Method for producing heparin-like substance, recombinant cell, and method for producing the same

Abstract

The purpose of the present invention is to provide a production method whereby a heparin-like substance is efficiently produced without using an animal-derived tissue. The present invention pertains to a method for producing a heparin-like substance, etc., said method comprising: (1) a step for preparing mammalian cells producing the heparin-like substance; (2) a step for preparing recombinant cells wherein a gene encoding the extracellular domain of syndecan is introduced into the heparin-like substance-producing mammalian cells prepared in step (1); and (3) a step for culturing the recombinant cells prepared in step (2) in a medium and collecting the heparin-like substance from the culture supernatant thus obtained.

Description

Technical Field

[0001] The present invention relates to a method for producing a heparin-like substance, a recombinant cell, and a method for producing the same.

Background Art

[0002] Heparin is an essential drug used in medical fields such as artificial dialysis and extracorporeal circulation as an anticoagulant. Heparin is used, for example, as a preventive or therapeutic agent for thromboembolism in an injectable form. Heparin is also used, for example, to form an anticoagulant surface on various experimental and medical devices such as kidney dialysis machines.

[0003] Heparin is a polysaccharide that is one of the four major subfamilies of glycosaminoglycans (hereinafter also abbreviated as GAG). GAG is a long polysaccharide consisting of repeating disaccharide units. GAG is mainly classified into four subfamilies: (1) heparan sulfate (hereinafter also abbreviated as HS) / heparin, (2) keratan sulfate (KS), (3) chondroitin / dermatan sulfate (CS / DS), and (4) hyaluronic acid or hyaluronan (HA).

[0004] The subfamilies of GAG differ from each other in terms of monosaccharide components, glycosyl linkages, and the position and degree of sugars. Except for HA, GAG is covalently bound to a core protein, and the one in which GAG is covalently bound to a core protein is known as a proteoglycan.

[0005] Heparin and HS are heterogeneous mixtures of acidic mucopolysaccharides, and heparin is a type of HS. Heparin / HS is a linear polysaccharide in which disaccharides of uronic acid (β-D-glucuronic acid and α-L-iduronic acid) and glucosamine (D-N-acetylglucosamine and D-N-sulfated glucosamine) form one unit, and they repeat the α- or β-1,4 bond dozens to hundreds of times. The molecular weight varies from 3 to 30 kDa depending on the chain length, with an average of 12 - 15 kDa. Modifications to heparin / HS include, for example, O-sulfation at the 2-position of uronic acid, O-sulfation at the 3- and 6-positions of glucosamine, and N-sulfation of the amino group at the 2-position.

[0006] Almost all mammalian cells produce GAGs that are incorporated into proteoglycans in cell-associated glycans present in the extracellular matrix to define tissue morphology and function. HS regulates cell growth and development by regulating growth factors such as the fibroblast growth factor (FGF) family, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). On the other hand, heparin is mainly produced by mast cells.

[0007] Heparin used in pharmaceuticals, etc., is mainly extracted and purified from porcine small intestine or bovine lung. However, heparin obtained from these sources is associated with several adverse events due to contamination with over-sulfated chondroitin sulfate (Non-Patent Document 1). Also, the isolation of heparin from animal tissues has a risk of transmission of disease pathogens such as viruses, bacteria, and Transmissible Spongiform Encephalopathy from animals to humans. Therefore, the development of new production technologies for heparin that do not use animal-derived tissues is desired. Although it is also possible to synthesize heparin by chemical or chemoenzymatic methods, it is difficult to produce a population of heterogeneous structures present in animal-derived heparin.

[0008] On the one hand, syndecan is a family of four cell surface proteoglycans with a retained plasma membrane domain and a cytoplasmic domain. The extracellular domain has covalently attached glycosaminoglycan chains. These are mainly heparan sulfate (HS), and also include chondroitin sulfate (CS) (Non-Patent Document 2).

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0010] As described above, there is a desire to develop a new production technology for heparin that does not use animal-derived tissues. The present inventors attempted to produce a heparin-like substance using mammalian cells that produce a heparin-like substance, and found that proteoglycans containing the heparin-like substance produced from the mammalian cells adhered to the cell membrane and were difficult to secrete into the culture supernatant. Therefore, a complicated purification process is required, which poses a major problem in the industrial production of heparin-like substances. Accordingly, an object of the present invention is to provide a production method for efficiently producing a heparin-like substance without using animal-derived tissues.

Means for Solving the Problems

[0011] The present inventors found that the above problems can be solved by introducing a gene encoding the extracellular domain of syndecan into mammalian cells that produce a heparin-like substance, and completed the present invention.

