WO1999011665A1 - Human thrombopoietin produced by established human cell lines, process for producing the same, and medicinal compositions containing the same - Google Patents

Abstract

Human thrombopoietin (TPO) similar to natural sugar chain structues produced by established human cell lines originating in human TPO-producing organs (for example, human liver-derived cells and human bone marrow stroma cells); a process for producing TPO which involves the step of expressing a foreign human TPO gene in an established human cell line; medicinal compositions containing TPO, such as remedies for thrombopenia; and human TPO having at least a sugar chain structure of the α-2,6-bond type sialic acid. This TPO can contribute to the improvement in depression of antigenicity, internal dynamics, etc.

Description

 Description: Human thrombopoietin produced by a human cell line, a method for producing the same, and a pharmaceutical composition containing the same Background of the invention

Field of the invention

 The present invention relates to a novel human thrombopoietin glycoprotein produced by a human cell line, in particular, a decrease in antigenicity and an improvement in body kinetics. The present invention relates to a human mouth bomboprotein glycoprotein, a method for producing the same, and a pharmaceutical composition containing the same. Disclosure of related technology

Thrombopoietin (TPR) is one of the site-to-site families of site power receptors.糖 It is a sugar protein cloned as a ligand of Ρ1 (de Sauvage et al., Nature (London) 369, 533-565, ( Bart 1ey, TD et al., Cell 77, 1117-1124, (1994)). All of these Mp1 ligands are derived from the serum and blood of animals with thrombocytopenia (for example, humans, mice, dogs, etc.). It has been detected in plasma, and its involvement in megakaryocyte formation and platelet formation has been confirmed.

 With the development of a therapeutic agent for thrombocytopenia in mind, the present inventors also showed an activity that promotes megakaryocyte production from megakaryocyte progenitor cells that are more highly purified than rat bone marrow. Was used as an index to purify lact TPO from rat plasma with thrombocytopenia, and based on the partial amino acid sequence, rat TPO cDNA and human TPO were further purified. He succeeded in cloning cDNA and obtaining large amounts of uniform human TPO by gene recombination technology (H. Miyazaki et al., Exp. Hemato 1. 22: 838, (1994)). The human TP0 successfully obtained by the present inventors has the same amino acid sequence as the factor obtained as the human Mp1 ligand described above. And have been determined (see Sequence Listing below: SEQ ID NO: 1).

The present inventors have proposed that the human TP0 be administered to a mouse with thrombocytopenia in which bone marrow suppression has been caused by administration of a cancer inhibitor or an immunosuppressant, or irradiation or BMT. Upon administration, a thrombocytopenia inhibitory effect, a thrombocytopenia promoting effect, and an increase in hematopoietic function were observed, and the human TP0 was effective in these thrombocytopenia. I have found this. Human TP 〇 has been reported previously on TP 中 in human plasma (Matsumoto, A. et al., 38th Annual Meeting of the ASH ( 1996)), but it has not yet been isolated and purified.Therefore, its structure, function, etc. are unknown, and various cells produce TPO. All were detected at the mRNA and ELISA levels. In human liver-derived cells (HepG2) (Hino, M. et al., Biochem Biophys Res Commun 217, 475-481, (1995)), constitutive mRNA expression was detected. Yes. In human fetal kidney cells (HEK), the activity of proliferating mp1-expressing cells (M0-7e, BaF3 / mp1) and TPO mRNA have been detected. However, it has also been isolated and has not been purified and its structure, function, etc., including its in vivo activity, are unknown.

On the other hand, recombinant human TPO using gene recombination technology is expressed in African monkey kidney cells (C0S-1). Kato, T. et al., J. Biochem 118, pp. 229-236, (1995)) and those expressed in Chinese hamster ovary cells (CH0) (Morita, H. et al., FEBS Lett. 395, 228-334, (1995); Kato, T. et al., Proc. Natl. Acad.

4669-4697, (1997)), expressed in E. coli (Ann, MF et al., Blood 86: 54-59, (1995)) and expressed in Baby Hamster kidney cells (BHK). (Ross, H. et al., Biochemistry 35, 14849-14861 (1996)), and those expressed in human fetal kidney cells (HEK293) (de Sauvage et al., Nature 369, 533). -538 (1994); Bartley, TD, et al., Cell 77, 1117-1124, (1994)).

 However, there has been no report on TP0 expressed in a human cell line derived from a TPO-producing organ so far.

 In addition, the TPO produced by the above-mentioned various non-human cell lines and the human TPO produced by the human cell lines differ in the structure of TP0 found in human blood. Nothing is known about it. Furthermore, at present, no report has been made on the relationship between the difference in the structure and the function of TPO.

Generally, in the case of a physiologically active glycoprotein such as TPO, there are organs that produce the protein in addition to the main TPO-producing organ, and the organ / organ. The produced glycoprotein may have a different structure depending on the type of cell from which it originated. Similarly, it is known that in the case of recombinant glycoproteins using genetic recombination technology, the sugar chain structure produced by the host cell used differs. Yes. In particular, when animal cells are used, the sugar chain structure to be added varies depending on the type (animal species, or the type of organ to be derived, cell line, etc.). However, the difference has little influence on the function of the protein, mainly on the antigenicity, pharmacokinetics and the like. In fact, there have been reports of the production of antibodies against many recombinant protein drugs (Steis et al., N. Eng. J. Med. 318, 1409-1413 (1988), etc.). However, this is structurally and functionally different from the natural body protein in the body, and therefore has better properties in terms of antigenicity and pharmacokinetics. A human T with

If it is a PO protein, it can be expected to further enhance its value as a pharmaceutical.

 Summary of the Invention

 The present invention relates to a human thrombopoietin produced by a human cell line.

(T P O)

In an embodiment of the present invention, TP0 is an expression product of an endogenous or exogenous human TPO gene in a human cell line. It has been isolated and purified. The human cell line may be a human cell line derived from a TPO-producing organ, for example, a human cell line such as JHH7 (FERM BP-6049) or HuH7 (FERM BP-6048). It is possible to enumerate cell-derived human bone marrow stromal cells.

 The TPO of the present invention has a sugar chain structure having an affinity for SSA-lectin column, or has at least an α2,6-bonded shear chain. It can have the sugar chain structure of luic acid. The amino acid sequence has, for example, the amino acid at positions 1 to 332 shown in SEQ ID NO: 1 in the sequence listing.

 The present invention also provides a pharmaceutical composition comprising, as an active ingredient, human as defined above. Specifically, the compound can be used as a platelet-increasing agent, a therapeutic agent for thrombocytopenia, or a therapeutic agent for thrombocytopenia. The medicament of the present invention, when used as a drug, can provide advantages such as a decrease in antigenicity and an improvement in body movement.

The present invention further relates to a method for expressing a human, which comprises expressing an exogenous human 遺 伝 gene in a human cell line derived from a 産生 産生-producing organ. Provide manufacturing method. Examples of the human cell line include the above-mentioned human liver-derived cells (eg, JHH7 (FERM BP-6049)). You can use human bone marrow stromal cells. A human TPO obtainable by such a method is also included in the present invention.

