and second purification steps of Example 2, respectively, and FIG. 1 shows a CM Sepharose column chromatogram in the first purification step.
  • FIG. 2 shows the results of Sephataryl S-200 column chromatography at the third stage of the purification process.
  • FIG. 3 shows the results of CM Sepharose column chromatography at the fourth stage of the purification step in Example 2.
  • FIG. 4 shows the results of phenyls-perlose column chromatography at the fifth stage of the purification step in Example 2.
  • FIG. 5 shows the results of isoelectric focusing on the sixth stage of the purification step in Example 2.
  • FIG. 6 shows the results of isoelectric focusing chromatography of Example 3.
  • FIG. 7— (a) and FIG. 7— (b) show the megakaryocyte amplification factor and the megakaryocyte amplification factor of IL-6 according to the present invention.
  • the activity is determined by liquid culture
  • the results evaluated by a method for measuring cetylcholinesterase activity (AchEfficiency) are shown.
  • Fig. 7-(a) shows the results of culture under serum-free conditions
  • Fig. 7-(b) shows the results of culture under conditions containing 15% DFP-treated horse serum (HS).
  • 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 known factors having similar activities. is there. 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.

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Abstract

Nouvelle protéine sensiblement pure d'amplification de mégacaryocytes, présentant un poids moléculaire de 25'000 ± 8'000 et de 28'000 ± 2'000 tel que déterminé respectivement par gel filtration et par électrophorèse sur gel de dodécyl sulfate de sodium et de polyacrylamide, ains qu'un point isoélectrique (pI) supérieur à 9 tel que déterminé par la chromatographie isoélectrique. On peut différencier de manière immunologique ladite protéine de l'érythropoïétine, de l'interleukine -1α, de l'interleukine-1β, de l'interleukine-6 et de l'interleukine-7 humaines, et elle ne présente aucune activité de facteur stimulant les colonies de mégacaryocytes, mais présente une activité d'activation de l'amplification de mégacaryocytes. On peut produire cette protéine à l'aide des techniques de culture cellulaire ou de génie génétique. Elle présente les activités d'activation de l'amplification de mégacaryocytes et d'augmentation des thrombocytes dans le sang périphérique. De ce fait, on peut l'utiliser pour la prophylaxie et le traitement de la thrombopénie.
PCT/JP1992/000372 1991-03-26 1992-03-26 Nouveau facteur d'amplification de megacaryocytes et son procede de production WO1992017500A1 (fr)

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JP3/60731 1991-03-26
JP3060731A JPH04295500A (ja) 1991-03-26 1991-03-26 新規な巨核球増幅因子とその製法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3328341B2 (ja) * 1991-12-27 2002-09-24 中外製薬株式会社 新規な巨核球増幅因子
WO1993016106A1 (fr) * 1992-02-07 1993-08-19 Asahi Kasei Kogyo Kabushiki Kaisha Nouvel amplificateur de megakaryocites et production
IL107366A (en) * 1992-10-23 2003-03-12 Chugai Pharmaceutical Co Ltd Genes coding for megakaryocyte potentiator

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63239298A (ja) * 1986-09-17 1988-10-05 マサチューセッツ　インスチチュート　オブ　テクノロジー 巨核球促進因子

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
JPS63239298A (ja) * 1986-09-17 1988-10-05 マサチューセッツ　インスチチュート　オブ　テクノロジー 巨核球促進因子

Non-Patent Citations (2)

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Title
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 262, No. 7, (1987), G. TAYRIEN et al., "Purification and properties of a Megakaryocytes Stimulatory Factor present both in the serum-free conditioned medium of human embryonic Kidney cells and in Thrombocytopenic plasma", p. 3262-3268. *
LEUKEMIA RESEARCH, Vol. 10, No. 4, (1986), O.S. HUAT et al., "Biochemical characterization of an in-vitro murine megakaryocyte growth activity: megakaryocyte potentiator", p. 403-412. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation

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AU1435092A (en) 1992-11-02

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