[0012] The present invention is as follows. 1. A method for producing a heparin-like substance, comprising the following steps (1) to (3). (1) A step of preparing mammalian cells that produce a heparin-like substance (2) A step of preparing recombinant cells in which a gene encoding the extracellular domain of syndecan (hereinafter abbreviated as SDC) is introduced into the mammalian cells that produce the heparin-like substance prepared in step (1) (3) A step of culturing the recombinant cells prepared in step (2) in a medium and recovering the heparin-like substance from the obtained culture supernatant 2. The mammalian cells that produce the heparin-like substance are cells into which at least one of a gene encoding bifunctional heparan sulfate N-deacetylase / N-sulfotransferase (hereinafter abbreviated as NDST2) and a gene encoding heparan sulfate ester glucosamine 3-sulfotransferase 1 (hereinafter abbreviated as Hs3st1) has been introduced. The production method according to 1 above. 3. The mammalian cells are Chinese hamster ovary (hereinafter abbreviated as CHO) cells. The production method according to 1 or 2 above. 4. NDST2 is a protein that contains any one of the amino acid sequences of the following (1a) to (1c) and has the function of NDST2. The production method according to 2 or 3 above. (1a) The amino acid sequence set forth in SEQ ID NO: 2 (1b) An amino acid sequence in which several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2 (1c) An amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 2 5. Hs3st1 is a protein that contains any one of the amino acid sequences of the following (2a) to (2c) and has the function of Hs3st1. The production method according to any one of 2 to 4 above. (2a) The amino acid sequence set forth in SEQ ID NO: 3 (2b) An amino acid sequence in which several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3 (2c) An amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 3 6. The production method according to any one of 1 to 5 above, wherein the extracellular domain of SDC contains any one of the following amino acid sequences (3a) to (3c) and has the function of the extracellular domain of SDC. (3a) The amino acid sequence set forth in SEQ ID NO: 1 (3b) An amino acid sequence in which several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1 (3c) An amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 7. The production method according to any one of 2 to 6 above, wherein NDST2 and Hs3st1 are derived from mouse or human. 8. The production method according to any one of 2 to 7 above, wherein the gene encoding NDST2 is any one of the following nucleotide sequences (1A) to (1D) and contains a nucleotide sequence encoding the amino acid sequence of a protein having the function of NDST2. (1A) The nucleotide sequence set forth in SEQ ID NO: 4 (1B) A nucleotide sequence in which several nucleotides are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 4 (1C) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 4 (1D) A nucleotide sequence having 80% or more homology with the nucleotide sequence set forth in SEQ ID NO: 4 9. The production method according to any one of 2 to 8 above, wherein the gene encoding Hs3st1 is any one of the following nucleotide sequences (2A) to (2D) and contains a nucleotide sequence encoding the amino acid sequence of a protein having the function of Hs3st1. (2A) The nucleotide sequence set forth in SEQ ID NO: 5 (2B) A nucleotide sequence in which several nucleotides are deleted, substituted or added in the nucleotide sequence set forth in SEQ ID NO: 5 (2C) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 5 A base sequence having 80% or more homology with the base sequence described in Array No. (2D) 5 10. The production method according to any one of 1 to 9 above, wherein the gene encoding the extracellular domain of SDC is any one of the following base sequences (3A) to (3D) and includes a base sequence encoding an amino acid sequence of a protein having the function of the extracellular domain of SDC. (3A) The base sequence described in SEQ ID NO: 6 (3B) A base sequence in which several bases from 1 are deleted, substituted or added in the base sequence described in SEQ ID NO: 6 (3C) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 6 (3D) A base sequence having 80% or more homology with the base sequence described in SEQ ID NO: 6 11. A recombinant cell in which a gene encoding the extracellular domain of SDC is introduced into a mammalian cell that produces a heparin-like substance. 12. The recombinant cell according to 11 above, wherein the mammalian cell that produces the heparin-like substance is a cell into which at least one of the gene encoding NDST2 and the gene encoding Hs3st1 is introduced. 13. The recombinant cell according to 11 or 12 above, wherein the mammalian cell is a CHO cell. 14. A method for producing a cell that secretes and produces a heparin-like substance, comprising the step of introducing a recombinant expression vector containing a gene encoding the extracellular domain of SDC into a mammalian cell that produces the heparin-like substance. [[Effect of the Invention]]

[0013] According to the method for producing a heparin-like substance of the present invention, by introducing a gene encoding the extracellular domain of syndecan into a mammalian cell that produces the heparin-like substance, the heparin-like substance can be secreted and produced in the culture supernatant, and the heparin-like substance can be recovered from the culture supernatant. This solves the problem that the heparin-like substance as a product adheres to the cell membrane and does not require a complicated purification process. Therefore, according to the production method of the present invention, the heparin-like substance can be efficiently produced without using animal-derived tissues.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0015] Hereinafter, unless otherwise specified, the terms used in this specification have the meanings commonly used in the art.

[0016] <Method for Producing Heparin-Like Substance> The method for producing a heparin-like substance of the present invention is characterized by including the following steps (1) to (3). (1) Step of preparing mammalian cells that produce a heparin-like substance (2) Step of preparing recombinant cells in which a gene encoding the extracellular domain of syndecan is introduced into mammalian cells that produce a heparin-like substance prepared in step (1) (3) Step of culturing the recombinant cells prepared in step (2) in a medium and recovering the heparin-like substance from the resulting culture supernatant The following describes each step.

[0017] Step (1): Step of preparing mammalian cells that produce a heparin-like substance Step (1) is a step of preparing mammalian cells that produce a heparin-like substance by introducing a gene encoding a protein required for heparin biosynthesis into mammalian cells serving as a host.

[0018] In the present invention, the "heparin-like substance" refers to a mixture of heparin and HS.

[0019] Examples of mammalian cells include Chinese hamster ovary cells (CHO cells) [Journal of Experimental Medicine, 108, 945 (1958); Proc. Natl. Acad. Sci. USA, 60, 1275 (1968); Genetics, 55, 513 (1968); Chromosoma, 41, 129 (1973); Methods in Cell Science, 18, 115 (1996); Radiation Research, 148, 260 (1997); Proc. Natl. Acad. Sci. USA, 77, 4216 (1980); Proc. Natl. Acad. Sci., 60, 1275 (1968); Cell, 6, 121 (1975); Molecular Cell Genetics, Appendix I, II (pp. 883-900)], CHO cells deficient in the dihydrofolate reductase gene (CHO / DG44 cells) [Proc. Natl. Acad. Sci. USA, 77, 4216 (1980)], CHO-K1 (ATCC CCL-61), DUKXB11 (ATCC CCL-9096), Pro-5 (ATCC CCL-1781), CHO-S (Life Technologies, Cat#11619), Pro-3, human umbilical vein endothelial cells (HUVEC), human umbilical artery endothelial cells (HUAEC), human lung microvascular endothelial cells (HLMVEC), human aortic endothelial cells (HAoEC), human coronary artery endothelial cells (HCAEC), human pulmonary artery endothelial cells (HPAEC), human embryonic kidney (HEK), rat myeloma cells YB2 / 3HL.P2.G11.16Ag.20 (also referred to as YB2 / 0), monkey cells COS cells, mouse myeloma cells NSO, mouse myeloma cells SP2 / 0-Ag14, Syrian hamster cells BHK or HBT5637 (Japanese Patent Laid-Open No. 63-000299), etc. From the perspective of production efficiency, among these, CHO cells, CHO / DG44 cells or CHO-K1 (ATCC CCL-61) are preferred, and CHO cells are more preferred.

[0020] Examples of proteins required for heparin biosynthesis include bifunctional heparan sulfate N-deacetylase / N-sulfotransferase (hereinafter abbreviated as NDST2), heparan sulfate ester glucosamine 3-sulfotransferase 1 (hereinafter abbreviated as Hs3st1), heparan sulfate 2-O-sulfotransferase, C5-epimerase, heparan sulfate 6-O-sulfotransferase, and the like. Among these, NDST2 and Hs3st1 are preferred. These proteins may be variant proteins as long as they have the activity required for heparin biosynthesis. These proteins are preferably derived from mammals, more preferably from mice or humans.

[0021] Examples of the proteins required for heparin biosynthesis include proteins that are not expressed in wild-type mammalian cells. Specifically, for example, when the mammalian cells are CHO cells, at least one of the genes encoding NDST2 and Hs3st1, which are required for heparin biosynthesis and not expressed in CHO cells, can be introduced to obtain CHO cells that produce heparin-like substances (Bail JY et al., Metab Eng14:81-90, 2012).