 The present invention further provides a human having at least an α2,6-linked sialic acid sugar chain structure. Here, human TP0 is an expression product of an exogenous human TP0 gene in a cell line, and can be isolated and purified. In addition, the cell line can be a CH0 cell into which a, 6—Sialyltransferase gene has been introduced.

 The present invention also introduces α2,6—sialyltransferase cDNA into CH0 cells and expresses the human TPO gene in the CHO cells. Including this, a method for producing humans having at least a sugar chain structure of α2,6-linked sialic acid, or humans in CHO cells At least alpha expression, including expressing the transgene and introducing the ci2,6—sialyltransferase cDNA into the CHO cells. It is intended to provide a method for producing human having a sugar chain structure of 2, 6-linked sialic acid.

 Brief description of the drawings

Figure 1 shows recChoTPO, nativeHepTPO (JHH7) and nativeH 4 is a photograph showing a comparison of molecular weight of epTPO (HuH7) by electrophoresis. Detection was performed by electroblotting and then reaction with anti-TP jerk IgG x RAG-HRP. 250 pg of TP II was used per lane.

 FIG. 2 is a photograph showing a comparison of the molecular weights of recHepTPO (JHH7 / 7 / c83), naiveveHepTPO (JHH7) and recChoTPO by electrophoresis. Detection was performed as in Figure 1.

 FIG. 3 shows a comparison of the reactivity of each TPO shown in the figure with the SSA or MAM lectin by the Lectin-ELISA method.

 FIG. 4 shows the in vitro activity of TP0 indicated in the figure by the FDCP-hMpl 635 assay.

 Figure 5 shows the TPO produced by clones CH0 + 2,6 ST / 19 / C4 and CH0 + 2,6 ST / 2c3, and the clone produced by clone CH029C14. The molecular weight with recChoTPO was determined by electrophoresis / Western blotting (anti-hTP0 anti-globin monoclonal antibody and horseradish peroxidase conjugated anti-goat IgG rabbit rabbit polyclonal antibody). This is a photograph compared with the above (using a linear antibody).

Figure 6 shows clones CH0 + 2 and 6 by the Lectin-ELISA method. The results of sialic acid analysis of TP0 produced by ST / 19 / c4 and CHO + 2,6 ST / 21 / c3 are shown.

 Figure 7 shows CHO cells transfected with cr2,6—Sialyltransferase cDNA by the FDCP-hMpl635 Atssay method (19c4 and 21c3 in the figure). The comparison of the in vitro activity of TPO obtained by expression in CHOD-cells (shown as HTCF in the figure) and CHOD-cells is shown.

 Detailed description of the invention

 The present inventors have confirmed that the major organ producing human TPO is the liver; it has been confirmed by mRNA leveling (Shimada, Y. et al., Exp.

Hemato, Vol. 23, pp. 1388-1396, (1995); Nomura, S. et al., Exp p. Hemato, Vol. 25, 565-572, (1995)). Attention was paid to the cells. Therefore, TPO (nativeP1 asmaTPO) partially purified from human plasma, human TPO-producing liver derived from the strain Yidani cells, TPO (nativeHepTPO) purified from human plasma, and the human Recombinant human TPO expressed in TPO-producing liver-derived cell lines

(recHepTPO) was prepared, and its sugar chain structure was compared with recombinant human TPO (recChoTPO) expressed in CHO. As a result, T cells purified from human TPO-producing liver-derived cell lines were PO (nativeveHepTPO) and recombinant human TPO (recHepTPO) expressed in the human TPO-producing liver-derived cell line have a binding pattern of N-linked sugar chains of CHO. It is different from the recombinant human TP0 expressed in E. coli, but rather, has the same power as TPO (nativePlasmaTPO) purified from human plasma. did.

 In other words, as described in the examples below, according to a lectin-ELISA assay using an anti-TPO monoclonal antibody and lectin, nativePlasmaTPO, nativveHepTPO, etc. And recHepTPO are SSA lectins characterized by the fact that the sialic acid addition mode at the sugar chain terminal is α2,6 linkage (Shibuya, N. et al., J. Biochem, 106). roll,

P. 1098-1103, (1989)) shows reactivity, but recChoTPO does not react, whereas MA is characterized by a sialic acid addition mode of α2,3 bond. All of these react with cutin (Wei-Chun, W. et al., J. Biol. Chem. 263, 4576-4585, (1988)). In other words, recChoTPO is very different from Τ α derived from human plasma, which has an α2,6-linked sialic acid addition structure in the N-linked sugar chain, whereas nativeHepTPO It can be said that recHepTPO is similar to TPO derived from human plasma.

Thus, according to the present invention, a novel human cell line is produced. A TPO will be provided. Preferably, a novel human cell line produced by a human cell line derived from a prostate-producing organ is provided. More preferably, the present invention provides a novel human protein having the above specific sugar chain structure.

 The novel human of the present invention provides a reduction in antigenicity, an improvement in pharmacokinetics, and a reduction in the effect of thrombocytosis during continuous administration.

As the human of the present invention, a human gene is introduced into a human cell line by using a gene recombination technique, expressed, and isolated and purified. Human TP0, which is an expression product of an exogenous human TPO gene in a human cell line, which is obtained by such a method, is mentioned. Alternatively, an endogenous human obtained by isolation and purification from a human cell line in which the human TP0 gene is expressed. Human TPO, which is an expression product of a TPO gene in a human cell line, may be used. Alternatively, human TPO, which is an expression product of endogenous human TPO gene, may contain a promoter such as a promoter in a human cell line having the human TPO gene. A method for activating and expressing the endogenous (eridog enous) human TPO gene (International Includes a human TP 0 as obtained by publication No. W096 / 29411).

 Examples of human cell lines include HL60 (ATCC accession number CC-240), Jurkat (ATCC accession number TIB-152), K562 (ATCC accession number TIB-152), and HeLa (at RIKEN depository). No. RCB0027), HepG2 (ATCC Deposit No. CKL 10741) and the like. Preferable are human cell lines derived from TPO-producing organs, such as human liver-derived cells and human bone marrow stromal cells. More preferably, a human-derived liver cell line such as JHH7, HuH7, HepG2 and the like can be mentioned. JHH7 was internationally deposited under the Budapest Treaty on August 11, 1997, under the accession number F ERM BP-6049 at the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry of Japan. ing . HuH7 has also been deposited with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of Japan under the accession number FERM BP-6048 on August II, 1997.

The human TPO of the present invention preferably has an α2,6-linked sialic acid sugar chain structure or at least at least SSA-Lecti η. [Official name: Sambucus siebo 1 dina aggu 1 utinin lect in or Elderberry Bark lect in (Si buya, et al., J. Biol. Che m 262: 1596-1616 (1987)], which has a sugar chain structure having an affinity. More preferably, the N-linked sugar chain has a sugar chain structure of ci 2,6 linked sialic acid or has an affinity for SSA-Lectin. Some of them have a sugar chain structure.