[0022] Examples of NDST2 include proteins that contain any one of the following amino acid sequences (1-a) to (1-c) and have the function of NDST2. (1-a) The amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_003626.1 (1-b) An amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_003626.1 (1-c) An amino acid sequence having 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence of NCBI accession number NP_003626.1

[0023] Examples of Hs3st1 include a protein that contains any one of the following amino acid sequences (2-a) to (2-c) and has the function of Hs3st1. (2-a) The amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence of NCBI accession number NP_034604.1 (2-b) An amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence of NCBI accession number NP_034604.1 (2-c) An amino acid sequence having 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence of NCBI accession number NP_034604.1

[0024] A polypeptide having an amino acid sequence in which one to several amino acids are deleted, substituted or added in the target amino acid sequence can be obtained, for example, by introducing site-specific mutations into DNA encoding a polypeptide containing the amino acid sequences of SEQ ID NOs: 1 to 3 using a site-directed mutagenesis method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987 - 1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985)].

[0025] The range of "one to several amino acids" in the "deletion, substitution, addition of one to several amino acids" of the amino acid sequence is not particularly limited. For example, if 100 amino acids in the amino acid sequence are taken as one unit, per this unit, it means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, more preferably about 1, 2, 3, 4 or 5 amino acids.

[0026] "Deletion of amino acids" means the deletion or disappearance of amino acid residues in the sequence. "Substitution of amino acids" means that the amino acid residues in the sequence are replaced by other amino acid residues. "Addition of amino acids" means that new amino acid residues are added so as to be inserted into the sequence.

[0027] Specific embodiments of "deletion, substitution, addition of one to several amino acids" include a mode in which one to several amino acids are replaced by other chemically similar amino acids. For example, when a certain hydrophobic amino acid is replaced by another hydrophobic amino acid, or when a certain polar amino acid is replaced by another polar amino acid having the same charge, etc. can be mentioned. Such chemically similar amino acids are known in the art for each amino acid.

[0028] Taking specific examples, non-polar (hydrophobic) amino acids include alanine, valine, glycine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, etc. Polar (neutral) amino acids include serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Basic amino acids having a positive charge include arginine, histidine, lysine, etc. Also, acidic amino acids having a negative charge include aspartic acid, glutamic acid, etc.

[0029] Examples of amino acid sequences having deletions, substitutions, additions, etc. of several amino acids from 1 in the amino acid sequence of the target protein include amino acid sequences having a sequence identity of a certain level or more with the amino acid sequence of the target protein. For example, amino acid sequences having a sequence identity of 60% or more, preferably 65% or more, preferably 70% or more, preferably 75% or more, preferably 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with the amino acid sequence of the target protein can be mentioned.

[0030] Examples of the gene encoding NDST2 include a gene having any one of the following base sequences (1-A) to (1-D) and including a base sequence encoding the amino acid sequence of a protein having the function of NDST2. (1-A) The base sequence described in SEQ ID NO: 4 or the base sequence of NCBI accession number NM_003635.3 (1-B) A base sequence in which several bases have been deleted, substituted, or added in the base sequence described in SEQ ID NO: 4 or the base sequence of NCBI accession number NM_003635.3 (1-C) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 4 or the base sequence of NCBI accession number NM_003635.3 (1-D) A base sequence having at least 60% or more homology, preferably 80% or more homology, and still more preferably 95% or more homology with the base sequence described in SEQ ID NO: 4 or the base sequence of NCBI accession number NM_003635.3

[0031] Examples of the gene encoding Hs3st1 include a gene having any one of the following base sequences (2-A) to (2-D) and including a base sequence encoding the amino acid sequence of a protein having the function of Hs3st1. (2-A) The base sequence described in SEQ ID NO: 5 or the base sequence of NCBI accession number NM_010474.2 (2-B) The nucleotide sequence described in SEQ ID NO: 5 or the nucleotide sequence of NCBI accession number NM_010474.2, wherein several nucleotides from 1 to several are deleted, substituted or added (2-C) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in SEQ ID NO: 5 or the nucleotide sequence of NCBI accession number NM_010474.2 (2-D) A nucleotide sequence having at least 60% or more homology, preferably 80% or more homology, more preferably 95% or more homology with the nucleotide sequence described in SEQ ID NO: 5 or the nucleotide sequence of NCBI accession number NM_010474.2

[0032] The nucleotide sequence that hybridizes under stringent conditions refers to the nucleotide sequence of DNA capable of hybridization obtained by colony hybridization method, plaque hybridization method, Southern blot hybridization method or DNA microarray method using DNA containing the target nucleotide sequence as a probe.

[0033] Specifically, the DNA base sequence can be identified by performing hybridization at 65°C in the presence of 0.7 to 1.0 mol / L sodium chloride using DNA derived from hybridized colonies or plaques, or a filter or slide onto which a PCR product or oligo DNA having the sequence has been immobilized [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University, (1995)], followed by washing the filter or slide at 65°C using 0.1 to 2x SSC solution (1x SSC solution consists of 150 mmol / L sodium chloride and 15 mmol / L sodium citrate).

[0034] Examples of DNA containing a nucleotide sequence that hybridizes under stringent conditions include DNA that shares a certain level of sequence identity with the nucleotide sequence of a DNA containing the nucleotide sequence of a target gene used as a probe. Examples include DNA that is at least 60% homologous to the target nucleotide sequence, preferably 80% homologous, and more preferably 95% homologous. Furthermore, examples include DNA containing a nucleotide sequence that has deletions, substitutions, or additions of one to several, preferably 1 to 40, preferably 1 to 35, preferably 1 to 30, preferably 1 to 25, preferably 1 to 20, more preferably 1 to 15, even more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and even more preferably 1, 2, 3, 4, or 5, bases per unit of the nucleotide sequence of the target gene.

[0035] "Base deletion" means that there is a deletion or disappearance of a base in the sequence, "base substitution" means that a base in the sequence is replaced by another base, and "base addition" means that a new base is added so as to be inserted.

[0036] Polymorphisms are often observed in the base sequences of genes encoding eukaryotic proteins. Genes used in the present invention also contain genes in which such polymorphisms have caused small-scale mutations in the base sequences.

[0037] The homology numerical value in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art, unless otherwise specified. For base sequences, examples include numerical values calculated using the default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)]. For amino acid sequences, examples include numerical values calculated using the default parameters in BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997), https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq].

[0038] As default parameters, G (Cost to open gap) is 5 when it is a nucleotide sequence and 11 when it is an amino acid sequence, -E (Cost to extend gap) is 2 when it is a nucleotide sequence and 1 when it is an amino acid sequence, -q (Penalty for nucleotide mismatch) is -3, -r (reward for nucleotide match) is 1, -e (expect value) is 10, -W (wordsize) is 11 residues when it is a nucleotide sequence and 3 residues when it is an amino acid sequence, -y [Dropoff(X) for blast extensions in bits] is 20 for blastn and 7 for programs other than blastn, -X (X dropoff value for gapped alignment in bits) is 15 and -Z (final X dropoff value for gapped alignment in bits) is 50 for blastn and 25 for programs other than blastn (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch).