 The human TPO of the present invention has the primary structure of its protein portion, namely, the amino acid sequence represented by SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. In the amino acid sequence shown in SEQ ID NO: 1, one of the amino acids has been modified (replacement, deletion, insertion, substitution, etc.) as long as it retains human TPO activity. And / or additions) to the amino acid sequence. Specifically, the TPO derivative described in International Publication No. W095 / 21919 (August 17, 1995) and the TPO derivative described in International Publication No. W096 / 25498 (August 22, 1996) are described. Modifications of amino acid residues such as those found in TPO derivatives, such as the TPO derivatives described in W095 / 18858 (July 13, 1995), may be mentioned. .

As used herein, "TPO activity" refers to the promotion of proliferation and differentiation of megakaryocyte progenitor cells and / or the stimulation of platelet production specifically in vivo. Is an activity that enhances. Further, according to the present invention, there is also provided a method for producing the above-mentioned novel human TP0.

 Methods for directly purifying and isolating human cell lines include those generally used for protein purification, such as ion-exchange chromatography. Graphite, lectin-figure chromatograph, dye-adsorbed chromatograph, hydrophobic interaction chromatograph Gel filtration chromatography, reversed-phase chromatography, phenolic chromatography, sulfated, sulfated Gel chromatograph, node chromatograph, metal chromatograph, metal chromatograph, A method combining at least one of isoelectric focusing chromatography, preparative electrophoresis, and isoelectric focusing There is. Also, the present invention includes a method that can be combined using the physicochemical properties of human TPO. In addition, τ

An antibody column using an antibody capable of recognizing PO can also be used. In addition, TP0 was found to be a Mp1 ligand (de Sauvage et al., Nature 369: 533-538 (1994); Bartley et al., Cell 77 Vol. 1177-1124 (1994); Kaushansky et al., Nature 369, 565-568 (1994)), M. It is also possible to prepare an affinity gel column by coupling P 1 to an activated gel, and use this to make TP. 0 can be purified. More specifically, the extracellular region of Mp1 (Mp1—X) was produced and prepared by a gene recombination method using CHO cells as a host. M pl — X columns are listed (BarUey et al., Supra (1994)).

 In addition, the present invention also includes a method for producing the human TPO of the present invention using a gene recombination technique. For example, transforming or transfecting a human cell line with a recombinant vector that incorporates the DNA encoding human TPO at the appropriate site in the vector. And a method for producing a human TPO of the present invention, which is characterized by separating and purifying the produced human TPO protein. In an embodiment of the present invention, the present invention relates to a human cell line derived from a TPO-producing organ, preferably a cell derived from a human liver (eg, a JHH7 cell) or a human cell line. A method for producing human TPO by introducing and expressing a human TPO gene into bone marrow stromal cells. The human cell lines used in these methods are as described above.

Transform or transfect these host cells Vectors used for this purpose include pSV2-neo (Southern and Berg; J. Mo 1. App 1. Genet.,, 327-341, 1982), pCAGGS (Niwa et al .; Gene, 108, 193-200, 1991), and cRI-SRα296 (Takebe et al .; MoI. Cell. Biol., 8, 466-472, 1988).

 These vectors include an origin of replication, a selection marker, and a promoter as needed, and are also required for vectors for eukaryotic cells. Accordingly, an RNA splice site, a polyadenylation signal, etc. are added.

 As the origin of replication, those derived from SV40, Adenovirus, or Sinophilus mavirus can be used.

 Promoters for gene expression include those derived from viruses such as retroviruses, poliomaviruses, adenoviruses, and SV40. Alternatively, chromosome-derived ones (eg, EF1-) can be used.

Selection markers include the neomycin (neo) resistance gene, the puromycin (pur) resistance gene, the thymidine kinase (TK) gene, and dihidide. (B) Folate reductase (DHFR) gene, Escherichia coli xanching guanfos folibosyltransferase (Ec0 gpt) Genes can be used.

 Preferably, as described in (4) of Example 1 described below, the hitheronge-factor 1-downstream of the alpha-promoter is preferable. The human TPO cDNA was placed in the cell, and the vector was further subjected to [] by applying the SV40 initial polygenylated signal, the DHFR gene, the SV40 replication origin, and the like. It is performed by transforming JHH7 cells using the Lance Fectum method (promega). The transformed JHH7 cells were transformed into the introduced exogenous human TP0 gene in addition to human TP0, an expression product of the endogenous human TPO gene. Will produce a TP0 based on it.

 Further, according to the present invention, there is also provided a pharmaceutical composition containing the human TPO of the present invention. The pharmaceutical composition of the present invention contains a stabilizing agent, a diluent, a solubilizing agent, a preservative, an antioxidant, an excipient, an isotonic agent and the like in accordance with the purpose of the preparation. It can be contained.

The pharmaceutical composition containing human TPO of the present invention is in a dosage form suitable for various administration routes including parenteral injection, parenteral administration, pulmonary administration, nasal administration, and oral administration. , Solutions, suspensions, tablets, pills, capsules, granules, freeze-dried preparations and the like. The pharmaceutical composition containing the human TPO of the present invention is usually administered in an amount of 0.05 μg / kg body weight to 1 mg / kg body weight as an active ingredient per day depending on the disease state, sex, administration route and the like. It can be administered once to several times.

 According to the present invention, there is provided a platelet-increasing agent comprising the human TPO of the present invention as an active ingredient and treating a large number of disease patients requiring an increase in platelets.

 In addition, chemotherapy and radiotherapy using anticancer and immunosuppressive drugs, or thrombocytopenia in patients undergoing bone marrow transplantation (BMT), PBSCT, and CBSCT Is provided.

 Furthermore, many diseases characterized by thrombocytopenia, for example, platelet depletion due to impaired platelet production and shortened platelet life (enhanced platelet destruction or increased platelet consumption). A therapeutic agent is provided.

For example, congenital Fanconi anemia, regenerative anemia associated with chemotherapy and radiation therapy, myelodysplastic syndrome, acute myeloid leukemia, or bone marrow formation such as bone marrow transplantation Insufficiency includes thrombocytopenia, which can be used to promote the recovery of platelets in such patients. In addition, thrombocytopenia due to shortened life of platelets and megakaryocytes, which is also useful for thrombocytopenia due to abnormal Ρ0 production, is, for example, idiopathic thrombocytopenic purpura. Disease, acquired immunodeficiency syndrome (AID)

S), is also useful for promoting platelet recovery in patients with disseminated intravascular coagulation, thrombotic thrombocytopenia, etc.

 Furthermore, TP0 is administered before surgery to increase own platelets, and the platelets are used as blood transfusion platelets during one's own operation, so-called autologous platelet transfusion. It is also useful for

 In addition, human TP0 of the present invention may also be due to, for example, transient platelet loss or damage due to other chemicals or drugs or therapeutic measures. The human TPOs of the present invention, which are also useful in the treatment of exposed platelet disorders, can be used to promote the release of new "intact platelets" in such patients. And TP

It has been revealed that one of the major producing organs of the liver is the liver, which indicates that various liver diseases that cause thrombocytopenia, such as biliary atresia and liver transplantation The clinical application of TPO administration in liver cirrhosis, hepatitis, etc. is also expected. Furthermore, it is also useful for restoring the ability of stored platelets to form a hemostatic thrombus.

Thus, the use of the pharmaceutical composition or therapeutic agent of the present invention. Thus, the antigenicity is further reduced, and the pharmacokinetics of the drug is further improved, so that a better platelet increasing effect, a therapeutic effect for thrombocytopenia or a therapeutic effect for thrombocytopenia is achieved. Obtainable

 The present invention further provides a human TP〇 having at least an α2,6-linked sialic acid sugar chain structure. Here, human TP〇 is an expression product of an exogenous human TP0 gene in a cell line, which may have been isolated and purified. The cell line is

Similar to CHO cells and ovarian cells, kidney-derived る 29,3 and α2 α6,6,6 which have low α2,6—sialyltransferase activity.

細胞 な ど の 細胞 細胞 細胞 細胞 細胞 細胞 細胞 細胞 細胞 α α 細胞 細胞 α α α α 細胞 α α α 細胞 α 細胞 細胞 α α α α α

 The present inventors have introduced rat α2,6—sialyltransferase cDNA into CHO cells,

3-linked and ct2,6-linked forms with both sialic acids added

An attempt was made to express TPO. In this case, the α2,6—Sialyltransferase cDNA can be introduced later into the CHO cells into which the human TPO gene has been introduced. Transformation may be performed, the order may be reversed, or the transformation may be performed at the same time. In addition, in the examples described later, C 遺 伝 0 cells The rat was introduced into the rat liver; 3 — galactoside α2, 6 — carolinoletransferrase — Entrezaccession

M 1 8 7 6 9), for example, those derived from mature rat brain (Entrezaccession No. L 2955 5 4) and mouse <3 — glass It is also possible to use the serial transfection cc 2, 6 — serial transfection (Entrezaccession No. D 1610 6).

 Although CHO cells originally do not have 2,6-sialyltransferase activity (Paulson, JC et al., J. Biol. Chem. 264, pp. 10931-10934). (1989)), and a powerful pro- cess such as the method of Lee, E. J. et al. (J. Bio 1. Chem. 264, pp. 13848-13855, (1989)). Α2, 3 — originally introduced into the cells by introducing the cDNA transfected with transgenic α2, 6 — located under the motor. Due to its quantitative superiority by competing with sialyltransferase activity, α2,6-linked sialic acid has α2,3-linked sialic acid. Τ Τ れ た will be produced on the same molecule as the type sialic acid, or will be added dominantly. Here, "α 2,

"6-linked sialic acid was predominantly added.""When measured by Lectin-ELISA as described in Example 3. It indicates that the response to the SSA-Lectin ELISA and the response to the MAM-Lecin ELISA is below the measurement limit. The TPO produced by the CHOD-strain has sialic acid added in an α2,3 type binding mode and also has sialic acid added in an α2,6 type binding mode. Although this has been confirmed, in fact, it has sufficient reactivity with the MAM-Lectin ELISA, but does not react at all with the SSA-Lectin ELISA. This reactivity was reversed by the gene transfer of 2,6—Sialyltransferase, and it was added in a 2,6—type binding mode that was not originally possessed. The fact that SSA—Lecin, which specifically binds to sialic acid, became responsive, suggests that 2,6—Sialyltransferases As a result of high expression by gene transfer, the sialic acid addition activity of 2,3 — sialyltransferase was overwhelmingly overwhelmed. Above this, it means that the position where sialic acid is originally added in α2,3 type binding mode was occupied by α2,6 type bonding mode sialic acid.

In a CHO cell into which a human Ο gene has been introduced, for example, CH transformed with an appropriate expression vector carrying DNA encoding human TP〇 can be used. 0 cells can be used. For example, a full-length human TPO deposited on January 31, 1995 with the Institute of Life Science and Industrial Technology, the Ministry of International Trade and Industry of Japan under the accession number FERM BP-4988. And a CHO cell line (CHO-DUKXBl1) transformed with a plasmid PDEF202-hTPO-Pl carrying the cDNA to be cloned.

 As described above, the present invention also provides a method for introducing α2, 6-sialyltransferase cDNA into CHO cells and subjecting the cells to the CHO cells. (EN) It is intended to provide a method for producing human TPO having at least an α2, 6-linked sialic acid sugar chain structure, including expression of a gene. Further, the present invention provides a method for expressing a human TPO gene in CHO cells and introducing a, 6-sialyltransferase cDNA into the CHO cells. It is intended to provide a method for producing human having at least an α2, 6-linked sialic acid sugar chain structure, which comprises.

 Example

 The present invention will be described more specifically with reference to the following Examples, but the present invention should not be limited by these Examples.

 Example 1>

(1) (Partial) Purification of Τ Ρ Ο from Human Plasma ... nat i veP 1 asmaT PQ

— Approximately 460 ml of human plasma stored at 80 ° C is thawed in running water, thawed at 135 OX g to remove insoluble precipitate, and 457 ml of the supernatant is added to an equal volume of lniM EDTA, 0. \% Tween 20 It was diluted with Du 1 becco's phosphate buffered saline (PBS) containing 0. \% NaN ₃ (DPBS: Cat. No. 05 913, manufactured by Nissui Pharmaceutical Co., Ltd.). For the purpose of inhibiting the protein degradation by protease, 914 ml of the 2-fold diluted human plasma was used to inhibit protease, Pefab1 ocSC ( MERK Cat.No.124839, final concentration 0.1 mM), trans-epoxysuccinyl-L-leucylamido (4-guaniai no) -butane (SIGMA Cat.No.E3132, final concentration 0.0 ImM) did. Using a SARTOBRAN 300 (Sartorius Cat. No. 5231307H5--00-B), pass through 0.22μ, and bind to an anti-human antibody NHS-activated Sepharose 4FF column (color The size was 0.5 cm X 5 cm, NHS-activated Sepharose FF Genole: Pharmacia Cat. No. 17-0906-01) was applied at a flow rate of 0.5 ral / min. At that time, in order to avoid non-specific adsorption to the anti-TPO antibody column,

Sepharose 4FF (column size: 0 O. 5 cmx O, 5 cm, Sepharose 4FF gel: Pharmaia Cat. No. 17-0149-01) <and normal Rabbit IgG binding NHS-ac ti vated Sepharose 4FF character (Calam size: 0.5 cra x 5 cm, NHS—activated Sepharose 4FF gel: Pharmacia Cat. No. 17—0906—01) as anti-TPO Connected to antibody column. Pli to case shea Yo down after termination, 1 mM EDTA, 0. 1% Tween 20, a 0. 1¾ NaN ₃ was washed mosquitoes ram in including DPBS off § chromatography, 280 nm absorbance (A _28.