[0039] Mammalian cells that produce heparin-like substances can be obtained by introducing a recombinant vector containing cDNA encoding the full length or a partial length of a protein required for heparin biosynthesis into mammalian cells serving as host cells.

[0040] As the recombinant vector, any one can be used as long as it can autonomously replicate or integrate into the chromosome in the host cell used and contains an appropriate promoter at a position where DNA encoding a polypeptide can be transcribed.

[0041] The recombinant vector does not necessarily require a transcription termination sequence, but it is preferable to arrange a transcription termination sequence immediately below the structural gene. Furthermore, the recombinant vector may contain a gene that controls the promoter.

[0042] As the recombinant vector, it is preferable to use a plasmid in which the Kozak sequence, which is a ribosome binding sequence, is appropriately arranged around the start codon.

[0043] In the base sequence of DNA, the bases can be substituted so as to be codons optimal for expression in the host, thereby improving the production rates of the target NDST2 and Hs3st1.

[0044] As the recombinant vector, any vector that can function in animal cells can be used. For example, pcDNA I, pcDM8 (Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 03-22979; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Patent Laid-Open No. 02-227075), pcDM8 [Nature, 329, 840 (1987)], pcDNA I / Amp (Invitrogen), pcDNA3.1 (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (International Publication No. 97 / 10354), N5KG1val (U.S. Patent No. 6001358), INPEP4 (Biogen-IDEC), and transposon vectors (International Publication No. 2010 / 143698) and the like can be mentioned.

[0045] As the promoter, any promoter that can function in animal cells can be used. For example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40, the promoter of retrovirus, the metallothionein promoter, the heat shock promoter, the SRα promoter, or the promoter or enhancer of Moloney murine leukemia virus can be mentioned. Further, the enhancer of the IE gene of human CMV may be used together with the promoter.

[0046] The recombinant vector may contain a selection marker. A "selection marker" is a gene that enables the selection of cells containing the gene. "Positive selection" refers to the process in which positive selection occurs and cells containing the selection marker are selected. Drug resistance is an example of a positive selection marker. Cells containing the marker survive in a culture medium containing the drug, while cells without the marker die. Examples of selection markers include drug resistance genes such as neo that confers G418 resistance, hygr that confers hygromycin resistance, and puro that confers puromycin resistance. Other positive selection marker genes include genes that enable the identification or screening of cells containing the marker. These genes include, among others, fluorescent protein (GFP and GFP-like chromophores, luciferase) genes, the lacZ gene, the alkaline phosphatase gene, and surface markers such as CD8. "Negative selection" refers to the process of exposing cells containing a negative selection marker to an appropriate negative selection drug to kill them. For example, cells containing the herpes simplex virus thymidine kinase (HSV-tk) gene [Wigler et al, Cell 11:223 (1977)] are sensitive to the drug ganciclovir (GAN). Similarly, the gpt gene makes cells sensitive to 6-thioxanthine.

[0047] As a method for introducing the recombinant vector into the host cell, any method for introducing DNA into animal cells can be used. For example, the electroporation method [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open No. 02-227075), or the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like can be mentioned.

[0048] Step (2): A step of preparing a recombinant cell in which a gene encoding the extracellular domain of syndecan is introduced into a mammalian cell that produces the heparin-like substance prepared in step (1). Step (2) is a step of preparing recombinant cells by introducing a recombinant vector containing a gene encoding the extracellular domain of syndecan into mammalian cells that produce the heparin-like substance prepared in step (1).

[0049] The structure of syndecan consists of an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Among these, the extracellular domain of syndecan contains a glycosaminoglycan binding site. It is preferable that the extracellular domain of syndecan has at least a glycosaminoglycan binding site. The extracellular domain of syndecan is preferably derived from a mammal, more preferably from a mouse or a human.

[0050] Examples of the extracellular domain of syndecan include proteins that contain any one of the following amino acid sequences (3-a) to (3-c) and have the function of the extracellular domain of syndecan. (3-a) The amino acid sequence set forth in SEQ ID NO: 1 or the amino acid sequence of NCBI accession number NP_001006947.1 (3-b) An amino acid sequence in which several amino acids are deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1 (3-c) An amino acid sequence having 60% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more homology with the amino acid sequence set forth in SEQ ID NO: 1

[0051] The amino acid sequence set forth in SEQ ID NO: 1 is the amino acid sequence from the N-terminus to the 1st to 229th amino acids of the amino acid sequence of NCBI accession number NP_001006947.1 (total length 310 amino acids).

[0052] Examples of the gene encoding the extracellular domain of syndecan include genes that have any one of the following base sequences (3-A) to (3-D) and contain a base sequence encoding the amino acid sequence of a protein having the function of the extracellular domain of syndecan. (3-A) The nucleotide sequence described in SEQ ID NO: 6 or the nucleotide sequence of NCBI accession number NM_001006946.1 (3-B) A nucleotide sequence in which several nucleotides from 1 are deleted, substituted or added in the nucleotide sequence described in SEQ ID NO: 6 or the nucleotide sequence of NCBI accession number NM_001006946.1 (3-C) A nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence described in SEQ ID NO: 6 or the nucleotide sequence of NCBI accession number NM_001006946.1 (3-D) A nucleotide sequence having at least 60% or more homology, preferably 80% or more homology, more preferably 95% or more homology with the nucleotide sequence described in SEQ ID NO: 6 or the nucleotide sequence of NCBI accession number NM_001006946.1

[0053] In step (2), a recombinant vector containing a cDNA encoding the extracellular domain of syndecan, which encodes the full length or a partial length of the extracellular domain of syndecan, is introduced into mammalian cells that produce the heparin-like substance prepared in step (1), thereby obtaining recombinant cells capable of secreting the heparin-like substance into the culture supernatant.

[0054] As the recombinant vector and the method for introducing the recombinant vector into the host cell, the same method as in step (1) is used.

[0055] (3) Step of culturing the recombinant cells prepared in step (2) in a medium and recovering the heparin-like substance from the resulting culture supernatant Step (3) is a step of culturing the recombinant cells prepared in step (2) in a medium to cause the recombinant cells to secrete and produce the heparin-like substance into the medium, and recovering the heparin-like substance from the resulting culture supernatant.

[0056] In step (3), the recombinant cells are preferably cultured under conditions that promote the production of the heparin-like substance. Specifically, for example, it is preferable to culture the recombinant cells in a medium that enables the production of the heparin-like substance by the recombinant cells and promotes the secretion of the heparin-like substance from the recombinant cells into the culture supernatant.

[0057] The medium preferably contains at least carbon, nitrogen, oxygen, and other nutrients, growth factors, buffers, cofactors, and any other substances sufficient to maintain the viability of the cells and enable the expression of the heparin-like substance. In embodiments where the gene encoding the heparin-like substance is under the control of an inducible promoter or includes an inducible promoter, the medium may further contain an inducer.

[0058] Examples of the medium include RPMI or DMEM supplemented with 10% fetal bovine serum (FCS), and tissue culture media supplemented with factors such as antibacterial agents, growth factors, and other cytokines (e.g., Cell Biology (Third Edition) A Laboratory Handbook, vol. 1, 2006, Elsevier Inc.). Specifically, for example, medium formulations known to those skilled in the art, such as RPMI, IMDM, DMEM, DMEM / F12, serum-free or low-serum EMEM, can be mentioned. These media may contain additional nutritional supplements such as antibiotics, lipids, transferrin, insulin, amino acids, and cofactors as needed.

[0059] From the perspective of promoting the production of the heparin-like substance, the medium preferably contains at least one selected from glucose, sulfate, and phosphate. The concentration of glucose in the medium is preferably usually 5 to 75 mM, more preferably 10 to 60 mM, and even more preferably 15 to 35 mM. The concentration of sulfate in the medium is preferably usually 0.5 to 50 mM, more preferably 10 to 50 mM, and even more preferably 30 to 50 mM. The concentration of phosphate in the medium is preferably usually 0.5 to 50 mM, more preferably 1 to 50 mM, and even more preferably 10 to 50 mM.

[0060] By generating and accumulating a heparin-like substance in the culture supernatant and collecting it from the culture supernatant, the heparin-like substance can be produced. The method of culturing recombinant cells in a medium can be carried out according to a conventional method. The culturing is usually carried out for 1 to 7 days under conditions such as a pH of 6 to 8, a temperature of 30 to 40 °C, and in the presence of 5% CO2.

[0061] The secretion and production of the heparin-like substance from recombinant cells can be confirmed by adding an enzyme solution containing heparin lyases I, II, and III to the culture supernatant for enzyme treatment and then quantifying by HPLC for unsaturated disaccharide analysis, as described below in the examples. Specifically, compared with the culture supernatant of control (wild-type) cells, when the amount of at least one selected from 2SNS6 in which three sulfate groups are bound to the amino group of glucosamine at the 6-position, 2SNS in which a sulfate group is bound to the 2-position of glucuronic acid and the amino group of glucosamine, NS6S in which two sulfates are bound to the amino group of glucosamine at the 6-position, and NS in which a sulfate group is bound to glucosamine N increases in the culture supernatant of recombinant cells, it is considered that a heparin-like substance is secreted and produced from the recombinant cells into the culture supernatant. The secretion and production of the heparin-like substance from recombinant cells can also be confirmed by measuring the amount of sGAG (sulfated GAG) in the culture supernatant, as described below in the examples.

[0062] In step (3), a proteoglycan, which is a glycoprotein containing a core protein bound to a heparin-like substance that is a GAG, is secreted from the recombinant cells into the culture supernatant. Isolation of the protein from the culture supernatant is carried out by a method conventionally known in the art. For example, a tag that facilitates the isolation of a heparin-like substance, such as an affinity tag, may be used. Examples of the tag include polyhistidine (His6 tag), nickel matrix, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), FLAG tag, or epitope tag.

[0063] Isolation of the heparin-like substance from the core protein is carried out by methods conventionally known in the art. For example, there may be mentioned an enzymatic digestion method using heparinase, treatment with sodium hydroxide or sodium borohydride.

[0064] The isolated heparin-like substance preferably contains disaccharides in which 50% or more, more preferably 60% or more, still more preferably 70% or more, and particularly preferably 80% or more are sulfated.

[0065] The isolated heparin-like substance contains, for example, the following (1) to (20). (1) 10 to 50% trisulfated disaccharide, 30 to 50% disulfated disaccharide, and 10 to 30% monosulfated disaccharide. (2) 10 to 40% trisulfated disaccharide, 35 to 45% disulfated disaccharide, and 10 to 25% monosulfated disaccharide. (3) 15 to 40% trisulfated disaccharide, 35 to 45% disulfated disaccharide, and 10 to 25% monosulfated disaccharide. (4) 5 to 25% trisulfated disaccharide, 30 to 50% disulfated disaccharide, and 10 to 20% monosulfated disaccharide. (5) 1 to 30% trisulfated disaccharide, 25 to 55% disulfated disaccharide, and 5 to 40% monosulfated disaccharide. (6) 15 to 40% trisulfated disaccharides. (7) 35 to 45% disulfated disaccharides. (8) 10 to 25% monosulfated disaccharides. (9) 10 to 50% trisulfated disaccharides. (10) 30 to 50% disulfated disaccharides. (11) 10 to 30% monosulfated disaccharides. (12) 15 to 30% trisulfated disaccharides. (13) 40 to 45% disulfated disaccharides. (14) 10 to 15% monosulfated disaccharides. (15) 5 to 25% trisulfated disaccharides. (16) 1 to 30% trisulfated disaccharides. (17) 30 to 50% disulfated disaccharides. (18) 25 to 55% disulfated disaccharides. (19) Disaccharides monosulfated at 10-20%. (20) Disaccharides monosulfated at 5-40%.

[0066] Heparin-like substances can include repeating structures of disaccharide units of various lengths. The heparin-like substance preferably contains any one of UA-GlcNAc(6S), UA(2S)-GlcNAc, UA-(2S)-GlcNAc(6S), UA-GlcNS, UA-GlcNS(6S), UA(2S)-GlcNS, UA(2S)-GlcNS(6S), and these may be present in any order in the heparin-like substance.

[0067] In the above, UA is a uronic acid residue (i.e., glucuronic acid or iduronic acid), Ac is acetyl, GlcNAc is N-acetylglucosamine, GlcNS is glucosamine-N-sulfate, 2S is 2-O-sulfate, and 6S is 6-O-sulfate.

[0068] <Recombinant cell> As one aspect of the present invention, there are provided recombinant cells in which a gene encoding the extracellular domain of SDC has been introduced into mammalian cells that produce heparin-like substances. The recombinant cells can be prepared by preparing mammalian cells that produce heparin-like substances and introducing a gene encoding the extracellular domain of syndecan into the mammalian cells, in the same manner as described above in steps (1) and (2) of the section <Method for producing heparin-like substances>.