) When the force dropped to about 0.2, the pre-charm was removed. The column was washed with 10 mM Sodium Phosphate pH 7.3 containing 0.5 mM NaCl to remove non-specifically adsorbed substances on the gel. After replacing the column with 150 mM NaCl, the TPO was eluted with 0.1 M Glycine-HCl pH 2.5 containing 150 mM NaCl and 5 mM CHAPS. After elution, each of the fractions to Chi's other neutralized Ri by the 1 M Na ₂ C0 _3, and subjected to high-sensitivity TP0-ELISA. Based on the results of ELISA, the TP0 eluted fraction was bulled out, and concentrated and exchanged by ultrafiltration. For ultrafiltration concentration, use an ULTRAFREE-MC 10K cut (Cat. No. UFC3LGC00, manufactured by MI LLI P0RE), centrifuge and concentrate at 4,000 X g, add DPBS several times, and repeat the concentration. Returned. Finally 500 1 of ImM EDTA, 0.1 ¾ Tween 20, 0.1%

Suspended in DPBS off § chromatography containing NaN _3, it was partially purified human plasma TP 0. A part of this partially purified TPO was subjected to gel filtration in the presence of SDS as follows. One-fifth volume of 250 raM Tris-HC1, pH 6.8, 50% Glycerol, 5¾ SDS, 10 mM EDTA was added to the partially purified TP〇, and the mixture was treated at 95 ° C for 5 minutes. Superose 6 HR gel permeation column equilibrated with DPBS containing 0.1% SDS and ImM EDTA (ø1 cm x 30 cm: Pharmacia Cat. No. 17-0537-01) The obtained fraction was subjected to TPO-ELISA, and the elution position of TPO was determined. As a result, human plasma-derived TPO was eluted in the main at almost the same position as CHO-expressed TPII (1-332a.a.), And was considered to have a molecular weight of about 80 kD. Although slightly, elution of partial-length TPO was observed at about 20 kD.

 (2) (partial) purification of TP0 from JHH7 cells ... nativeHep TPO (JHH7)

Place JHH7 cells in a culture flask with a culture area of 225 cm2, and add 5% with 1: 1 Dulbecco's modified Eagle's medium Noham F12 (DF) (Gibco) containing 10% FCS. The cells were cultured in a 37% carbon dioxide incubator at 37 ° C until they became confluent. After that, this was replaced with serum-free DF medium, and after culturing for 4 days, the supernatant was recovered. A 4 L supernatant (TP0 concentration 55 pg / nil) obtained from the above culture was divided into approximately 1 L portions, and then vigorously equilibrated with 5 mM potas i urn phosphate pH 6.8. Agarose (manufactured by Seikagaku Corporation) was applied to a column filled with 5 ml. After washing with about 250 ml of 5 mM potasium phosphate pH 6.8, elution was carried out with 15 ml of 0.4 M N-acetylgalactosamine, 5 mM potassium phosphate pH 6.8. The eluate was concentrated with UF15-Okcut (Millipore) to obtain a TPO sample (1.0 ml, 159 ng / ml).

 (3) (Partial) purification of TPO from HuH7 cells: nativeHep TPO (HuH7)

Put the HuH7 cells in a culture flask with a culture area of 225 cm2 and in a DF medium containing 10% FCS in a 5% carbon dioxide incubator at 37'C until confluent. And cultured. After that, this was replaced with serum-free DF medium, and cultured for 4 days, after which the supernatant was collected. A 1.9 L supernatant (147 pg / ml) was divided into approximately 1 L portions, and the WGA-a garose (Biochemical) was equilibrated with 5 mM potassium phosphate pH 6.8. (Manufactured by Kogyo Co., Ltd.) into a column filled with 5 ml. After washing with about 250 ml of 5 mM potasium phosphate pH 6.8, 0.4 MN-acetylgalactosamine, 5 mM potasumum pho It was eluted in 15 ml of sphate pH 6.8. The eluate was concentrated with UF 15-10kcut (manufactured by Millipore) to obtain a TP0 sample (1.5 m and 77 ng / m1).

 (4) Expression and purification of TP0 in JHH7 cells ... recHe PTP0 (JHH7 / 7 / C83)

 JHH 7 cells are grown in a 6 cm-diameter plate (Falcon) in a DMEM medium containing 10% FCS, and this is transfected. It was transformed by PDEF202-h-TPO-Pl (Reviewed Japanese Patent Application Laid-Open No. 8-228781) using the method (promega).

 The CH0 cells (CH0-DUKB II) transformed by the plasmid pDEF202-h-TP0-P1 were produced on January 31, 1995 by the Ministry of International Trade and Industry of Japan. Deposited with the Biotechnology Research Institute under the accession number FER M-BP-4988.

S3 Chi, _P DEF202-hTP0-Pl bra scan Mi de 10 g, pEFneo plus Mi de 1 mu g to 500 l Solut ion A was added to a DMEM medium, and 500 mu preparative to D Micromax E Micromax medium ΐ 6 μm of a mixture of the solution factor (1 mg / 4001 EtOH) 51 and Solition し supplemented with 51 and 5001 serum-free DΜ Ε Μ medium Add 1 ml to the diameter plate and place in a 5% carbon dioxide incubator for 6 hours and 37 hours Was. Next, 3.5 ml of DMEM medium was added, and serum was added to a final concentration of 10%. Then, the mixture was again placed in a 37 t :, 5% CO 2 incubator. Time left. The production of pEFneo brassmid was performed as follows. That is, the plasmid pRc / CMV (Invitrogen) is treated with the restriction enzyme EcoRl-BamHl, and the smaller fragment containing the neomycin resistance gene is recovered by agarose-single gel electrophoresis. After collecting and blunt-ending the ends with T4 polymerase (Takara Shuzo), the fragment and plasmid pEF18S (Japanese Patent Application Laid-Open No. 8-228781) were used as restriction enzymes. The vector DNA treated with Smal is ligated with T4 DNA ligase (Takara Shuzo). Of the two types of plasmids thus obtained, the neomycin resistance gene is inserted in the same direction as the ergonge-factor-promoter. The other was selected as pEFneo.

Next, this was stripped and dispersed with 0.05% tribcine, 53mM EDTA solution, divided into 10 24-well plates, and selected with DMEM + 10% FCS medium containing 4mg / mgG418. went. The cells that formed the colony were subjected to FDCPhMpl 635 / MTS assay (Morita H. et al., FEBS Lett. 395, 228-334, (1995)). You have selected a knee. Limited to the selected colony 8/03884 Cloning was performed by the boundary dilution method to obtain a single clone.

 The JHH7 cell line (JHH7 / 7 / C83), transformed with PDEF202-hTPO-P1 containing human TPO cDNA, was dated August 11, 1997, according to the Ministry of International Trade and Industry. It has been deposited internationally under the Budapest Treaty under the accession number FERM BP-6050 at the Institute of Biotechnology and Industrial Technology.

 Put this recombinant JHH7 cell line (JHH7 / 7 / C83) in a culture flask with a culture area of 225 cm2, and in a DF medium containing 10% FCS in a 5% CO2 incubator at 37 ° C. The cells were cultured until they became confluent. Thereafter, this was replaced with a serum-free DF medium, and cultured for 4 days, after which the supernatant was collected.

 A volume of 8.97 L of the supernatant (TP0 concentration 0.55 ^ § /! 111) was mixed with pell icon-2c assette lOkcut (Millipore, catalog number P2B0 10A05) in 899 ml (TP0 concentration 5.7 β g / ml). Protease inhibitor, Leupep tin (final concentration 0.0 ImM), trans-epoxysucciny 1 -L- for the purpose of inhibiting protein degradation by protease Leucylamido (4-guanid ino) -butane (final concentration 0.0 ImM) was added.

After decomposing into 225 ml pieces, 5 mM pot as i urn phosphate WGA-agarose (manufactured by Seikagaku Corporation) equilibrated at pH 6.8 was applied to four columns filled with 5 ml. After washing each with about 250 ml of 5 mM potasium phosphate pH 6.8, each of 0.4 M N-acetyl phosphate, 5 mM p01asium phosphate pH 6.8 and eluted with 15 ml (TPO concentration 73 μ _g / ml).

 The eluate was concentrated with UF15-lOkcut (Millipore) and changed to 5 mM potasium phosphate pH6.8 (5.0 ml, TPO concentration 1084 g / m1).

 This was injected into a Hydroxyl Apatite type 1 (BioRad 1 cm diameter, bed height 10 cm) column, and added at a flow rate of ΙπΠ / min. After the addition was completed, 5 mM potasium phosphate pH 6.8 was injected into the column to continue the bow I, and 50 ml of the unadsorbed fraction (TP0 concentration 54 / ig / ml) was collected. The pH was adjusted to 8.5 by adding 1 M Tris-base solution thereto.

Next, this fraction was developed on a Q-Sepharose HP (Pharmacia Biotech, catalog number 5 mm diameter, bed height 10 cm) column. That is, using 20 mM Tris-HCl pH 7.5 as a developing solvent A and 20 mM Tris-HCl pH 7.5 containing 1 M NaC 1 as a developing solvent B, pre-equilibration was performed with a 20 mM Tris-HCl buffer pH 7.5. Samples were added to the column at a flow rate of 0.3 ra1 / min. After the addition is completed, increase the flow rate from 0.3 ra min to 0.3. Raise to 5 ml / min, spread from 0% B to 22% B with a linear concentration gradient, and add 1.0 ml (2 mi π) to a Polypropylene tube. collected.

 Each fraction was measured by ELISA, and the TP0 fraction was identified. As a result, the distribution of TPO was found in tube numbers 34 to 55 (0.116 to 0.2 M in NaCl concentration). The TPO fraction was recovered, and TPO fraction FA (22 ml) was collected. It was decided. This was concentrated with UF15-1 Okcut (Millipore), and when the volume of the solution reached 4.1 ml during the process, the TP0 concentration was measured by ELISA (1143 tg / ml). ml). The concentration was further increased to 2001.

Separation was performed using Superdex 200HR (Pharmacia Biotech, 1 cm in diameter, 30 cm bed height) as the final step. Immediately after the column was replaced with phosphate buffered saline (PBS), 200 µl of FA concentrated sample was injected at a flow rate of 0.3 ml / min. After the injection, PBS was continuously added to the column, and a fraction of 600 μl (3πιίη) was prepared after 15 minutes from the start of the sample addition. This was subjected to silver staining, anti-human TPO polyclonal antibody, and refraction was measured by the EUSA method. As a result, tubes Nos. 13, 14, and 15 (from the start of addition) were measured. The total amount of TP ◦ was found to be 7.2 to 9.0 m 1). Issued. When these three fractions were collected and analyzed for composition using the AccQTag method, it was found that the concentration was approximately 733 mg / m 1 (1.8 ml). (table 1 ) . At this time, the amount of TP0 measured by the ELISA method was about 1 mg / ml, which was almost the same as the composition analysis value. In other words, it can be said that the ELISA used here shows the same reactivity to recHepTPO as standard TP0.

Table 1 recHe _P TPO (JHH7 / 7 / c83)

 CBB ELISA

 AccQ "TAG method [rag / ral]

Example 2> Measurement of molecular weight

For the purpose of measuring the molecular weight of various TPOs obtained in Example 1, SDS-PAGE and western blotting were performed. According to a conventional method, using microslab gel (manufactured by Dai-ichi Kagaku Chemical, 4.0% to 20% gradient polyacrylamide gel), SDS gel The electrophoresis was performed at room temperature at a constant current of 10 mA and then at 20 mA for about 2 hours. The molecular weight marker used was Breastend Prod-Imaichi (New England Biolabs).

 Immediately after the end of the electrophoresis, a constant current of 150 mA was used for 1 hour using a semi-dry transfer device (Wetfor transfer device model KS-8640, manufactured by Marisol). Then, it was electrically transferred to a PVDF film (Millipore). 0.3 M Tris, 20% methanol, pH 10.4 for anode, 25 mM Tris, 20% methanol, pH 10.4 for transfer membrane solution, 25 mM Tris, 40 mM ammonia for catholyte solution Bronic acid and 20% methanol were used.

After the transferred membrane was washed with a TTBS solution for 5 minutes, the membrane was blocked for 1 hour with a block ace (Yukishiro). After washing this again with TTBS for 5 minutes, add anti-human TPO goat antibody to 90% TTBS, 10% block ace, 0.05% BSA solution and shake for 1 hour. did. Then, the membrane was again washed twice with TTBS twice for 5 minutes, and then an anti-goat and a baoxidase conjugated antibody were added to the TTBS solution and shaken for 1 hour. Thereafter, the membrane is washed four times in a TTBS solution for 10 minutes, and the ECL is developed. The color was developed on a system (Amersham) and exposed to a film (Amersham).

 The apparent molecular weights in electrophoresis are the same for recChoTPO (CH0), nativveHepTPO (JHH7, HuH7), and recHepTPO (JHH7 / 7 / c83). This is significantly different from the predicted molecular weight of 35 KDa from the amino acid sequence, and it is easy to see that this is the increase in molecular weight due to glycosylation. Is estimated.

Example 3> Sialic acid analysis by Lect in- EU SA

 Lectin-ELISA can be applied to a solid phase by the method reported so far (Raf ferty, B. et al., J. Endoer i. 145, 527-533). By using a specific antibody against the target protein, the target protein is adsorbed, and the biotinylated receptor is allowed to react with the target protein. The color is developed by the reaction of the virgin, and the absorbance is measured.

That is, at a concentration of 10 ^ g / ml, an anti-human mouse monoclonal antibody, TN-1 (Tahara, T. et al., Br. J. Hemato 157, 783-788, (1995)) was dispensed into a 96-well plate, 100 1 per well, and the mixture was stirred and left at 4 ° C for about 10 hours. This is applied to each hole 35 After washing four times with 0 μl of Τ Τ Β S solution, agarose (SSA-) bound with the same lectin that is used for detection in advance is preliminarily used. Agarose or MAM-Agarose (both manufactured by Seikagaku Corporation) was mixed by inversion for 4 hours, and 1% BSA / TTBS solution from which lectin reaction product was removed was dispensed into each well. Stirred at room temperature for 1 hour. Subsequently, discard this and allow to dry for 30 minutes, then dispense ΙΟΟμΙ of the sample obtained by adding the sample to the above-mentioned 1% BSA / TTBS solution at various concentrations in 3 wells at each concentration. Then, the mixture was stirred for 4 hours. The plate was washed again five times with 350 μl of TTBS solution per each hole, and then lectin-piotin (manufactured by Seikagaku Corporation) was added to SSA-Biotin. In the case of MAM-Piotin, add to the 0.5% BSAZTTBS solution to a final concentration of 0.2 g / ml. Then, the mixture was stirred for 2 hours. Wash the plate again 5 times with 3501 TTBS solution per well, then 30 minutes before avidin-alkaline phosphatase / avigin — Piotin solution (manufactured by DAK0) was added to each well at a concentration of 1 μm 10 ml, a 0.25% BSATTBS solution was added, and 100 μl was dispensed per well and stirred for 1 hour. Finally, the plate is washed 6 times with 350 β1 TTBS solution per each hole, and the alkaline phosphor Chromogenic kit (manufactured by DAK0) Add 基質 μ1 substrate solution for 10 minutes, add Ιμ 発 sensitizing color solution, and stir for 5 minutes. (Manufactured by Kogyo Co., Ltd.) and absorbance at 492 nm, and as a control, 630 nm. Based on the measured values, the temperature-dependent curve of the absorbance at that value and concentration, that is, the reactivity of various TPOs with lectin was blocked. I did it.