[0069] <Method for producing cells that secrete and produce heparin-like substances> As one aspect of the present invention, there is provided a method for producing cells that secrete and produce heparin-like substances, which includes the step of introducing a recombinant expression vector containing a gene encoding the extracellular domain of SDC into mammalian cells that produce heparin-like substances. The production method can be carried out by preparing mammalian cells that produce heparin-like substances and introducing a gene encoding the extracellular domain of syndecan into the mammalian cells, in the same manner as described above in steps (1) and (2) of the section <Method for producing heparin-like substances>.

[0070] <Pharmaceutical composition> As one aspect of the present invention, there is provided a pharmaceutical composition comprising a heparin-like substance or a fragment thereof produced by the method described herein. In this aspect, the heparin-like substance or a fragment thereof may be bound to a core protein. The pharmaceutical composition may contain other therapeutic agents. Further, the pharmaceutical composition can be formulated by using, for example, a conventional vehicle or diluent, and a type of pharmaceutical additive (such as an excipient, a binder, a preservative) suitable for the desired administration method.

[0071] Examples of the form of the pharmaceutical composition include sterile aqueous injection solutions. Sterile aqueous injection solutions can be formulated according to known techniques using appropriate dispersing or wetting agents and suspending agents. The sterile aqueous injection solution may be, for example, a sterile injection solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic saline.

[0072] The dosage form of the pharmaceutical composition is not particularly limited and can be in a variety of dosage forms. Examples of dosage forms for administering the pharmaceutical composition include, for example, administration in the form of tablets, capsules, sachets, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, etc., intravenous administration or injection, pastes, emulsions or solutions. Further, for example, transdermal administration by a patch mechanism or an ointment is included. Any of these may be modified into a sustained release and / or controlled release formulation.

[0073] Pharmaceutically acceptable carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrants and carriers. Typically, a pharmaceutically acceptable carrier is chemically inert to the active compound and has no adverse side effects or toxicity under the conditions of use. The nature of the pharmaceutically acceptable carrier may vary depending on the particular dosage form used and other characteristics of the composition.

[0074] <Methods of Treatment and Prevention> In one aspect of the present invention, there is provided a method of treating or preventing a condition related to or caused by blood coagulation or blood coagulation in a subject in need thereof, comprising administering a heparin-like substance or a fragment thereof produced by the method described herein. In this aspect, the heparin-like substance or a fragment thereof may be bound to a core protein. Conditions related to or caused by blood coagulation or blood coagulation include, for example, acute coronary syndrome, atrial fibrillation, deep vein thrombosis or pulmonary embolism.

[0075] The subject refers to a mammal. Mammals include, for example, humans, primates, domestic animals (e.g., sheep, cows, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory test animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captured wild animals (e.g., foxes, deer). Mammals are typically humans or primates, more typically humans.

[0076] The dosage and administration time are preferably those that provide a therapeutic benefit in the treatment, prevention or management of a condition related to blood coagulation or blood coagulation. The specific dosage and administration time that are effective may vary depending on factors such as the condition of the subject, medical history, physical build, weight, age, etc.

Examples

[0077] Examples are shown below, but the present invention is not limited to the following examples.

[0078] 1. Evaluation of heparin production ability in CHO / NH-SRGN cells and CHO / NH-SDC cells <Preparation of CHO / NH-SRGN cells and CHO / NH-SDC cells> Based on Bail JY et al., Metab Eng14:81-90, 2012, DNA encoding NDST2 (base sequence represented by SEQ ID NO: 4) and DNA encoding Hs3st1 (base sequence represented by SEQ ID NO: 5) were co-introduced to obtain clone #3 of CHO / NH cells (CHO-NH#3 cells).

[0079] The vector PB513B-1_SRGN-IRES-EGFP used for the preparation of CHO / NH-SRGN cells is a vector based on the PiggyBac Transposon Vector PB513B-1 and has a Chinese hamster EF1α promoter, an IRES element, and a mycHis tag sequence at the C-terminus. In the vector PB513B-1_SRGN-IRES-EGFP, DNA encoding hSRGN (base sequence represented by SEQ ID NO: 7) was introduced under the control of the EF1α promoter so as to co-express with the EGFP gene as a screening marker and the puromycin resistance gene.

[0080] CHO / NH-SRGN cells into which DNA encoding human serglycin (SRGN) (base sequence represented by SEQ ID NO: 7) was introduced using the vector PB513B-1_SRGN-IRES-EGFP, and CHO / NH-SDC cells into which DNA of the extracellular domain of human syndecan (SDC) (base sequence represented by SEQ ID NO: 6) was introduced using the vector PB513B-1_SDC-IRES-EGFP were prepared.

[0081] The vector PB513B-1_SDC-IRES-EGFP used for the production of CHO / NH-SDC cells is a vector based on the PiggyBac Transposon Vector PB513B-1 and has a Chinese hamster EF1α promoter, an IRES element, and a mycHis tag sequence at the C-terminus. In the vector PB513B-1_SDC-IRES-EGFP, DNA encoding SRGN (base sequence represented by SEQ ID NO: 7) was introduced under the control of the EF1α promoter so as to co-express with the EGFP gene, which is a screening marker, and the puromycin resistance gene.

[0082] The culture supernatants of each cell prepared as described above were analyzed to evaluate the heparin production ability.

[0083] <Measurement of the amount of sGAG in the culture supernatants of CHO / NH-SRGN cells and CHO / NH-SDC cells> After culturing each cell in a proliferative state, it was seeded in a 6-well plate at 0.6×10 6 cells / well, and the culture supernatant collected 3 days later without medium change was used as a sample, and the amount of sGAG was measured by sGAG assay. The sGAG assay refers to an assay for colorimetric quantification of the total amount of sGAG in a solution using a dye (1,9-dimethylmethylene blue) that specifically binds to sulfated sugar chains. The results are shown in Figure 1.

[0084] <Analysis results of sugar chain structure by HPLC> Since heparin production into the culture supernatant was suggested by sGAG assay, the sugar chain structure of the same sample was analyzed by HPLC. The sGAG quantification results of the samples used for the analysis are shown in Figure 2. For the sample of CHO / NH-SDC cells, since the concentration was low and it was not possible to confirm whether there were sufficient sugar chains for analysis, a concentration operation using a spin column was performed.

[0085] For the confirmation of heparin production, first, 20 μL of the sample solution was directly taken, 10 μL of buffer, and 10 μL of an enzyme solution containing 10 mU each of heparinases I, II, and III were added, followed by enzymatic treatment at 37 °C for 3 hours. After the reaction, the enzyme was inactivated at 100 °C for 5 minutes, freeze-dried, 20 μL of water was added, and quantification was performed by HPLC for unsaturated disaccharide analysis. The results are shown in Figures 3(A) and (B).

[0086] In Figures 3(A) and (B), WT represents wild-type CHO cells, #3 represents CHO-NH#3 cells, SRGN represents CHO-SRGN cells in which DNA encoding SRGN (base sequence represented by SEQ ID NO: 7) was introduced into CHO cells using the vector PB513B-1_SRGN-IRES-EGFP, #3SRGN represents CHO / NH-SRGN cells, and SDC represents CHO-SDC cells in which DNA of the extracellular domain of SDC (base sequence represented by SEQ ID NO: 6) was introduced into CHO cells using the vector PB513B-1_SDC-IRES-EGFP.

[0087] As shown in Figures 3(A) and (B), in the culture supernatants of SRGN-introduced cells (SRGN, #3SRGN), no increase in the detection of heparin-like substances was detected compared to WT or #3. On the other hand, in the culture supernatants of SDC-introduced cells (SDC, #3SDC), the production of heparin-like substances was increased compared to WT or #3. From the above results, it was considered that CHO / NH-SDC cells were more suitable for the production of heparin-like substances, and cloning and analysis in each clone were performed.

[0088] 2. Measurement results of the amount of GAG after serum-free adaptation Since a sugar chain structure characteristic of heparin was observed, cloning of CHO / NH-SDC cells and analysis in each clone were performed.

[0089] <Cloning and serum-free adaptation> CHO / NH-SDC cells were cultured using a serum-containing medium until they adhered, and then cloned using a collagen-coated 96-well plate by the limiting dilution method. At this time, for culturing from single cells, considering the toxicity to the cells, the culture was carried out without adding the drug. The collagen coating was scaled up to 24- and 6-well plates, and multiple clones were cultured, and 36 clones were established. Thereafter, each cell was seeded at 0.6×10 6 cells / well in a normal 6-well plate, and the culture supernatant collected 3 days later without medium change was used as a sample, and the amount of sGAG in the culture supernatant was measured. Serum-free adaptation was performed on the top 6 clones (#5, #20, #26, #29, #30, #34).

[0090] <Measurement of sGAG amount, sGAG production rate, and SDC amount after serum-free adaptation> After serum-free adaptation, each cell was seeded at 0.6×10 6 cells / well in a normal 6-well plate, and the culture supernatant collected 3 days later without medium change was used as a sample, and the amount of sGAG in the culture supernatant was measured. The results are shown in Fig. 4. Also, the results of calculating the production rate of sGAG are shown in Fig. 5, and the results of quantifying the amount of SDC in the culture supernatant by ELISA are shown in Fig. 6, respectively.

[0091] As shown in Fig. 6, the 6 clones evaluated had a secretory production ability of SDC equal to or higher than that of CHO / NH-SDC cells, and #5 was more than 1.5 times that of CHO / NH-SDC cells and showed the highest expression level.

[0092] <Analysis of transgene expression level by qRT-PCR> Each cell was seeded at 0.6×10 6 cells / well in a 6-well plate, and RNA was extracted and cDNA was prepared from the cells cultured for 3 days without medium change. The expression levels of the transgenes (NDST2, Hs3st1, and SDC) were quantified by qRT-PCR. Fig. 7(A) shows the expression level of the NDST2 gene, Fig. 7(B) shows the expression level of the Hs3st1 gene, and Fig. 7(C) shows the expression level of the SDC gene, respectively.

[0093] As shown in FIGS. 7(A) to 7(C), while high expression levels of each gene were confirmed in #5, no expression was observed in #26. #26 also showed lower activity compared to other clones in anticoagulant activity, suggesting that the ATIII recognition specific sequence was formed by NDST2 and Hs3st1.

[0094] 3. Analysis of sugar chain structure by HPLC The sugar chain structures of heparin-like substances produced by various CHO cells were analyzed by HPLC.

[0095] <Analysis conditions> As a pretreatment of the sample, 400 μL of the culture supernatant of various CHO cells was centrifuged at 10,000 rpm for 10 minutes, and 400 μL of double distilled water (DDW) was added to the supernatant and freeze-dried. Then, 20 μL was taken from 40 μL of the sample, treated with Heparin lyase I, II, and III, 80 μL of DDW was added, and 20 μL was subjected to HPLC.

[0096] The treatment with specific degrading enzymes was as follows. 20 μL of acetate buffer [0.1 M CH3COONa, 10 mM (CH3COO)2Ca (pH 7.0)], 5 μL of 0.5 mU / μL Heparinase, 5 μL of 0.5 mU / μL Heparitinase I, and 5 μL of 0.5 mU / μL Heparitinase II were added simultaneously and incubated at 37°C for 16 hours to decompose heparin-like sugar chains into unsaturated disaccharides. After heating in a boiling water bath for 3 minutes to inactivate the enzyme, freeze-drying was performed and then dissolved in water for use in the analysis of unsaturated disaccharides by HPLC.

[0097] <Disaccharide composition analysis of HS and CS> The disaccharide composition analysis of HS and CS was performed using the apparatus and analysis software shown below. Eluent pump: LIQUID CHROMATOGRAPHY LC-10Ai manufactured by Shimadzu Corporation Detector: Intelligent Fluorescence Detector FP-920S manufactured by JASCO Pump for post-reaction: MINICHEMI PUMP NP-FX(II)-1U manufactured by Nippon Precision Science Co., Ltd. Integrator: Chromato Integrator D-2500 manufactured by Hitachi, Ltd. Injector: sample injector (model 7725) manufactured by Reodyne Column: Senshu Pak DOCOSIL (4.6 mm i.d. x 150 mm) manufactured by Senshu Science Column oven: L-7300 manufactured by Hitachi, Ltd. Dry reaction tank: Dry Reaction Bath DB-5 manufactured by Shimamura Instruments Co., Ltd. Analysis software: Chromato-PRO manufactured by Runtime Instruments Co., Ltd.

[0098] The results of quantifying sGAG in the samples used for analysis by HPLC are shown in Fig. 8, and the chromatograms of the unsaturated disaccharides derived from standard heparin (HS-Standards) and the samples obtained from the samples are shown in Fig. 9. Also, the results of quantifying the content of heparin-like sugar chains and the ratio of each sugar chain in sulfation by HPLC are shown in Figs. 10(A) and (B), respectively. Table 1 shows the concentration and ratio of sulfated sugar chains (HS) in the heparin-like substances detected.

[0099]

Table 1

[0100] As shown in Table 1, the degree of sulfation of the sugar chains detected from the samples obtained from the prepared clones (#5, #20, #26, #29, #30, #34) was comparable to that of heparin, and the production of heparin-like sugar chains was significantly increased compared to the controls (CHO-S, SDC).

[0101] <Measurement of anti-factor Xa activity> Measurements were performed using the Test Team Heparin S measurement kit (http: / / www.info.pmda.go.jp / tgo / pack / 13A2X00197218081_A_01_10 / ) manufactured by Sekisui Medical Co., Ltd. The standard heparin was prepared using medical heparin (101 IU / mg). The measurement principle was outlined below. Reagent name and ingredients: 1. Substrate: N-benzoyl-L-isoleucyl-L-glutamyl (γ-OR)-glycyl-L-arginyl-p-nitroanilide hydrochloride (S-2222) 2. Antithrombin III agents: Antithrombin III (human origin) 3. Factor Xa preparation: Factor Xa (bovine origin) 4. Buffer solution: 2-amino-2-hydroxymethyl-1,3-propanediol buffer solution 5. Normal plasma preparation: normal human plasma

[0102] Anticoagulation verification The clones with high sulfation levels (#20, #26, #29, #30, and #34) in the HPLC results were examined for anticoagulant activity (anti-FXa activity) using the calibration curve shown in Figure 11. The results are shown in Table 2.

[0103] [Table 2]

[0104] As shown in Table 2, anti-FXa activity of approximately 0.1 IU per mL of medium was confirmed in samples obtained from #20, #29, #30, and #34. This result suggests that the introduction of NDST2 and Hs3st1 resulted in the inclusion of an antithrombin-binding sequence in the heparin-like sugar chains in the culture supernatant.

[0105] Although the present invention has been described in detail using specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese Patent Application (Japanese Patent Application No. 2019-182467) filed on October 2, 2019, the entire contents of which are incorporated herein by reference.

Claims

1. A method for producing a heparin-like substance, comprising the following steps (1) to (3). (1) A step of preparing mammalian cells that produce a heparin-like substance (2) A step of preparing recombinant cells in which a gene encoding an extracellular domain of syndecan (hereinafter abbreviated as SDC) is introduced into mammalian cells that produce the heparin-like substance prepared in step (1) (3) A step of culturing the recombinant cells prepared in step (2) in a medium and recovering the heparin-like substance from the obtained culture supernatant

2. The mammalian cells that produce the heparin-like substance are cells into which at least one of a gene encoding bifunctional heparan sulfate N-deacetylase / N-sulfotransferase (hereinafter abbreviated as NDST2) and a gene encoding heparan sulfate ester glucosamine 3-sulfotransferase 1 (hereinafter abbreviated as Hs3st1) has been introduced. The production method according to Claim 1.

3. The mammalian cells are Chinese hamster ovary (hereinafter abbreviated as CHO) cells. The production method according to Claim 1 or 2.

4. The mammalian cells are cells into which at least a gene encoding NDST2 has been introduced, and NDST2 is a protein that contains any one of the following amino acid sequences (1a) to (1c) and has the function of NDST2. The production method according to Claim 2 or 3. (1a) The amino acid sequence set forth in SEQ ID NO: 2 (1b) An amino acid sequence in which several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2 (1c) An amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2

5. The mammalian cells are cells into which at least a gene encoding Hs3st1 has been introduced, and Hs3st1 is a protein that contains any one of the following amino acid sequences (2a) to (2c) and has the function of Hs3st1. The production method according to any one of Claims 2 to 4. (2a) The amino acid sequence set forth in SEQ ID NO: 3 (2b) An amino acid sequence in which several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 3 (2c) An amino acid sequence having sequence identity of 90% or more with the amino acid sequence set forth in SEQ ID NO: 3

6. The production method according to any one of claims 1 to 5, wherein the extracellular domain of SDC comprises any one of the following amino acid sequences (3a) to (3c) and has the function of the extracellular domain of SDC. (3a) The amino acid sequence set forth in SEQ ID NO: 1 (3b) An amino acid sequence in which one to several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 1 (3c) An amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1

7. The production method according to any one of claims 2 to 6, wherein the mammalian cell is a cell into which at least one of the genes encoding mouse- or human-derived NDST2 and the gene encoding Hs3st1 has been introduced.

8. The production method according to any one of claims 2 to 7, wherein the mammalian cell is a cell into which at least the gene encoding NDST2 has been introduced, and the gene encoding NDST2 is any one of the following base sequences (1A) to (1D) and comprises a base sequence encoding an amino acid sequence of a protein having the function of NDST2. (1A) The base sequence set forth in SEQ ID NO: 4 (1B) A base sequence in which one to several bases are deleted, substituted or added in the base sequence set forth in SEQ ID NO: 4 (1C) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 4 (1D) A base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 4

9. The production method according to any one of claims 2 to 8, wherein the mammalian cell is a cell into which at least the gene encoding Hs3st1 has been introduced, and the gene encoding Hs3st1 is any one of the following base sequences (2A) to (2D) and comprises a base sequence encoding an amino acid sequence of a protein having the function of Hs3st1. (2A) The base sequence set forth in SEQ ID NO: 5 (2B) A base sequence in which one to several bases are deleted, substituted or added in the base sequence set forth in SEQ ID NO: 5 (2C) A base sequence that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 5 (2D) A base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 5

10. The production method according to any one of claims 1 to 9, wherein the gene encoding the extracellular domain of SDC has a base sequence of any one of the following (3A) to (3D) and includes a base sequence encoding an amino acid sequence of a protein having the function of the extracellular domain of SDC. (3A) The base sequence set forth in SEQ ID NO: 6 (3B) A base sequence in which one to several bases are deleted, substituted or added in the base sequence set forth in SEQ ID NO: 6 (3C) A base sequence that hybridizes with a base sequence complementary to the base sequence set forth in SEQ ID NO: 6 under stringent conditions (3D) A base sequence having 90% or more sequence identity with the base sequence set forth in SEQ ID NO: 6

11. A recombinant cell in which a gene encoding the extracellular domain of SDC has been introduced into a mammalian cell that produces a heparin-like substance (excluding CHO-K1 cells).

12. The recombinant cell according to claim 11, wherein the mammalian cell that produces the heparin-like substance is a cell into which at least one of the gene encoding NDST2 and the gene encoding Hs3st1 has been introduced.

13. The recombinant cell according to claim 11 or 12, wherein the mammalian cell is a CHO cell (excluding CHO-K1 cells).

14. A method for producing a cell that secretes and produces a heparin-like substance, comprising the step of introducing a recombinant expression vector containing a gene encoding the extracellular domain of SDC into a mammalian cell that produces a heparin-like substance (excluding CHO-K1 cells).

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