 Fig. 3 shows that the reactivity to SSA-LectinELISA shows similar tendency for nativefasmaTPO, nativeHepTPO (JHH7) and nativeHepTPO (HuH7). In particular, nativeP1 asmaTPO, nativeHepTPO (JHH7) and nativeHepTPO (HuH7) show high reactivity, while recChoTPO is below the detection limit. The reactivity to MAM-LectinE LISA was similar for nativePlasmaTPO, nativeHepTPO (JHH7), and nativeHepTPO (HuH7), whereas recChoTPO and recHepTPO (JHH7 / 7 / c83) showed similar tendencies. Low reactivity, low power.

Example 4> In vivo activity

 In vivo activity is calculated in principle based on the platelet count when various samples are administered to mice.

That is, various specimens of various concentrations suspended in PBS are applied to 8-week-old BALB / c mice for 4 consecutive days. 3 counted from the end date On the day, collect blood samples and count platelets. The children of the door-out 1 OOmU was defined as the concentration in blood platelet count Ru example given the 3 X 106 platelets / μ _1.

Various T [rho This filtrate recHepTPO to have hands compared to the A tree Se I method Ο is 4. 5 X 10 ⁴ U / mg with a Li, lower values as compared to 9. 0 X 10 ⁴ U / mg of recChoTPO And it became.

Example 5> In vitro activity (FDCP-hMpl 635 atsushi)

 FDCP-hMpl 635 cells in which full-length human MP1 has been forcibly expressed in FDC / P2 cells subcultured in the presence of TP0 (Morita, H. et al., FEBS Lett. 395: 228-334) , (1995)) were collected, washed thoroughly, and resuspended in IMDM culture medium containing I0% FCS. Pour FDCP-hMpl 635 cells so that the number of cells per well of a 96-well flat bottom plate for tissue culture will be 2.5 x 103 cells, and use the standard and test samples as well. In addition, the final volume was 200 μl / well. The plate was placed in 5% carbon dioxide and incubated at 37 ° C for 3 days. Four hours before the third day of culture, add MTSZPMS solution (promega) 251 to each well, and after completion of the culture, read the absorbance at 492 nm on a plate (Seikagaku Corporation). It was measured.

For recombinant TPO derived from various cells, the composition of TPO Analytical values, TPO from plasma or non-recombinant liver cells are described in ELISA (Tahar, T. et al., Br. J. Hemato 157, 783-788, (1995)). Based on the values obtained, it was found that all of them showed the same activity (Fig. 4).

Example 6〉 Preparation of 2, 6 — serial transfection enzyme expression vector (pE Fneo — a2, 6 ST)

After sufficiently cutting pEF neo plasmid described in (4) of Example 1 with XbaI, XhoI is allowed to act for a short time continuously. As a result, a partially truncated fragment of XbaI-XhoI was obtained. In this case, since there are three Xhol sites, three types of fragments containing the SV40 replication origin (0 ri) can be obtained. The target fragment is the second longest, but it is not possible to connect the three types of mixture with the target fragment, and later p EF neo Sequencer using 5 '— CCTCAGACAGTGGTTCAAAG-3' (SEQ ID NO: 2), which encodes the position near the 3 'end of the EF-1α promoter of the It was possible, and indeed, to eliminate the other two possibilities. The target fragment here is ^ 1 galactosides α-2, 6-derived from rat liver. 5 'end including the entire length of the cDNA sequence (Genbank M 18769 / We in stein, J. et al., J. Biol. Chem. 262, 17735-17743) Is a DNA fragment having an XhoI site and an Xbal site at the 3 'end.

 This vector is the replication initiation region of SV40,

— Shot factor 1 — α promoter, SV40 initial polyadenylation site, and SV40 promoter region, neomycin resistance Gene, including the SV40 initial polyadenylation site and the β-lactamase gene (Amp ^r ); One downstream of the motor is connected to 9 galactosides cr2, 6-serial transfection cDNA.

Example 7> Introduction of a 2, 6 — serial transfection enzyme expression vector into CHO cells

Already in that we not express human full length TPO CH 0 2 9/1 4 strains (2 0 0 n M main source preparative les xenon one preparative resistance) to 3 X 1 0 ⁵ flop les over the cells 6 cm diameter （(Fa1c0n company) 10% 中 α Minimum essential medium containing fetal serum (α-Μ Ε Ε (-)), thymidine and hypoxanthine added ), And transfer it to a transfector. Transformation was carried out by the plasmid method (Promega), and the expression vector of α2,6—sialyltransferase was introduced.

That is, p EF neo —α2,6ST plasmid 15S prepared in Example 6 was mixed with a-MEM (—) 500 μ1 added to 15S. And 1 mg of the transfection factor dissolved in 400 μl of ethanol was added to 5 il and α-MEM (-) 5 Ό 0 β 1 was added and mixed. After mixing, the mixture was allowed to stand at room temperature for 10 minutes. During this time, the cells cultured on a 6 cm-diameter plate should be replaced with α-MEM (—) not containing fetal calf serum. Was added dropwise to this DNA solution flop les over preparative was about 6 hours of culture in C 0 ₂ Lee emissions queue Beta scratch. 2.5 ml of α-MEM (—) and 501 fetal fetal serum were added to the plate, and the culture was continued for 1 day. Serum-containing selection medium

(-MEM (-), thymidine, hypoxanthine-free, and dienecin (Gibocco) added) were selected. The selection was performed after the cells were treated with Tribcine, followed by a 6 cm diameter plate.

After dividing one plate into 10 plates of 24 holes, the culture is continued by exchanging the selective medium 2-3 days later and continuing the culture. did . At this time, the final concentration was 0.5 mg / m2 for the first 2 days. Selective medium containing 1 Geneticsin for 3 days

Change to a selective medium containing 1 mgm 1 of dienecin, and then replace the medium with a final medium containing about half the final concentration of dienecin. As a result, the dienetin concentration was eventually reduced to 0.2 mg Z ml. The concentration of the meso-rexet was maintained at 200 nM during this period. For the holes in which cells have grown, the amount of human TPO in the culture supernatant of TPO — ELISA and that of α2 in SSA — lectin ELISA are calculated as follows.

The relative amount of sialic acid added in the 6-type binding mode was measured. In its one second human T [rho Omicron activity and _alpha 2, 6-linked sheet also addition of A Le acids found we were in the culture supernatant, the © El has newly

The cells can be split 1:15 in a selection medium containing 200 mg of ηΜ meso-rexet 2 mg / m 1 of sci- enticin and culture can be continued. In addition, as a result of cloning by growing cells resistant to lysiegenicin, a final 200 nM meso-rexetate 2 was obtained. Clones resistant to mg / m 1 of geneticin CHO + 2, 63, 19 19, 4 and 〇1 "10 +2,

6 S T / 2 1 / c 3

<Example 8> Confirmation of expression For the purpose of measuring the molecular weights of various TPOs obtained in Example 7, SDS-PAGE / Western blotting was performed by the method described in Example 2. Tahara, T. et al.'S method (Br

J. Hematol. 5 / 783-788 (1995)), clone CHO + 2, 6ST / 1 obtained from the expression level measured based on the expression level.

9 c4 and THO produced by CHO + 2, 6ST / 21 / c3

The apparent molecular weight in the electrophoresis of P O is recChhoTP

O CHO 2 9 c 14) and exists near 8 O KDa

(See Figure 5) This is the molecular weight 3 predicted from the amino acid sequence.

Unlike 5 KDa, it is easy to assume that this is an increase in molecular weight due to glycosylation

Example 9> Sialic acid analysis by lectin-ELISA—Sialic acid analysis was performed on TP0 obtained in Example 8 by the method described in Example 3 ( (Fig. 6)

 According to the results, CHO + 2, 6 ST

/ 19 Zc4 is a cell that produces Τ Ρ with a predominantly added sialic acid in the α 2, 6 — binding mode, CHO + 2,

6 STZ 2 1 / c 3 produces Ρ 0 in which both α 2, 3-and α 2, 6-linked sialic acids are added on the same molecule. It turned out to be a cell.

Example 10> Activity confirmation at i n V i tr 0

 For TP0 obtained in Example 7, the invitro activity was confirmed by the FDCP—hMpl6335 Atssay method described in Example 5.

In a 96-well plate, 2.5 X 10 ³ FDCP hMPl 6 35 cells were incubated with various concentrations of TPO at 37 ° C for 3 days, and then 2 Add 20 μl of MTS solution to the well. After a further 4 hour incubation, the activity of the sample was detected as absorbance at 492 nm. Figure 7 shows the results.

 These results indicate that both TPO expressed in CHOD-cells and TPO expressed in CHO cells transfected with a2,6-sialyltransferase gene are FDCP h The propagation activity of Mp1665 was almost equivalent. In other words, even if the binding mode of sialic acid changes from α2,3 type originally found in CHO cells to α2,6 type, the cell growth activity remains the same regardless of the ratio. It is the same.

Claims

The scope of the claims

 1. Human thrombopoietin (TP0) produced by a human cell line.

 2. The human TPO is an expression product of an exogenous human TPO gene in a human cell line, which is isolated and purified. The human TPO described in 1.

 3. The human TPO according to claim 2, wherein the human cell line is a human cell line derived from a TPO-producing organ.

 4. The human TPO according to claim 3, wherein the human cell line derived from a TPO-producing organ is a human liver-derived cell.

 5. The human TPO according to claim 4, wherein the human liver-derived cell is J type 7 (FERM BP-6049).

 6. The human TPO according to claim 4, wherein the human liver-derived cell is HuH7 (FERM BP-6048).

 7. The human TP III according to claim 3, wherein the human cell line derived from a TPO-producing organ is a human bone marrow stromal cell.

8. It is characterized in that human TPO is an expression product of an endogenous human TP0 gene in a human cell line, which has been isolated and purified. A human TPO according to claim 1.

9. The human bacterium according to claim 8, wherein the human cell line is a human cell line derived from a TPO-producing organ.

 10. The human TPO according to claim 9, wherein the human cell line derived from a TPO-producing organ is a human liver-derived cell.

 11. The human TPO according to claim 10, wherein the human TPO has a cell strength derived from human liver, J type 7 (FERM BP-6049).

 12. The human TP に according to claim 10, wherein the human liver-derived cell is HuH7 (FERM BP-6048).

 13. The human TPO according to claim 9, wherein the human cell line derived from a TPO-producing organ is a human bone marrow cell.

 14. The method according to claim 1, wherein the saccharide has a sugar chain structure having an affinity for SSA-lectin column.ト Τ Ρ.

 15. The method according to claim 14, wherein the amino acid sequence of the human amino acid is 1 to 332 of SEQ ID NO: 1.

 16. The hin according to any one of claims 1 to 13, characterized in that it has a sugar chain structure of at least ct2,6-linked sialic acid. TP 〇.

17 7. Amino acid sequence of human TP 0 1 to 332 of SEQ ID NO: 1 The human TP 〇 according to claim 16, characterized in that:

 18. A pharmaceutical composition comprising the human TP〇 according to any one of claims 1 to 17 as an active ingredient.

 19. A platelet increasing agent comprising human TPO according to any one of claims 1 to 17 as an active ingredient.

 20. A therapeutic agent for platelet damage, comprising the human TPO according to any one of claims 1 to 17 as an active ingredient.

 21. A therapeutic agent for thrombocytopenia, comprising the human TPO according to any one of claims 1 to 17 as an active ingredient.

 22. The platelet increasing agent according to claim 19, wherein the antigenicity is reduced.

23. The therapeutic agent for platelet damage according to claim 20, wherein the antigenicity is reduced.

 24. The therapeutic agent for thrombocytopenia according to claim 21, wherein the antigenicity is reduced.

 25. The platelet-increasing agent according to claim 19, which has improved pharmacokinetics.

 26. The therapeutic agent for platelet damage according to claim 20, wherein the pharmacokinetics are improved.

27. The thrombocytopenia according to claim 21 whose pharmacokinetics has been improved. Therapeutic agent.

 28. A method for producing human TPO, which comprises expressing an exogenous human TPO gene in a human cell line derived from a TPO-producing organ.

 29. A method for producing human TPO, comprising expressing an exogenous human TP〇 gene in human liver-derived cells.

 30. A method for producing human TPO, comprising expressing an exogenous human TPO gene in human bone marrow stromal cells.

 31. A method for producing human TPO, comprising expressing an exogenous human TPO gene in JHH7 (FERM BP-6049).

32. A human TP0 obtainable by the method according to any one of claims 28 to 31.

 33. At least a human with an α2,6-linked sialic acid sugar chain structure.

 34.t is a product of the expression of exogenous human TPO gene in a cell line, which is isolated and purified. A human TPO according to claim 33.

35. The cell line is characterized in that the cell line is a CHO cell into which the α2,6—Sialyltransferase—gene has been introduced. The hit finger 0 according to claim 34.

 36. Including transfection of α2,6—Sialyltransferase cDNA into CHO cells and expressing the human TPO gene in the CHO cells And a method for producing human having at least an α2,6-linked sialic acid sugar chain structure.

 37. Expression of the human Ο gene in CHO cells and introduction of α2,6—Sialyltransferase cDNA into the CHO cells. A method for producing a human having at least an α2,6-linked sialic acid sugar chain structure, comprising: