WO2007102432A1 - Procede pour la production d'une composition de glycoproteine - Google Patents

Procede pour la production d'une composition de glycoproteine Download PDF

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Publication number
WO2007102432A1
WO2007102432A1 PCT/JP2007/054067 JP2007054067W WO2007102432A1 WO 2007102432 A1 WO2007102432 A1 WO 2007102432A1 JP 2007054067 W JP2007054067 W JP 2007054067W WO 2007102432 A1 WO2007102432 A1 WO 2007102432A1
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seq
rna
dna
sequence represented
cell
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PCT/JP2007/054067
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Japanese (ja)
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Katsuhiro Mori
Mitsuo Satoh
Yutaka Kanda
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2007102432A1 publication Critical patent/WO2007102432A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01129—Endo-alpha-sialidase (3.2.1.129)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a cell into which a small interfering RNA (hereinafter abbreviated as siRNA) that suppresses sialidase activity, a method for producing a glycoprotein molecule using the cell, and the The present invention relates to a glycoprotein composition obtained by the production method.
  • the present invention also relates to a method for suppressing sialidase activity using siRNA.
  • Sialidase is a kind of exo-type a-glycosidase that catalyzes the initial reaction of glycan degradation by removing sialic acid, a non-reducing terminal force of glycans added to glycoproteins.
  • the function of many glycoprotein molecules changes when sialic acid residues are also removed from the non-reducing end of sugar chains added to glycoproteins by sialidase. So far, sialidase derived from bacteria and viruses has been used to elucidate the function of sialidase. For details on how sialic acid is eliminated in the body of mammals, The power was not analyzed.
  • Non-patent Document 1 Non-patent Document 1
  • Non-patent Documents 2 and 3 Mammalian endogenous sialidase is localized in the cytoplasm (Non-patent document 4), lysosomal lumen (Non-patent document 5), lysosomal membrane (Non-patent document 6) and plasma membrane (Non-patent document 6). Enzymatic properties such as substrate specificity are different.
  • cytoplasmic sialidase gene (NEU2 sialidase) as the first mammalian sialidase was revealed (Non-patent document 7), followed by lysosomal sialidase (NEU1) (Non-patent document 8) and plasma membrane sialidase (NEU3) (non-patent document 7).
  • the gene of Patent Document 9) has been cloned.
  • amino acid sequences of various sialidases including sialidases originating from microorganisms
  • the homology is not so high, but the presence of multiple aspartic acid box (Ser-X-Asp-X-Gly-X-Thr-Trp) sequences, the Phe-Arg-Ile-Pro sequence near the N-terminus, and Commonly found amino acid sequences are conserved, such as the Va ⁇ Gly-X-Gly sequence located between two aspartate box sequences.
  • amino acid residues involved in sialidase activity are highly conserved and have high similarity in amino acid conformation.
  • the crystal structure of Salmonella LT2-derived sialidase is similar to the crystal structure of human NEU2 sialidase (Non-patent Document 11).
  • NEU2 sialidase gene encodes a single-chain polypeptide consisting of 1679 bases and 379 amino acid residues.
  • NEU2 sialidase has two aspartic acid box sequences, is rich in cysteine residues and j8 sheet structure, and is known to have a three-dimensional structure similar to that of microorganism-derived sialidase.
  • This enzyme is present in the cytoplasm, works near neutral pH, and uses oligosaccharides, gandariosides, and glycoproteins as substrates. In particular, it is reported that it is highly expressed in testis and skeletal muscle and plays an important role in differentiation of skeletal muscle cells (Non-Patent Document 12).
  • Non-patent document 13 genes from Chinese hamster ovary (CHO) cells (Non-patent document 13), human skeletal muscle (Non-patent document 14), mouse brain (Non-patent document 15) and mouse thymus (Non-patent document 16) Isolated!
  • glycoproteins that are considered to be applied to pharmaceuticals are produced using genetic recombination techniques, and are derived from mammalian cells, such as CHO cells derived from Chinese nomster ovary tissue and mouse myeloma. Manufactured using cells as host cells.
  • mammalian cells such as CHO cells derived from Chinese nomster ovary tissue and mouse myeloma.
  • a sugar chain that can exhibit optimal pharmacological activity is not always added.
  • the addition of a non-reducing terminal sialic acid in the sugar chain may have a significant effect on the pharmacological activity of the administered glycoprotein, so it is important to produce and provide glycoprotein pharmaceuticals with controlled sialic acid modification. It is.
  • glycoprotein drugs in which sialic acid addition is important for the expression of pharmacological activity include erythroboyetin (EPO) and tissue-type plasminogen activator (t-PA).
  • Non-Patent Documents 17 to 19 The reason for this is that when sialic acid is eliminated, a galactose residue is exposed on the non-reducing end side, and is captured and rapidly degraded by the nascent glycoprotein receptor (galactose receptor) localized in the liver. Yes (Non-patent document 20).
  • Glycoproteins are secreted and produced by culturing transformants, which are established by stably incorporating cDNAs encoding these glycoproteins into host cells, under appropriate culture conditions for an appropriate period.
  • a variety of sugar chains are added to glycoproteins secreted into the culture medium in terms of production cell power based on the sugar chain-modifying mechanism inherent in the cells.
  • the non-reducing terminal side of mature sugar chains is negative. It is modified with a charged sialic acid residue.
  • the rate of carotage varies depending on the production strain and culture method, and it is difficult to protect the non-reducing ends of all sugar chains with sialic acid.
  • one of the main reasons for the reduction of the sialic acid addition rate is the involvement of sialidase in the production cells.
  • Non-patent Document 21 In the production of glycoprotein, sialidase, particularly NEU2 sialidase, leaks and accumulates in the culture medium from the cytoplasm of the production cells (Non-patent Document 21), and the glycoprotein sugar chain accumulated in the culture medium is also sial. The elimination of acid has become a component (Non-patent Document 22). This has been verified by production studies of multiple types of recombinant glycoproteins using CHO cells as hosts (Non-patent Documents 23 and 24).
  • Culture engineering techniques that have been developed so far to prevent the reduction of sialic acid addition rate include optimization of various culture parameters (Non-patent Document 25, Patent Document 1) and inhibition of sialidase. Examples thereof include a method of adding a substance having activity to a medium component (Non-patent Document 24, Patent Document 2).
  • the optimization of the culture parameters is carried out with the aim of maintaining the viability of the production cells at the highest possible level for a long period of time in the culturing process, since leakage of cytoplasmic power of sialidase occurs with decreasing cell viability.
  • This method a slight improvement in the rate of sialic acid addition to the glycoprotein is observed, but this is not a sufficient amount.
  • a method of adding a sialidase inhibitor as a medium component includes adding a specific inhibitor such as N-acetyl-2-2,3-dehydro-2-deoxyneuraminic acid (Neu5Ac2en).
  • a specific inhibitor such as N-acetyl-2-2,3-dehydro-2-deoxyneuraminic acid (Neu5Ac2en).
  • methods for adding relatively high concentrations of copper ions have been developed.
  • the economic burden related to the preparation of the culture medium and waste liquid treatment is large. ing.
  • non-naturally occurring compounds such as Neu5Ac2en are used in the manufacture of pharmaceuticals, consider the safety of these substances themselves and the derivatives that these substances change during culture. Must-have.
  • Non-patent Document 3 The method of inhibiting NEU2 sialidase mRNA splicing by antisense is to specifically hybridize with NEU2 sialidase mRNA based on the NEU2 sialidase cDNA sequence of the cloned CHO cells.
  • Non-Patent Document An anti-sense RNA is designed, a DNA construct for stable expression of this anti-sense RNA is introduced into glycoprotein-producing cells, and a method that can suppress the expression of NEU2 sialidase has been developed (Non-Patent Document) 26).
  • the NEU2 sialidase antisense RNA expression vector is stably introduced into CH 0 cells that produce thread-reversed DNase, thereby suppressing cytoplasmic sialidase activity to 40% and sializing the product DNase. Increases acid addition rate by up to 37%. Inhibition rate of cytoplasmic sialidase activity is insufficient.
  • Non-patent literature l Trends Biochem. Sci "10, 357-360 (1985); Glycobiology, 3, 201-217 (1993)
  • Non-Patent Document 2 J. Biochem., 107, 794-798 (1990)
  • Non-Patent Document 3 Glycoconjugate Journal vol.17,301-306 (2000)
  • Non-Patent Document 4 Journal of Biological Chemistry vol.260,6710-6716 (1985)
  • Non-Patent Document 5 European Journal of Biochemistry vol.141, 75-81 (1984)
  • Non-Patent Document 6 Journal of Biochemistry vol.107,787-793 (1990)
  • Non-Patent Document 7 Journal of Biological Chemistry vol.268,26435- 26440 (1993)
  • Non-Patent Document 8 Genes and Development vol.10,3156- 3169 (1996)
  • Non-Patent Document 9 Journal of Biological Chemistry vol.274,5004-5011 (1999)
  • Non-Patent Document 10 Pro ⁇ Natl.Acad.Sci.USA vol.90,9852--9856 (1993)
  • Non-patent literature 11 Journal of Biological Chemistry vol. 280, 469-475 (2005)
  • Non-patent literature 12 Biochem.Biophys.Res.Commun.vol.221, 826-830 (1996)
  • Non-patent literature 13 Glycobiology vol.4 , 367-373 (1994)
  • Non-Patent Document 14 Genomics vol.57, 137-143 (1999)
  • Non-patent document 15 Biochem.Biophys.Res.Commun.vol.258, 727-731 (1999)
  • Non-patent document 16 Biochem.Biophys.Res.Commun.vol.286, 250-258 (2001)
  • Non-Patent Document 18 European Journal of Biochemistry 194, 457 (1990)
  • Non-Patent Document 19 Journal of Biological Chemistry 265, 12127 (1990)
  • Non-Patent Document 20 Blood 73, 84 (1989)
  • Non-Patent Document 21 Glycobiology vol.3, 455-463 (1993)
  • Non-Patent Document 22 BIO / TECHNOLOGY vol.13, 692 (1995)
  • Non-Patent Document 23 Biotechnology Progress vol.12, 559-563 (1996)
  • Non-Patent Document 24 Biotechnology Progress vol.20, 864-871 (2004)
  • Non-Patent Document 25 Cytotechnology vol.15, 217-221 (1994)
  • Non-Patent Document 26 Biotechnology and Bioengineering vol.60, 589-595 (1998) Patent Document 1: US Pat. No. 5,705,364
  • Patent Document 2 US Pat. No. 6,528,286
  • Patent Document 3 Japanese Translation of Japanese Patent Publication No. 8-510133
  • the object of the present invention is to bind siRNA and glycoprotein compositions that suppress the expression of sialidase.
  • the amount of sialic acid contained in the sugar chain is to provide a cell into which the siRNA has been introduced, a method for producing a glycoprotein composition using the cell, and use thereof.
  • the present invention relates to the following (1) to (20).
  • the small interfering RNA is a small interfering RNA comprising the RNA selected from the group consisting of the following (a) to (d) and its complementary RNA (1) ) The cell described.
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 consecutive base sequences in the base sequence represented by SEQ ID NO: 1;
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 consecutive base sequences in the base sequence represented by SEQ ID NO: 2;
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 base sequences consecutive in the base sequence represented by SEQ ID NO: 3;
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 consecutive base sequences in the base sequence represented by SEQ ID NO: 4.
  • the small interfering RNA is a small interfering RNA comprising the RNA selected from the group consisting of the following (a) to (h) and its complementary RNA (1) ) The cell described.
  • RNA whose expression suppresses the expression of sialidase in the cell; (e) RNA consisting of the base sequence represented by SEQ ID NO: 45;
  • nucleotide sequence represented by SEQ ID NO: 51 one or several bases at both ends of the nucleotide sequence may be deleted, substituted, inserted and Z or added, and introduced into cells. RNA that suppresses the expression of sialidase in the cell.
  • sialidase is a protein encoded by a DNA selected from the following group forces (a) to (h):
  • sialidase is a protein from which the following group forces (a) to (1) are selected.
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having a sialidase activity.
  • mouse myeloma cell line SP2 / 0-Agl4 cells (d) mouse myeloma cell line SP2 / 0-Agl4 cells; (e) Syrian hamster kidney tissue-derived BHK cells;
  • the glycoprotein composition comprising the steps of culturing the cell according to (8) above in a medium, producing and accumulating a glycoprotein composition in the culture, collecting the glycoprotein composition from the culture, and purifying the composition. Manufacturing method.
  • the glycoprotein composition is characterized in that the glycoprotein composition has an increased amount of sialic acid per molecule of glycoprotein compared to the glycoprotein composition produced by the parent cell line (9) The method described in 1.
  • glycoprotein composition is a glycoprotein composition having a longer blood half-life than the glycoprotein composition produced by the parent cell.
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 consecutive base sequences in the base sequence represented by SEQ ID NO: 1;
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 consecutive base sequences in the base sequence represented by SEQ ID NO: 2;
  • RNA corresponding to DNA consisting of a base sequence represented by 10 to 40 base sequences consecutive in the base sequence represented by SEQ ID NO: 3;
  • RNA consisting of the base sequence represented by SEQ ID NO: 45;
  • a recombinant DNA obtained by integrating a DNA corresponding to the RNA described in (13) or (14) above and a complementary DNA thereof into a vector.
  • the method comprises culturing the transformant according to (17) above in a medium, producing and accumulating a glycoprotein composition in the culture, collecting the glycoprotein composition from the culture, and purifying the sugar.
  • a method for producing a protein composition comprises culturing the transformant according to (17) above in a medium, producing and accumulating a glycoprotein composition in the culture, collecting the glycoprotein composition from the culture, and purifying the sugar.
  • the present invention relates to a cell into which siRNA that suppresses sialidase activity has been introduced, a method for producing a glycoprotein molecule using the cell, and a glycoprotein composition obtained by the production method.
  • the present invention also provides a method for suppressing sialidase activity using siRNA.
  • the production method of the present invention can provide a glycoprotein composition in which the amount of sialic acid added is increased as compared with conventional glycoprotein compositions.
  • FIG. 1 shows the production of plasmid FT81ib3 / pBS.
  • FIG. 2 shows the production of plasmid FT81ib3 / pAGE.
  • FIG. 3 is a diagram showing the production of plasmids pSiA to pSi38.
  • FIG. 4 is a diagram showing production of plasmid pT7 Blue Sialidase.
  • FIG. 5 shows the production of plasmid pAGELucia.
  • FIG. 6 is a diagram showing the production of plasmid pKANTEXLucia!
  • FIG. 7 is a diagram showing reporter activity when a chimeric reporter gene and a sialidase siRNA expression vector are co-introduced into CHO / DG44 cells.
  • FIG. 8 is a diagram showing the sialidase activity of a cell lysate when a sialidase siRNA expression vector is introduced into CHO / DG44 cells.
  • a cell into which siRNA that suppresses the sialidase activity of the present invention is introduced (hereinafter referred to as "the cell of the present invention") is a siRNA introduced so as to suppress the enzyme activity of intracellular sialidase. If it is a cell, such a cell is also included.
  • Sialidase is an enzyme that catalyzes a reaction in which the non-reducing end of a sugar chain added to a glycoprotein also removes sialic acid.
  • Sialidase activity refers to the activity of catalyzing a reaction that removes the non-reducing terminal strength sialic acid of a sugar chain added to a glycoprotein.
  • the siRNA that suppresses the sialidase activity is a double-stranded RNA comprising RNA and its complementary RNA force and capable of reducing the amount of intracellular sialidase mRNA. If it is! /, It can be something! / ...
  • the length of the RNA is preferably 1 to 50, more preferably 10 to 40, still more preferably 10 to 30, and most preferably 15 to 30.
  • specific RNA is preferably 1 to 50, more preferably 10 to 40, still more preferably 10 to 30, and most preferably 15 to 30.
  • RNA corresponding to DNA comprising a base sequence represented by 10 to 40 base sequences in the base sequence represented by SEQ ID NO: 1;
  • RNA corresponding to DNA comprising a base sequence represented by 10 to 40 base sequences in the base sequence represented by SEQ ID NO: 2;
  • RNA corresponding to DNA comprising a base sequence represented by 10 to 40 base sequences in the base sequence represented by SEQ ID NO: 3;
  • RNA corresponding to DNA consisting of a base sequence represented by a continuous 10 to 40 base sequence in the base sequence represented by SEQ ID NO: 4;
  • RNA consisting of the base sequence represented by SEQ ID NO: 22;
  • (k) RNA consisting of the base sequence represented by SEQ ID NO: 51;
  • RNA that suppresses the expression of sialidase In the base sequence represented by SEQ ID NO: 51, one or several bases are deleted, substituted, inserted and Z or added, and the base sequence is also introduced into the cell. RNA that suppresses the expression of sialidase.
  • a nucleotide sequence in which one or several bases are deleted, substituted, inserted and Z or added is:
  • the nucleotide sequence of SEQ ID NO: 22, 34, 45 or 51 is the nucleotide sequence in which the 5 ′ end and one or several bases at the Z or 3 ′ end are deleted, substituted, inserted and Z or added.
  • the length of the base sequence is preferably 1 to 50, more preferably 10 to 40, still more preferably 10 to 30, and most preferably 15 to 30 continuous RNA.
  • siRNA As a method for introducing siRNA, any method can be used as long as siRNA can be introduced into cells, but a genetic engineering technique is preferred.
  • siRNA, precursor RNA, or a DNA molecule that is a cocoon of these can be transiently introduced into a cell, or a siRNA or precursor RNA cocoon of a DNA molecule can be stably integrated into the host genomic DNA. Methods.
  • siRNA, precursor RNA, or their DNA molecules are expressed in cells.
  • a recombinant vector by inserting the prepared DNA fragment or full length downstream of the promoter of an appropriate expression vector, and the recombinant vector is transformed into a host suitable for the recombinant vector. Examples include a method of introducing into a cell and a method of directly introducing into a cell a double-stranded RNA designed based on the base sequence of sialidase without using an expression vector.
  • the designed siRNA, precursor RNA, or a DNA molecule of these types can be transcribed.
  • examples thereof include a method of preparing an expression vector containing a promoter at a position where it can be introduced, introducing the expression vector into a host cell suitable for the expression vector, and integrating it into a host chromosome.
  • the sialidase includes a protein encoded by the following DNA (a), (b), (c), (d), (e), (1), (g) or (h), or (0 , (J), (k), (1), (m), (n), (o), (p), (q), (r), (s) or (t) proteins It is done.
  • (0) a protein having an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 7 and having a sialidase activity;
  • (P) a protein having amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 8, and has a sialidase activity;
  • the DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3 or 4, or a part thereof DNA obtained by using the Koguchi-one 'hybridization method or the Southern blot hybridization method, etc., using the above fragment as a probe, specifically, DNA derived from colonies or plaques Using a fixed filter, 0.7 to 1M sodium chloride was present, and after carrying out noise hybridization at 65 ° C, 0.1 to 2 times concentrated SSC solution (composition of 1 times concentrated SSC solution) Can be obtained by washing the filter under 65 ° C conditions using 150 mM sodium chloride and 15 mM sodium citrate).
  • Hybridization is molecular cloning (A Molecular manual, A Laboratory manual, second edition, Cold Spring Haroor Laboratory Press, 1989). 'Molekiyura 1 ⁇ ' Neolon 1 ⁇ (Current Protocols in Molecular Biology), John Wiley & Sons, 1987-1997 (hereinafter 'Protocol's In'Molecular ⁇ ⁇ ⁇ Biology), Dneue Claw -DNA Cloning 1: Can be performed according to the method described in Core Techniques, A Practica 1 Approach, Second Edition, Oxford University (1995), etc. Specific examples of DNA that can be hybridized include sequencing. DNA having at least 60% or more homology with the nucleotide sequence represented by No. 1, 2, 3 or 4, preferably 70% or more, more preferably 80% or more, more preferably 90% Furthermore, particularly preferably 95% or more, most preferably be mentioned a DNA having a homology of 98% or more.
  • the amino acid sequence represented by SEQ ID NO: 5, 6, 7 or 8 has an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and / or added, and has a sialidase activity.
  • Proteins are molecular 'Crowung 2nd edition, current' profile Conoréles 'in' molecular ⁇ ⁇ ⁇ Biology, Nucleic 'acid' research (Nuclei c Acids Research), 10, 6487 (1982), Proceedings' National 'Academic' Science USA (p roc . Natl. Acad.
  • site-directed mutagenesis is introduced into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 5, 6, 7 or 8. It means a protein that can be obtained by doing.
  • the number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. However, deletion, substitution, or The number is such that it can be added, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
  • the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5, 6, 7 or 8 and having sialidase activity is SEQ ID NO: 5, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, most at least 80% or more of the protein having the amino acid sequence described in 6, 7 or 8
  • a protein having a homology of 99% or more and having a sialidase activity is preferable.
  • homology values described in the present invention are values calculated using a homology search program known to those skilled in the art. Examples include numerical values calculated using default parameters in 'Ob' Molecular Biology (J. Mol. Biol), 215, 403 (1990)].
  • BLAST2 Nucleic Acid Res., 25, 3389 (1 997)]; Genome Res., 7, 649 (1997); http: // www. ncbi.nlm.nih.gOv/Education/BLASTinfo/infomation3.html] and numerical values calculated using default parameters.
  • Default parameter 1 is 5 if G (Cost to open gap) is a base sequence, 11 if it is an amino acid sequence, 2 if -E (Cost to extend gap) is a base sequence, 1 if it is an amino acid sequence , -Q (penalty for nucleotide mismatch) force 3, r (reward for nucleotide match) force 1, e (expect value) force 10, one W (wordsize) force base 11 3 residues for amino acid residues, 20 if y (Dropoff (X) for blast extemsions in bits) is blastn, 25 for programs other than blastn (http: ⁇ www.ncbi. nlm.nih.gov/blastcgihelp.html) 0 Another example of amino acid sequence analysis software is FASTA [Methods in Enzymology, 183, 63 (1990)].
  • the cells of the present invention include yeast, animal cells, insect cells, plant cells and the like, but animal cells are preferred.
  • yeast examples include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth mouth, Genus Trichospolon, Schumi-omyces, etc. That power S.
  • Animal cells include human cells, such as Namalwa cells, monkey cells, COS cells, Chinese cells, CHO cells, which are Muster cells, and HBT5637 (Japanese Patent Laid-Open No. 63-299). Rat myeloma cells, mouse myeloma cells, Syrian omster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.
  • Insect cells include Spodoptera frugiperda ovarian cells S19, Sf21 (Current 'protocol ⁇ Norez in'more ⁇ ' Neolon ⁇ Baculovirus Expression Vectors, A Laooratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani's ovary cells, High 5 (Invitrogen), and the like.
  • Plant cells include tobacco, potatoes, tomatoes, carrots, soybeans, rape, alfalfa, rice, wheat, barley, green moss, duckweed, and the like.
  • animal cells include Chinese, mustar ovarian tissue-derived cells (CH 2 O cells), rat myeloma cell line YB2Z0 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2Z0—Agl4 cell, and Syrian nomus muster.
  • Kidney tissue-derived B HK cells (ATCC CCL-10), MDCK (ATCC CCL-34), PER-C6 TM, human NM-F9 cells, human HEK293 cells, Hypridoma cells, human leukemia cell line Namalva cells, embryonic Examples thereof include stem cells and fertilized egg cells.
  • CHO cells include CHO-K1 strain (ATCC CCL-61), CHO / dhfr- strain (ATCCCRL-9096), Pro5 strain (ATCC CR L-1781), and commercially available CHO-S strain (Cat # manufactured by Invitrigen). 11619), or substrains obtained by acclimating these strains to serum-free medium.
  • Rat myeloma cells For example, mouse cells such as Y3 Agl.2.3. (ATCC CRL-1631), YO (ECACC No: 85110501), YB2 / 0 (ATCC CRL 1662), NSO (ATCC CR L-1827), Sp2 / 0 (ATCC CRL—1581) and the like, myeloma cells or hybrid cells of the myeloma cell line, or substrains (ATC C CRL—1581.1) in which these strains are conditioned to a serum-free medium or the like.
  • human cells include PER. C6 (ECACC 9602 2940), or sub-strains obtained by acclimating these strains to a serum-free medium.
  • cells having the same properties as these cells obtained by subjecting these cells to mutation treatment or cell fusion with B cells obtained by immunizing mammals other than humans with antigens are also available. It is contained in the animal cell in this invention.
  • the cell in the present invention includes a lectin that recognizes a sugar chain structure in which N-glycosidic complex type sugar chain reducing terminal N-acetylyldarcosamine 6-position and fucose 1-position are a-linked.
  • a composition comprising a resistant cell, for example, a glycoprotein molecule having an N-glycoside-bonded complex sugar chain, of all N-glycoside-bonded complex sugar chains contained in the composition, Examples thereof include cells having the ability to produce a glycoprotein composition in which the proportion of sugar chains in which fucose is not bound to N-acetyl darcosamine at the sugar chain reducing end is 20% or more. Examples of such cells include cells in which the activity of at least one protein listed below has been reduced or inactivated.
  • N-glycoside-linked complex sugar chain reducing terminal N-acetylcylcosamine is located at the 6-position of fucose (cells with reduced or inactivated activity of enzyme protein involved in X-linked sugar chain modification) Examples thereof include cells in which a gene encoding intracellular a 1,6-fucosyltransferase has been knocked out (WO02 / 31140, WO03 / 85107, WO03 / 85102).
  • Enzyme proteins involved in the synthesis include any enzyme involved in the synthesis of sugar nucleotide GDP-fucose, which is the source of fucose to sugar chains in the cell.
  • Fucose Enzymes involved in the synthesis include enzymes that affect the synthesis of intracellular sugar nucleotide GDP-fucose.
  • the intracellular sugar nucleotide GDP-fucose is supplied by the de novo synthesis pathway or the Salvage synthesis pathway. Therefore, all enzymes involved in these synthetic pathways are included in enzymes involved in the synthesis of intracellular GDP-fucose.
  • Enzymes involved in the de novo synthesis pathway of intracellular sugar nucleotides GDP-fucose include GDP-mannose 4,6-dehydratase (GDP-mannose 4,6-dehydratase; hereinafter referred to as GM D), (JDP — Keto—ri— deoxymannose 3,5— epimerase, 4,6— reductase (LJDP—keto-deoxymannose 3,5-epimerase, 4,6-reductase; hereinafter referred to as Fx) .
  • GDP-beta-L-fucose-pyrophosphorylase As an enzyme involved in the Salvage synthesis pathway of intracellular sugar nucleotide GDP-fucose, GDP-beta-L-focose pyrophosphorylase (GDP-beta-L-fucose-pyrophosphorylase; hereinafter referred to as GFPP) , Fucokinase and the like.
  • Examples of the enzyme that affects the synthesis of intracellular sugar nucleotide GDP-fucose include the above-mentioned substances that affect the activity of enzymes involved in the intracellular sugar nucleotide GDP-fucose synthesis pathway, and substances that serve as substrates for the enzyme. Enzymes that affect structure are also included.
  • N-glycoside bond complex type N-glycoside bond complex type N-glycoside bond complex type N-glycosyl bond complex type Any enzyme involved in the reaction in which the 6-position of N-acetylyldarcosamine at the sugar chain reducing end and the 1-position of fucose are a-linked is included.
  • N-glycosidic complex type N-acetylyldarcosamine 6-position of the sugar chain reducing end and the enzyme involved in ⁇ -bonding of fucose 1-position are: Any enzyme that affects the reaction in which the 6th position of acetyltilcosamine and the 1st position of fucose bind to a is included. Specifically, ⁇ -1, 6-fucosyltransferase is a-L-fucosidase.
  • Proteins involved in transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus include intracellular sugar nucleotide GDP-protein involved in transport of fucose to the Golgi apparatus, or intracellular sugar nucleotide GDP- Any protein that affects the reaction of transporting fucose into the Golgi is included.
  • proteins involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus include the GDP-fucose transporter.
  • proteins that affect expression As a protein that affects the reaction of transporting intracellular sugar nucleotide GDP-fucose into the Golgi apparatus, it affects the activity of the above-mentioned proteins involved in transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus. Also included are proteins that affect expression.
  • V or a deviation method may be used as long as it can reduce or eliminate the target enzyme activity. it can.
  • N-glycoside-linked sugar chain N-acetylyldarcosamine 6-position and fucose 1-position of fucose are selected for strains that are resistant to lectins that recognize sugar-linked glycan structures. It is done.
  • a lectin that recognizes a sugar chain structure in which the 6-position of N-glycidyl darcosamine at the reducing end of N-glycoside-linked sugar chain and the 1-position of fucose are a-linked is any lectin that can recognize the sugar chain structure.
  • Any lectin can be used. Specific examples include Lentil lectin LCA (Lentil Agglutinin derived from Lens Culinaris), Endumen lectin PS A (Peasum sativum-derived Pea Lectin), Broad bean lectin VFA (Agglutin in derived from Vicia faba), Hiratiyawantake Lectin AAL (Lectin from Aleuria aurantia) etc. it can.
  • a cell resistant to lectin refers to a cell whose growth is not inhibited even when an effective concentration of lectin is given.
  • the effective concentration is not less than the concentration at which cells before the genome gene modification (hereinafter referred to as parent strain) cannot grow normally, and preferably the same concentration at which cells before the genome gene modification cannot grow.
  • the concentration is more preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
  • the effective concentration of a lectin whose growth is not inhibited may be appropriately determined according to the cell line.
  • the effective concentration of a normal lectin is 10 / zg / mL to 10 mg / mL, preferably 0.5 mg / mL to 2.0. mg / mL.
  • a method for producing a glycoprotein composition using the cells of the present invention specifically, a method for producing using a host cell into which a gene encoding a glycoprotein has been introduced, a gene encoding a glycoprotein was introduced.
  • a method of producing non-human embryonic stem cells or fertilized egg cells into early embryos of non-human animals and then using the generated transgenic non-human animals, or introducing a gene encoding a glycoprotein And a method of producing using a transgenic plant produced from a plant callus cell.
  • the present invention also relates to a method for producing a glycoprotein composition in which the glycoprotein composition has an increased amount of sialic acid per molecule as compared to the glycoprotein composition produced by the parent cell line.
  • the amount of sialic acid added per molecule of glycoprotein of the glycoprotein composition produced by introducing the siRNA that suppresses sialidase activity of the present invention was produced from the cells before the siRNA was introduced. Increased compared to the amount of sialic acid added per molecule of glycoprotein in the glycoprotein composition.
  • a glycoprotein composition having an increased amount of sialic acid per molecule of glycoprotein is 80 to 90% or more, preferably 95% or more of the number of molecules to which sialic acid is added per molecule of glycoprotein. More preferably, it refers to a glycoprotein composition in which sialic acid is bound to 100%.
  • the method for measuring sialidase activity is not particularly limited, but is a method using a fluorescent substrate such as 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid ammonium (4MU-Ne5AC) (Molecular Biotechnology). vol.15, 69 (2000)), Proceedings of the society for experimental biology and medicine vol.169, 50 1-505 (1982)) and a method using a lectin (Biol Pharm. Bull, vol. 17, 29-33 (1994)).
  • a fluorescent substrate such as 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid ammonium (4MU-Ne5AC) (Molecular Biotechnology). vol.15, 69 (2000)), Proceedings of the society for experimental biology and medicine vol.169, 50 1-505 (1982)) and a method using a lectin (Biol Pharm. Bull, vol. 17, 29-33 (1994)).
  • the parent cell line refers to a cell before introduction of siRNA that suppresses sialidase activity.
  • the glycoprotein composition refers to a composition containing a glycoprotein molecule having a sugar chain in which a sialic acid addition site is present at the non-reducing end.
  • sugar chains of glycoproteins are divided into two chains: sugar chains that bind to asparagine (N-glycoside-linked sugar chains) and sugar chains that bind to serine, threonine, etc. (0-glycoside-linked sugar chains). Broadly divided into types. These are collectively referred to as glycoside-linked sugar chains.
  • N-glycoside-linked sugar chains have a variety of structures. [Biochemical Experimental Methods 23-Glycoprotein Sugar Chain Research Methods (Academic Publication Center) Atsuko Takahashi (1989)], V Even in the case of misalignment, it has a common core structure shown in the following structural formula (I).
  • the end of the sugar chain that binds to asparagine is referred to as the reducing end, and the opposite side is referred to as the non-reducing end.
  • the N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, and a galactose-N-acetylcylcosamine (hereinafter referred to as Ga ⁇ GlcNAc) branch to the non-reducing end of the core structure.
  • a complex type (hereinafter also referred to as a complex type) having a structure such as sialic acid or bisecting N-acetylcylcosamine on the non-reducing end side of Ga to GlcNAc. Examples include a hybrid type having both a high mannose type and a complex type branch on the non-reducing terminal side of the structure.
  • the reducing end of N-acetylylgalatatosamine is bonded to the hydroxyl group of serine or threonine, and further, galactose, N-acetylyldarcosamine, N-acetylgalato Samine, sialic acid, or a sugar chain to which sialic acid is bonded, xylose is a sugar chain having j8 bond to the hydroxyl group of serine, galactose is bonded to the hydroxyl group of hydroxylysine and j8 bond Sugar chain and the like.
  • a sugar chain in which xylose is bonded to the hydroxyl group of serine by / 3 usually has a plurality of sugars bonded to the 4-position of the xylose, and a linear polysaccharide composed of two sugars is bonded to the end of the bonded sugar. is doing.
  • Examples of the substance having such a sugar chain structure include cartilage proteodalycan.
  • Examples of the substance having a sugar chain structure in which galactose is ⁇ -bonded to the hydroxyl group of hydroxylysine include collagen.
  • sugar constituting the sugar chain examples include ⁇ -acetylyl darcosamine, ⁇ -acetyl galatatosamine, mannose, galactose, fucose, sialic acid, xylose, and arabinose. May be combined in order.
  • the glycoprotein composition refers to a composition having a glycoprotein molecular force having a ⁇ -glycoside-linked sugar chain or a 0-glycoside-linked sugar chain. Since there are many sugar chains that bind to glycoproteins and their sugar chain structures are diverse, there are many combinations of sugar chains in the sugar chains of glycoproteins. Accordingly, the glycoprotein composition of the present invention includes a composition composed of glycoprotein molecules combined with a single sugar chain structure, and a glycoprotein molecule combined with a plurality of different sugar chain structures. The number of sugar chains can be one or more! /.
  • the amount of sialic acid added per molecule of glycoprotein of a glycoprotein composition produced from a cell into which siRNA that suppresses sialidase activity of the present invention has been introduced is the amount of sugar produced from the cell prior to the introduction of siRNA. Increased compared to the amount of sialic acid added per molecule of glycoprotein in the protein composition.
  • a glycoprotein composition having an increased amount of sialic acid per molecule of glycoprotein exhibits improved physiological activity when administered in vivo as compared to the glycoprotein composition produced by the parent cell.
  • Physiological activity includes affinity between glycoprotein and receptor, half-life time of glycoprotein in the body, changes in tissue distribution after administration in blood, interaction with proteins necessary for expression of pharmacological activity, etc. Can be given.
  • the composition comprises glycoprotein molecules having N-glycoside-bonded complex-type sugar chains, and all N-glycosides contained in the composition Of the combined complex-type glycans, fucosyl is linked to the N-acetylyldarcosamine at the sugar chain reducing end.
  • a glycoprotein composition in which the proportion of sugar chains to which no sugar is bound is 20% or more has further excellent physiological activity.
  • an antibody composition comprising an antibody molecule having an N-glycoside-bonded complex sugar chain in the Fc region, wherein the N-glycoside-bonded complex sugar chain is an N-acetylyldarco at the reducing end of the sugar chain.
  • An antibody composition having a sugar chain, in which fucose is bound to samine has a higher ADCC activity.
  • fucose is bound, and as a sugar chain, fucose is not bound to N-acetylcylcosamine on the reducing end side in the chemical formula shown above. Also, the structure of the sugar chain at the non-reducing end can be quite good!
  • the fact that fucose is not bound to N-acetylyldarcosamine at the sugar chain reducing end means that fucose is not substantially bound.
  • the glycoprotein composition to which fucose is not substantially bound specifically refers to a case where the glycoprotein composition is such that fucose is not substantially detectable in the sugar chain analysis described in 3 below. . To the extent that it cannot be detected substantially means that it is below the detection limit of measurement.
  • glycoproteins produced by the cells of the present invention include glycoproteins described below, glycoprotein fragments thereof, fusion proteins obtained by fusing two or more glycoproteins or glycoprotein fragments, and the like.
  • Glycoproteins include antibodies, erythropoietin (EPO) [J. Biol. Chem., 252, 5558 (19 77)], thrombopoietin (TPO) [Nature, 369, 533 (1994)] tissue-type plasminose Genactivator, prokinase, thrombomodulin, antithrombin III, a1 antitrypsin, C1 inhibitor, haptoglobin, activated protein C, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood coagulation factor X, blood coagulation Factor XI, blood coagulation factor XII, blood coagulation factor XIII, prothrombin complex, fibrinogen, albumin, gonadal stimulating hormone, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, Activin, bone morphogenetic factor, granulocyte colony stimulating factor (G—C SF
  • soluble interleukin 4 examples include receptors, tumor necrosis factor at, DnaseU galactosidase, oc darcosidase, darcocelle brosidase, hemoglobin, transferrin and the like.
  • V may be an antibody having an antigen binding property! /, But an antibody that binds to a tumor-related antigen, an antibody that binds to an antigen associated with allergy or inflammation, or a cardiovascular disease An antibody that binds to an antigen, an antibody that binds to an antigen associated with an autoimmune disease, or an antibody that binds to an antigen associated with a virus or bacterial infection is preferred, and the antibody class is preferably IgG.
  • Anti-GD2 antibodies Anticancer Res., 13, 331-33 6, 1993
  • anti-GD3 antibodies Anticancer Immunol. Immunother., 36, 260-266, 1993
  • Anti-GM2 antibody Anti-GM2 antibody
  • Anti-HER2 antibody Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992
  • anti-CD52 antibody Nature, 332, 323) -327, 1988
  • anti-MAGE antibody British J.
  • anti-insulin-like growth factor receptor antibody J. Neurosci. Res., 40, 647-659, 1995
  • anti-PMSA antibody J Urology, 160, 2396-24 01, 1998)
  • anti-vascular endothelial growth factor antibody Cancer Res., 57, 4593-4599, 1997)
  • anti-vascular endothelial growth factor receptor antibody Oncogene, 19, 2138- 2146, 2000
  • anti-CA125 antibody anti-17-1A antibody, anti-integrin ⁇ 3 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD19 antibody
  • Examples thereof include an anti-EGF receptor antibody (Immunology Today 21 (8), 403-410 (2000)), an anti-CD10 antibody (American Journal of Clinical Pathology, 113, 374-382, 2000) and the like.
  • Anti-interleukin 6 antibody (Immunol. Rev., 127, 5-24, 1992), anti-interleukin 6 receptor antibody (Mole) are antibodies that bind to antigens related to allergy or inflammation. cular Immunol, 31, 371-381, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127, 5-24, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562-567, 1991), anti-interleukin 4 receptor antibody (J. Immunol.
  • anti-tumor necrosis factor antibody Hybridoma, 13, 183-190, 1994
  • anti-tumor Necrosis factor receptor antibody Molecular Pharmacol, 58, 237-245, 2000
  • anti-CCR4 antibody Nature, 400, 776-780, 1999
  • anti-chemokine antibody J. Immunol. Meth., 174, 249-257, 1994
  • anti-chemokine receptor antibody J. Exp. Med., 186, 1373-1381, 1997)
  • anti-IgE antibody anti-CD23 antibody
  • anti-CD11a antibody Immunology Today, 21 (8), 403-410) (2000)
  • anti-CRTH2 antibody J
  • antiplatelet-derived growth factor receptor antibody J. Biol. Chem., 272, 17400-17404, 1997) or anticoagulant factor antibody (Circulation, 101, 1158-1164, 2000) .
  • antibodies that bind to antigens associated with autoimmune diseases include anti-self DNA antibodies (Immunol. Letters, 72, 61-68, 2000).
  • antibodies that bind to antigens associated with viral or bacterial infection include anti-gpl20 antibodies
  • anti-CD4 antibody J. Rheumatology, 25, 2065-2076, 1998), anti-CCR4 antibody, anti-verotoxin antibody (J. Clin. Microbiol, 37, 396-399, 1999), autoimmune disease (psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, etc.), anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 Examples thereof include antibodies, anti-CD4 antibodies, anti-integrin ⁇ 4 j87 antibodies, anti-CD40L antibodies, anti-IL-2 receptor antibodies (Immunology Today, 21 (8), 403-410 (2000)).
  • An antibody is a protein produced in a living body by an immune reaction as a result of stimulation with a foreign antigen, and has an activity that specifically binds to an antigen, as long as it is a molecule containing the Fc region of an antibody. , Such molecules are also included.
  • Specific examples include antibodies, antibody fragments, and fusion proteins containing the Fc region.
  • antibodies examples include antibodies produced by gene recombination techniques, ie, antibody expression vectors into which antibody genes have been inserted, in addition to antibodies secreted by hybridoma cells prepared from spleen cells of immunized animals after immunizing animals with antigens. And the like, which are obtained by introducing the protein into a host cell.
  • Specific examples include antibodies produced by Hypridoma, human chimeric antibodies, humanized antibodies, human antibodies, and the like.
  • a hyperidoma is obtained by cell fusion of a B cell obtained by immunizing a mammal other than a human with an antigen and a myeloma cell derived from a rat or mouse. It means a cell producing a monoclonal antibody having a desired antigen specificity.
  • the human chimeric antibody is a non-human animal antibody heavy chain variable region (hereinafter, heavy chain is referred to as H chain, variable region is referred to as HV or VH as V region) and antibody light chain variable region (hereinafter referred to as light chain).
  • CL the light chain constant region of the human antibody
  • animals other than humans any mouse, rat, mouse, muster, rabbit, etc. can be used as long as it is possible to produce a hyperidoma.
  • a human chimeric antibody For a human chimeric antibody, cDNAs encoding VH and VL are obtained from a hybridoma producing a monoclonal antibody, and inserted into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively.
  • a human chimeric antibody expression vector can be constructed and introduced into a host cell for expression and production.
  • the CH of the human chimeric antibody may be any of those belonging to human immunoglobulin (hereinafter referred to as “hlg”), but is preferably of the hlgG class, and more preferably of hlgG1, hIgG2, hIgG3, Any of the subclasses such as hIgG4 can be used.
  • hlg human immunoglobulin
  • any ⁇ class or ⁇ class can be used as long as it belongs to hlg.
  • the humanized antibody is also referred to as a complementarity determining region (hereinafter referred to as CDR) grafted antibody.
  • CDR complementarity determining region
  • a humanized antibody means an antibody obtained by grafting the VH and VL CDR amino acid sequences of a non-human animal antibody into appropriate positions of the VH and VL of a human antibody.
  • the human ⁇ antibody constructs a cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of the non-human animal antibody to the VH and VL CDR sequences of any human antibody.
  • a humanized antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding the antibody CL, and the humanized antibody is expressed and produced by introducing the expression vector into the host cell. it can.
  • CH of the human rabbit antibody if belonging to hlg! /, It may be something! /, But those of hlgG class are preferred, and hIgGl, hIgG2, hIgG3 belonging to hlgG class, Any subclass such as hIgG4 can be used.
  • CL of human rabbit antibody may be any as long as it belongs to hlg, and those of ⁇ class or ⁇ class can be used.
  • a cell into which siRNA that suppresses sialidase activity of the present invention is introduced (hereinafter abbreviated as “the cell of the present invention”) can be prepared, for example, as follows.
  • RNAi RNA interferance gene construct having an appropriate length including a portion encoding a sialidase or a portion of an untranslated region is designed.
  • a thread recombination vector is prepared by inserting the prepared DNA fragment or full length downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • the cell of the present invention can be obtained by selecting a transformant using as an index the mRNA amount and activity of the introduced sialidase, or the glycoprotein molecule on the produced glycoprotein molecule or the glycoprotein on the cell surface. .
  • any yeast cell, animal cell, insect cell, plant cell or the like having a target sialidase gene can be used. Specifically after Examples of the host cell described in 2. above.
  • the above host cell can be autonomously replicated! /, Or can be integrated into a chromosome, and contains a promoter at a position where the designed RNAi gene can be transcribed. Used. Specifically, an expression vector that is transcribed by RNA polymerase III or a recombinant vector suitable for various host cells described in 2. below can be used.
  • sialidase cDNA and genomic DNA examples include the methods described below.
  • Total RNA or mRNA is prepared from the tissues or cell strength of various host cells.
  • a cDNA library is prepared from the prepared total RNA or mRNA.
  • a degenerative primer is prepared, and a gene fragment encoding sialidase is obtained by PCR using the prepared cDNA library as a template.
  • a cDNA library can be screened to obtain DNA encoding sialidase.
  • Human or non-human animal tissue or cell mRNA may be commercially available (for example, manufactured by Clontech), or human or non-human animal tissue or cell force may be prepared as follows. .
  • mRNA is prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (manufactured by Invitrogen) or Quick Prep mRNA Purification Kit (manufactured by Pharmacia). Togashi.
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA.
  • the cDNA library can be prepared by the method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or a commercially available kit. Examples thereof include a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies: ⁇ ), ZAP-cDNA by nthesis Kit (STRATAGENE).
  • any phage vector or plasmid vector can be used as long as it can autonomously replicate in Escherichia coli K12.
  • ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
  • ⁇ ZAP II manufactured by STRATAGENE
  • gtl0, gtl l [DNA cloning, A Practical A pproach], 1, 49 (1985)]
  • TriplEx (Clontech )
  • pT7 T318U Pharmacia
  • pcD2 [Mole. Cell. Biol., 3, 280 (1983)]
  • pUC18 Gene, 33, 103 (1985)].
  • Escherichia coli is preferably used as a host microorganism for preparing a cDNA library. Specifically, Escherichia coli XL1—Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia E. coli YlO 88 [Science, 222, 778 (1983)], Escherichia coli Yl 090 [Science, 222, 778 (1983)], Escherichia coli NM522 [Jananore 'Ob' Molecular ⁇ ⁇ ⁇ Biology 0. Mol.
  • this cDNA library can be used as it is for the subsequent analysis, it was developed by Kanno and others to reduce the percentage of incomplete-length cDNA and to obtain full-length cDNA as efficiently as possible.
  • Oligo cap method [Gene, 138, 171 (1994); Gene, 200, 149 (1997); protein nucleic acid enzyme, 41, 603 (1996); experimental medicine, 11, 2491 (1993); It may be prepared by using cDNA cloning (Yodo Co., Ltd.) (1996); Gene library preparation method (Yodo Co., Ltd. (1994)).
  • a degenerative primer specific to the 5'-end and 3'-end base sequences predicted to encode the amino acid sequence is prepared, Obtaining a gene fragment encoding sialidase by amplifying DNA using the prepared cDNA library as a saddle and using the PCR method [PCR Protocols, Academic Press (1990)] Can do.
  • the obtained gene fragment is a DNA encoding sialidase.
  • Sanger et al.'S dideoxy method [Procedidas • Ob The National Academia ⁇ ⁇
  • a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems), or the like, Proc. Natl. Acad. Sci. USA, 7 4, 5463 (1977)] Can be confirmed.
  • colony hybridization or plaque hybridization (molecular clones) from cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. -2nd edition) can be used to obtain the salidase DNA.
  • cDNA synthesized from mRNA contained in tissues or cells of human or non-human animals can be obtained using a cDNA library as a template and PCR. By using this amplification, it is possible to obtain cDNAs of various enzymes related to the addition of sialic acid.
  • the base sequence of the obtained DNA encoding the sialidase can be determined by a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [Procedidas' Ob 'The' National * By analyzing using a base sequence analyzer such as Obci Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1 977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) The base sequence of the DNA can be determined. [0077] Based on the determined base sequence of cDNA, a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL, and DDBJ. It can be confirmed that the gene encodes sialidase.
  • a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL, and DDBJ. It can be confirmed that the gene encodes sialidase.
  • Examples of the base sequence of the gene encoding sialidase obtained by the above method include the base sequences set forth in SEQ ID NOs: 1, 2, 3 or 4.
  • Lidase cDNA Based on the determined DNA base sequence! / And by chemically synthesizing with a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin Elmer) using the phosphoramidite method, Lidase cDNA can also be obtained.
  • a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin Elmer) using the phosphoramidite method
  • Examples of the method for preparing the genomic DNA of sialidase include the methods described below.
  • genomic DNA of sialidase can be obtained by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker TM Kits (CLONTECH).
  • Examples of methods for selecting transformants using sialidase activity as an index include the following methods.
  • cells can be selected using as an index the change in traits resulting from the suppression of sialidase activity.
  • a method of selecting a transformant using the sugar chain structure of the produced glycoprotein as an index can be mentioned. Specifically, the method described in 3. below can be mentioned.
  • RNA gene for suppressing the amount of mRNA of the sialidase gene can be prepared by a conventional method or using a DNA synthesizer.
  • RNA gene construct is [Nature, 391, 806 (1998); Procedurals of the National Academia Science (Proc. Natl. Acad. Sci. USA) , 95, 15502 (1998); Nature, 395, 854 (1998); Proceedings 'Ob' The National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci. USA) , 96, 5049 (1999); Cell, 95, 1017 (1998); Procedures' Ob The National Academia ⁇ ⁇ Ob Science (Proc. Natl. Acad. Sci. USA ), 96, 1451 (1999); Procedurals of the National Sciences (Proc. Natl. Acad. Sci. USA), 95, 13959 (1998); ⁇ ⁇ It can be designed according to the description of Cell's Biology (Nature Cell Biol), 2, 70 (2000).
  • the cell of the present invention can be obtained by directly introducing into a cell a double-stranded RNA designed based on the base sequence of sialidase without using an expression vector.
  • Double-stranded RNA can be prepared by a conventional method or using an RNA synthesizer. Specifically, among the complementary RNA base sequences of sialidase cDNA and genomic DNA, it corresponds to 1 to 50 bases, preferably 10 to 40 bases, more preferably 10 to 30 bases, and even more preferably 15 to 30 bases. Based on the sequence information of the oligonucleotide having a sequence, it can be prepared by synthesizing an oligonucleotide (antisense oligonucleotide) corresponding to a sequence complementary to the oligonucleotide. The oligonucleotide and antisense oligonucleotide may be synthesized independently, or connected with a spacer nucleotide that does not interfere with double-stranded RNA formation! /! /.
  • oligonucleotide derivatives examples include oligo RNA and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
  • Oligonucleotide derivatives include phosphodiester bonds in oligonucleotides. Oligonucleotide derivative converted to phosphothioate bond, phosphodiester bond in oligonucleotide converted to N3, -P5, phosphoramidate derivative converted to phosphoramidate bond, substituted with uracilca SC-5 propynyluracil in oligonucleotide Oligonucleotide derivatives, oligonucleotide derivatives substituted with uracilca C-5 thiazoleuracil in the oligonucleotide, oligonucleotide derivatives substituted with cytosine in the oligonucleotide with C-5 propylcytosine, in the oligonucleotide Oligonucleotide derivatives in which the cytosine is replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in
  • the glycoprotein composition was obtained from Molecular 'Crowning 2nd Edition, Current' Protocols 'In' Molecular 1 ⁇ Nylon 1 ⁇ , Antibodies, A Laboratory manual, old Spring Harbor Laboratory, 1988 (hereinafter referred to as Antibodies).
  • Monoclonal Antibodies principles and practice, Third Edition, Acad. Press, 1993 (hereinafter abbreviated as Monoclonal Nanoreantibodies), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter, For example, the cell of the present invention can be used as a host cell and expressed in the host cell as described below.
  • a cDNA encoding a glycoprotein is prepared, and a DNA fragment of an appropriate length containing a portion encoding the molecule is prepared.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA into the downstream of the promoter of an appropriate expression vector.
  • a transformant producing a glycoprotein molecule can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • any yeast cell, animal cell, insect cell, plant cell, etc. can be used as long as it can express the target gene.
  • the follicle is raised.
  • An expression vector that can replicate autonomously in the above host cell or can be integrated into a chromosome and contains a promoter at a position where DNA encoding the target glycoprotein molecule can be transcribed is used. It is done.
  • the cDNA can be prepared from a tissue or cell of a human or non-human animal using a probe or primer specific for the target glycoprotein according to the cDNA preparation method described in 1. above. it can.
  • yeast When yeast is used as a host cell, examples of expression vectors include YEP13 (ATC C 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419) and the like.
  • any promoter can be used as long as it can be expressed in yeast strains.
  • promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
  • any method for introducing DNA into yeast can be used.
  • the Elect Mouth Position Method [Methods. Enzymol., 194, , 182 (1990)]
  • Spheroplast method Proceedings' Ob The National de force of science ( proc . Natl. Acad. Sci. USA), 84, 1 929 (1978 )]
  • Lithium Acetate Method Lithium Acetate Method [Journal ⁇ Ob 'Battereriology 0. Bacteriology), 15 3, 163 (1983)], Proceedings' Ob The 'National' Academia 1 'Ob' Science (Proc. Natl Acad. Sci. USA), 75, 1929 (1978)].
  • examples of expression vectors include pcDNAU pcD M8 (sold by Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechno logy, 3, 133, ( 1990)], pAS3-3 [JP-A-2-227075], pCDM8 [Nature, 32 9, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGElO 3 [Journal 'Ob' Biochemistry, 101, 1307 (1987)], pAGE21 0 etc. can be raised.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • CMV cytomegalovirus
  • SV40 early promoter SV40 early promoter
  • retrowinores examples thereof include promoters, meta-mouthone promoters, heat shock promoters, SRa promoters, and the like.
  • any method for introducing a recombinant vector any method can be used as long as it is a method for introducing DNA into animal cells.
  • the electoral position method [Cytote chnology, 3, 133 (1990) , Calcium phosphate method [Japanese Patent Laid-Open No. 2-227075], lipofussion method [Proc. Natl. Acad. Sci. USA], 84, 7413 (1987) ], Injection method [Murpurating 'The' Mouse Embryo Laboratories Manual], method using particle gun (Patent No. 2606856, Patent No.
  • the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
  • Examples of gene transfer vectors used in the method include pVL1392, pVLl, and the like.
  • baculoviruses include the ability to use autora, californica nuclear polyhedrosis virus and the like, which are viruses that infect night stealing insects.
  • baculoviruses include the ability to use autora, californica nuclear polyhedrosis virus and the like, which are viruses that infect night stealing insects.
  • a method for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing a recombinant virus for example, the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proceedings] 'Ob The National'Academia's Ob Science ( proc . Natl. Acad. Sci. USA), 84, 7413 (1987)].
  • expression vectors include Ti plasmids and tobacco mosaic virus vectors.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
  • CaMV cauliflower mosaic virus
  • any method for introducing a recombinant vector any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium Japanese Patent Laid-Open No. 59-140 885, Kaisho 60-70080, WO94 / 00977
  • Elect Mouth Position Method Japanese Patent Laid-Open No. 60-251887
  • a method using a particle gun Japanese Patent No. 25 17813
  • Japanese Patent No. 25 17813 Japanese Patent No. 25 17813
  • secretory production can be carried out according to the method described in Molecular Cloning 2nd edition, etc. in addition to direct expression.
  • the transformant obtained as described above is cultured in a medium, and the desired glycoprotein composition is produced and accumulated in the culture, and the glycoprotein composition is collected from the culture.
  • a composition can be produced.
  • the method of culturing the transformant in the medium can be performed according to a usual method used for culturing host cells.
  • the medium As a medium for culturing a transformant obtained using a eukaryote such as yeast as a host, the medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the organism, and the culture of the transformant is performed. If the medium can be efficiently used, the difference between natural and synthetic media can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphates, and other ammonium or organic acid salts. Nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts use is made of monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate, and the like. be able to.
  • the culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culture is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • antibiotics such as ampicillin or tetracycline to the medium during culture.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the culture medium.
  • RPMI 1640 medium commonly used as a medium for culturing transformants obtained using animal cells as a host [The Journal of the American American Medical Association (The Journal of the American Medical Association), 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959) ], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Whitten medium [Genetic engineering experiment] Manual-Transgeneic • How to make a mouse (Kodansha) Motoya Katsuki (1987) An added medium or the like can be used.
  • Cultivation is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and the presence of 5% CO.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
  • Common media for culturing transformants obtained using insect cells as hosts include the commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, and ExCell405. (Both manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] and the like can be used.
  • Cultivation is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.
  • antibiotics such as gentamicin may be added to the medium as needed during the culture.
  • Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do.
  • a medium for culturing the transformant commonly used Murashige 'and' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. It is possible to use the prepared medium.
  • Cultivation is usually carried out for 3 to 60 days under conditions of pH 5 to 9 and 20 to 40 ° C.
  • antibiotics such as kanamycin and hygromycin may be added to the medium as needed during the culture.
  • a yeast, animal cell, or plant cell-derived transformant having a recombinant vector incorporating a DNA encoding a glycoprotein molecule is cultured according to a normal culture method to produce a glycoprotein composition.
  • the glycoprotein composition can be produced by accumulating and collecting the glycoprotein composition from the culture.
  • the glycoprotein composition can be produced in a host cell, secreted outside the host cell, or produced on the outer membrane of the host cell.
  • the method can be selected by changing the structure of the glycoprotein molecule to be produced.
  • the glycoprotein composition When the glycoprotein composition is produced in the host cell or on the host cell outer membrane, Luson et al. [Journal ⁇ Ob ⁇ Biological ⁇ Chemistry (J. Biol. Chem.), 264, 17619 (1989)], Roe et al. [Proceedings 'Ob The National'' Ob . Science ( proc . Natl. Acad. Sci. USA), 86, 8227 (1989); Gene' Development (Genes Develop.), 4, 1288 (1990)], or JP 05-336963, By applying the method described in WO94 / 23021 etc., the glycoprotein composition can be actively secreted out of the host cell.
  • a DNA encoding a glycoprotein molecule and a DNA encoding a signal peptide appropriate for the expression of the glycoprotein molecule are inserted into an expression vector, and the expression vector is used as a host.
  • the target glycoprotein molecule can be actively secreted outside the host cell.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • an animal individual transgenic non-human animal or plant individual (transgenic plant) into which the gene has been introduced is created.
  • a glycoprotein composition can be produced using these individuals.
  • the transformant is an animal or plant individual, it is bred or cultivated according to a usual method to produce and accumulate the glycoprotein composition, and the glycoprotein composition is collected from the animal individual or plant individual.
  • the glycoprotein composition can be produced.
  • -A non-human animal ⁇ ! Can be produced by producing and accumulating a glycoprotein composition in the animal and collecting the glycoprotein composition from the animal.
  • the Examples of the production 'accumulation site in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192). ), And eggs.
  • Any promoter can be used as long as it can be expressed in animals.
  • a mammary cell specific promoter a chick casein promoter, an e casein promoter, an e lactoglobulin promoter, whey acidity.
  • a protein promoter or the like is preferably used.
  • glycoprotein composition As a method for producing a glycoprotein composition using an individual plant, for example, a known method for a transgenic plant introduced with DNA encoding a glycoprotein molecule [tissue culture, 20 (199 4); tissue culture]. , 21 (1995); Trends in Biotechnology, 15, 45 (1997)], and the glycoprotein composition is produced and accumulated in the plant. A method for producing a glycoprotein composition by collecting the glycoprotein composition can be mentioned.
  • a glycoprotein composition produced by a transformant into which a gene encoding a glycoprotein molecule has been introduced for example, when the glycoprotein composition is expressed in a dissolved state in a cell
  • the cells are collected by centrifugation, suspended in an aqueous buffer solution, and then disrupted with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as tilaminoethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical), and cation using resin such as S-Sepharose FF (Pharmacia) Exchange chromatography method, hydrophobic chromatography method using resins such as butyl sepharose and ferrule sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, isoelectric point
  • a purified preparation of the glycoprotein composition can be obtained by using a method such as electrophoresis such as electrophoresis alone or in combination.
  • the glycoprotein composition when expressed by forming an insoluble substance in the cell, the cell is similarly collected, disrupted, and centrifuged to insoluble the glycoprotein composition as a precipitate fraction. Collect body. The recovered insoluble material of the glycoprotein composition is dissolved with a protein denaturant. The solubilized solution is diluted or dialyzed to return the glycoprotein composition to a normal three-dimensional structure, and then a purified preparation of the glycoprotein composition can be obtained by the same isolation and purification method as described above. Togashi.
  • the glycoprotein composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation similar to the above, and glycoproteins are obtained from the culture supernatant by using the same isolation and purification method as described above. A purified preparation of the composition can be obtained. If the host cell already has the ability to express the glycoprotein composition, the cell has the ability to express the glycoprotein composition using the method described in 1. above. After the preparation, the cells are cultured, and the desired glycoprotein composition is collected from the culture and purified, whereby the glycoprotein composition can be produced.
  • the sugar chain structure of a glycoprotein molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein.
  • neutral sugars such as galactose, mannose, and fucose
  • amino sugars such as N-acetyldarcosamine
  • acidic sugars such as sialic acid
  • neutral sugar or amino sugar can be liberated by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
  • composition ratio can also be analyzed by a fluorescent labeling method using 2-aminoviridine. Specifically, a sample subjected to acid-branching decomposition according to a known method [Agricultural 'and' Biological Chemistry (Agric. Biol. Chem.), 55 (1), 283-284 (1991)] Fluorescent labeling can be performed by 2-aminoviridylation, and HPLC analysis can calculate the composition ratio.
  • Acid sugar composition analysis As a method for analyzing the composition of the sugar chain added to the glycoprotein, acid sugar is liberated by acid hydrolysis of the sugar chain with hydrochloric acid or sulfuric acid, and the released acid sugar is labeled. Thus, the sugar composition ratio can be analyzed.
  • monosaccharides that constitute sugar chains added to glycoproteins include sialic acid, neutral sugars, and amino sugars.
  • the stability of monosaccharides released to acids is limited to amino sugars, neutral sugars, and sialic sugars. The order is acid. Therefore, since sialic acid is completely decomposed under the conditions for cleaving the glycosidic bond of amino sugar and neutral sugar, analysis of sialic acid and analysis of amino acid and neutral sugar are usually performed separately.
  • the glycoprotein composition is hydrolyzed to release sugar chains from glycoprotein molecules, and the fluorescent labeling of sugar chains with 2-aminoviridine (hereinafter abbreviated as “ ⁇ ”) [Journal After 'Ob' biochemistry (J. Biochem.), 95, 197 (1984)], the glycan is separated from excess PA reagent by gel filtration and reverse phase chromatography is performed. Next, normal phase chromatography is performed for each peak of the separated sugar chain. Based on these results, a 2D glycan map was plotted and the glycan standard (TaKaRa), literature [Analytical's Biochem., 171, 73 (1988)] The sugar chain structure can be estimated by comparing the spots with. [0122] Furthermore, mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain map method.
  • ⁇ 2-aminoviridine
  • a purified glycoprotein composition is labeled with a compound such as a radioisotope, and the binding reaction with the receptor or interacting protein of the labeled glycoprotein composition is strong.
  • a method for quantitatively measuring the thickness It is also possible to measure protein-protein interactions using various devices such as Biacore's BIAcore series (J. Immnunol. Methods, 145, 229 (1991)). Intermolecular interaction experiment method, Yodosha (1996)).
  • the pharmaceutical containing the glycoprotein composition of the present invention can be administered alone as a prophylactic or therapeutic agent. Usually mixed with one or more pharmacologically acceptable carriers. However, it is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
  • the route of administration includes oral administration where it is desirable to use the most effective treatment, or parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous. In the case of a glycoprotein preparation, intravenous administration is desirable.
  • the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Preparations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol, and fructose, Daricols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, P- It can be produced using preservatives such as hydroxybenzoates and the like, flavors such as stove belly flavors and peppermint as additives.
  • Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. It can be produced using a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • preparations suitable for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both.
  • a powder injection can be prepared by freeze-drying a glycoprotein composition according to a conventional method and adding sodium chloride thereto.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the spray is prepared using a carrier that does not irritate the glycoprotein composition itself or the recipient's oral cavity and airway mucosa and disperses the glycoprotein composition as fine particles to facilitate absorption. Is done.
  • the carrier include lactose and glycerin.
  • preparations such as aerosols and dry powders are possible.
  • the components exemplified as additives for oral agents can also be added.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, weight, etc.
  • the amount of active ingredient is usually 1 ⁇ / 13 ⁇ 4 to 200 g / kg per day for an adult. is there.
  • methods for measuring the biological activity of glycoprotein compositions include in vitro experiments, glycoprotein receptor binding activity measurement methods, glycoprotein composition enzyme activity measurement methods, glycoprotein receptor expression methods.
  • examples include in vitro tests such as methods for measuring proliferation / differentiation promoting activity using cell lines, or in vivo tests using human disease model animals.
  • plasmid pBluescriptll KS (+) was dissolved in 30 L of NEBuffer for EcoRI (New England Biolabs), and 10 units of restriction enzymes EcoRI and XhoI (New England Biolabs) were added. Digestion reaction was performed at 37 ° C for 2 hours. After the reaction, add 13 w L of sterile water, 5 ⁇ L of 10 X Alkaline Phosphatase Buffer and 1 unit of Alkaline Phosphatase E. coli 75 to the reaction solution, and remove at 37 ° C for 1 hour.
  • reaction solution was subjected to agarose gel electrophoresis, and an EcoRI-Xhol fragment derived from about 2.9 Kb of plasmid pBluescriptll KS (+) was collected using RECOCHIP (manufactured by Takara Bio Inc.).
  • DNA EcoRI-Xhol fragment obtained above (approx. 250 bp) 8 ⁇ plasmid pBluescriptll KS (+) derived EcoR to Xhol fragment (approx. 2.9 Kb) 2 les Ligation High (Toyobo) 10 ⁇ L mixed And reacted at 16 ° C for 2 hours.
  • Escherichia coli DH5 ⁇ strain (manufactured by Invitrogen) was transformed using the reaction solution, and plasmid DNA was isolated from the obtained ampicillin meta clone using QIAprep spin Mini prep Kit (manufactured by Qiagen).
  • the obtained plasmid is hereinafter referred to as FT81ib3 / pBS (Fig. 1).
  • plasmid pAGE249 was dissolved in 30 ⁇ l of NEBuffer 1 (New England Biolabs), and 10 units of restriction enzymes Nael and XhoI (New England Biolabs) were added at 37 ° C. The digestion reaction was performed for 6 hours. After the digestion reaction, sterilized water 22 L, 10 X Alkaline Phosphatase Buffer 6 ⁇ L and Alkaline Phosphatase E.coli C75 (manufactured by Takara Bio) 1 Units were added and dephosphorylation was performed at 37 ° C for 1 hour.
  • the reaction solution was subjected to agarose gel electrophoresis, and a Nael-Xhol fragment derived from about 4.4 Kb of plasmid pAGE249 was recovered using RECOCHIP (Takara Bio Inc.).
  • PAGE249 is a derivative of pAGE248 [Journal 'Ob' Biological. Chemistry (J. Biol. Chem., 269, 14730 (1994))].
  • dhfr dihydrofolate reductase gene
  • PAGE249_seq FW SEQ ID NO: 8
  • pAGE249-seq RV SEQ ID NO: 9
  • the inserted DNA sequence is included in plasmid FUT8shRNA / lib2B / pP UR or plasmid FUT8shRNA / lib3 / pPUR
  • the RNA-val promoter ⁇ Short hairpin RNA expression unit V was consistent with the sequence corresponding to the EcoR and Xhol fragment.
  • the resulting plasmid is hereinafter referred to as FT81ib3 / pAGE ( Figure 2).
  • a synthetic oligo DNA was prepared that forms a double-stranded DNA cassette containing the target sequence of siRNA against the Chinese nomstar NEU2 sialidase gene.
  • NEU2 sialidase cDNA sequence represented by SEQ ID NO: 1 (GeneBank, U06143), and Reynolds et al. (Nature Biotechnology, 22, 326 (2004))
  • SEQ ID NO: 1 GeneBank, U06143
  • Reynolds et al. London Biotechnology, 22, 326 (2004)
  • Condition 1 GC content is 30% to 50%. Add 1 point if satisfied.
  • At least 3 bases A or U are added to the 15th to 19th 5 bases of the sense strand. included. If satisfied, add 1 point for every 1 base of A or U.
  • Condition 4 19th force A of the sense strand. Add 1 point if satisfied.
  • a double-stranded DNA cassette was designed for the selected target sequence by the following procedure. Double-stranded DNA cassettes are also generated in the order of 5 'end force by cleavage of the restriction enzyme Sad, the 3' overhanging end, the antisense coding DNA complementary to the DNA sequence corresponding to SEQ ID NOs: 11-55 and the restriction enzyme Kpnl 3 'Has a protruding end.
  • the 5 ′ end of the double-stranded DNA was phosphorylated and the target sequence shown in SEQ ID NO: 11 was used, and the base sequence of the sense strand (hereinafter referred to as SiAl) of the synthetic oligo DNA designed was SEQ ID NO: 56, the antisense strand.
  • the base sequence of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 12 (hereinafter referred to as SiaBl) is shown in SEQ ID NO: 57 as the base sequence of SIAA2 (hereinafter referred to as SiaA2).
  • the base sequence is SEQ ID NO: 59
  • the base sequence of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 13 (hereinafter SiaCl) is the base sequence of SEQ ID NO: 60
  • the base sequence of the antisense strand (hereinafter SiaC2) is SEQ ID NO:
  • the base sequence of the sense strand (hereinafter referred to as SiaD1) of the synthetic oligo-DNA designed by the target sequence shown in SEQ ID NO: 14 is represented by SEQ ID NO: 62
  • the base sequence of the antisense strand (hereinafter referred to as SiaD2) is represented by SEQ ID NO: 63.
  • Synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 65 the base sequence of the sense strand of the oligo DNA (hereinafter SiaEl) is SEQ ID NO: 64
  • the base sequence of the antisense strand (hereinafter SiaE2) is SEQ ID NO: 65
  • the nucleotide sequence of the sense strand (hereinafter referred to as SiaFl) is SEQ ID NO: 66, antisense.
  • the base sequence of the strand (hereinafter referred to as SiaG2) is the base sequence of the sense strand (hereinafter referred to as SiaGl) of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 67.
  • the base sequence of the sense strand (hereinafter referred to as SialFW) of the synthetic oligo DNA designed as shown in SEQ ID NO: 69 and the target sequence shown in SEQ ID NO: 18 is the base sequence of SEQ ID NO: 70 and the antisense strand (hereinafter referred to as SialRV).
  • Sia2FW the nucleotide sequence of the sense strand (hereinafter referred to as Sia2FW) of the synthetic oligo DNA designed in accordance with the target sequence shown in SEQ ID NO: 71 and SEQ ID NO: 19, and the nucleotide sequence of the antisense strand (hereinafter referred to as Sia2RV).
  • the base sequence of the sense strand (hereinafter referred to as Sia3FW) of the synthetic oligo DNA designed in accordance with the target sequence shown in SEQ ID NO: 20 is represented by SEQ ID NO: 74, and the base sequence of the antisense strand (hereinafter referred to as Sia3RV). No.
  • the base sequence of the sense strand (hereinafter referred to as Sia4 FW) of the designed synthetic oligo DNA is represented by SEQ ID NO: 76
  • the base sequence of the antisense strand (hereinafter Sia4RV) is represented by SEQ ID NO: 77
  • the target sequence represented by SEQ ID NO: 22
  • the base sequence of the sense strand (hereinafter referred to as Sia5FW) of the designed synthetic oligo DNA is represented by SEQ ID NO: 78
  • the base sequence of the antisense strand (hereinafter Sia5RV) is represented by SEQ ID NO: 79
  • the target sequence represented by SEQ ID NO: 23 the base sequence of the sense strand (hereinafter referred to as Sia4RV) of the designed synthetic oligo DNA is represented by SEQ ID NO: 78
  • the base sequence of the antisense strand (hereinafter Sia5RV) is represented by SEQ ID NO: 79
  • the base sequence of the sense strand (hereinafter Sia7FW) is SEQ ID NO: 82
  • the base sequence of the antisense strand (hereinafter Sia7RV) is SEQ ID NO: 83
  • the sense strand of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 25 (Sia8FW)
  • Sia9FW The base sequence of the sense strand (hereinafter referred to as Sia9FW) of the synthetic oligo DNA designed for column No.
  • the base sequence of the antisense strand (hereinafter referred to as Sia8RV) is shown in SEQ ID NO: 85 and the target sequence shown in SEQ ID NO: 26 as SEQ ID NO: 86.
  • the base sequence of the sense strand (hereinafter referred to as SialOFW) of the synthetic oligo DNA designed by connecting the base sequence of the antisense strand (hereinafter referred to as Sia9RV) to the target sequence shown in SEQ ID NO: 87 and SEQ ID NO: 27 is shown in SEQ ID NO: 88.
  • the base sequence of the antisense strand (hereinafter referred to as SialORV) is shown in SEQ ID NO: 89, and the base sequence of the sense strand (hereinafter referred to as Sial 1 FW) of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 28 is shown in SEQ ID NO: 90.
  • the base sequence of the sense strand (hereinafter referred to as Sial2FW) of the synthetic oligo DNA designed according to the target sequence shown in SEQ ID NO: 91 and the target sequence shown in SEQ ID NO: 29 as the base sequence of the strand (hereinafter referred to as SiallRV) is represented by SEQ ID NO: 92 Sial2RV)
  • the base sequence is SEQ ID NO: 93
  • the base sequence of the sense strand (hereinafter Sial3FW) of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 30 is SEQ ID NO: 94
  • the base sequence of the antisense strand (hereinafter Si al3RV) is SEQ ID NO: 95
  • the base sequence of the antisense strand here
  • the base sequence of the synthetic oligo DNA sense strand (hereinafter referred to as Sial6FW) designed for the target sequence shown in number 33 is shown in SEQ ID NO: 100
  • the base sequence of the antisense strand (hereinafter referred to as Sial6RV) is shown in SEQ ID NO: 101
  • the target shown in SEQ ID NO: 34 In the array
  • the synthesized oligo DNA sense strand (hereinafter referred to as Sial7FW) has the nucleotide sequence of SEQ ID NO: 102
  • the antisense strand hereinafter referred to as Sial7RV
  • the synthetic oligo designed for the target sequence shown in SEQ ID NO: 35 has the nucleotide sequence of SEQ ID NO: 103
  • the base sequence of the DNA sense strand (hereinafter referred to as Sial8FW) is SEQ ID NO: 104
  • the base sequence of the antisense strand (hereinafter referred to as Sial8RV) is SEQ ID NO: 105
  • the sense strand of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 36 ( The base sequence of Sial9FW) is SEQ ID NO: 106
  • the base sequence of the antisense strand (hereinafter Sial9RV) is SEQ ID NO: 107
  • the target sequence shown in SEQ ID NO: 37 is the base of the sense strand (hereinafter Sia20FW) of the synthetic oligo DNA designed.
  • the base sequence of the synthetic oligo DNA sense strand (hereinafter referred to as Sia21FW) designed by SEQ ID NO: 108, the nucleotide sequence of the antisense strand (hereinafter referred to as Sia20RV) as SEQ ID NO: 109, and the target sequence shown as SEQ ID NO: 38 SEQ ID NO: 110, the base sequence of the antisense strand (hereinafter referred to as Sia21RV) in SEQ ID NO: 111, and the base sequence of the sense strand (hereinafter referred to as Sia22FW) of the synthetic oligo DNA designed using the target sequence shown in SEQ ID NO: 39 as SEQ ID NO: 112, the nucleotide sequence of the antisense strand (hereinafter referred to as Sia22RV) is shown in SEQ ID NO: 113, and the nucleotide sequence of the synthetic oligo DNA sense strand (hereinafter referred to as Sia23FW) designed according to the target sequence shown in S
  • the base sequence of the synthetic oligo DNA sense strand (hereinafter Sia25FW) designed for the target sequence shown in SEQ ID NO: 117 and the target sequence shown in SEQ ID NO: 42 (hereinafter Sia24RV) is shown in SEQ ID NO: 1. 18.
  • the nucleotide sequence of the sense strand (hereinafter referred to as Sia26FW) of the synthetic oligo DNA designed according to the target sequence shown in SEQ ID NO: 119 and the target sequence shown in SEQ ID NO: 43 as the nucleotide sequence of the antisense strand (hereinafter Sia25RV) is SEQ ID NO: 120.
  • the base sequence of the sense strand (hereinafter referred to as SiA27FW) of the synthetic oligo DNA designed with the target sequence shown in SEQ ID NO: 121 and the target sequence shown in SEQ ID NO: 44 as the base sequence of the sense strand (hereinafter referred to as Sia26RV) is SEQ ID NO: 122 as the antisense strand.
  • the base sequence of the synthetic oligo DNA sense strand (hereinafter referred to as Sia28FW) designed according to the target sequence shown in SEQ ID NO: 123 and the target sequence shown in SEQ ID NO: 45 is referred to as SEQ ID NO: 124 and the antisense strand (hereinafter referred to as Sia28RV).
  • Sia29FW Is the nucleotide sequence of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 46 (hereinafter referred to as Sia29FW), the nucleotide sequence of SEQ ID NO: 126, and the nucleotide sequence of the antisense strand (hereinafter referred to as Sia29RV).
  • SEQ ID NO: 1 In 27, the base sequence of the synthetic oligo DNA sense strand (hereinafter Sia30FW) designed for the target sequence shown in SEQ ID NO: 47 is SEQ ID NO: 128, and the base sequence of the antisense strand (hereinafter Sia30RV) is SEQ ID NO: 129.
  • the base sequence of the synthetic oligo DNA sense strand (hereinafter referred to as Sia31FW) designed according to the target sequence shown in SEQ ID NO: 48 is shown in SEQ ID NO: 130
  • the base sequence of the antisense strand (hereinafter referred to as Sia31RV) is shown in SEQ ID NO: 131
  • the base sequence of the sense strand (hereinafter Sia32FW) of the synthetic oligo DNA designed for the target sequence shown in 49 is shown in SEQ ID NO: 132
  • the base sequence of the antisense strand (hereinafter Sia32RV) is shown in SEQ ID NO: 133
  • the target sequence shown in SEQ ID NO: 50 The base sequence of the sense strand (hereinafter referred to as Sia33FW) of the synthetic oligo DNA designed for SEQ ID NO: 134
  • the base sequence of the antisense strand (hereinafter referred to as Sia33RV) is shown in SEQ ID NO: 135, and
  • the sense sequence of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 136 is shown in SEQ ID NO: 136 for the base sequence of the sense strand (hereinafter Sia34FW) of the Rigo DNA, SEQ ID NO: 137 in the base sequence of the antisense strand (hereinafter Sia34RV).
  • the base sequence of the strand (hereinafter referred to as Sia35FW) is SEQ ID NO: 138, the base sequence of the antisense strand (hereinafter referred to as Sia35RV) is shown in SEQ ID NO: 139, and the synthetic oligo DNA sense strand designed by the target sequence shown in SEQ ID NO: 53 (
  • the base sequence of Sia36FW) is represented by SEQ ID NO: 140
  • the base sequence of the antisense strand hereinafter Sia36RV
  • the sense strand of the synthetic oligo DNA (hereinafter referred to as the target sequence shown by SEQ ID NO: 54).
  • Sia37FW is the base sequence of SEQ ID NO: 142
  • the antisense strand (hereinafter Sia37RV) is the base sequence of SEQ ID NO: 143
  • the sense strand of the synthetic oligo DNA designed for the target sequence shown in SEQ ID NO: 55 ( The base sequence of Sia38FW) is shown in SEQ ID NO: 144
  • the base sequence of the antisense strand (hereinafter Sia38RV) is shown in SEQ ID NO: 145.
  • the designed synthetic oligo DNA was synthesized according to a conventional method (Molequila I. Cloning 2nd edition).
  • the synthetic oligo DNA synthesized in this section (2) was inserted into the siRNA-type DNA insertion region of FT81ib3 / pAGE obtained in this section 1 (1).
  • the synthetic oligo DNA was annealed according to the following procedure (Fig. 3). 200 pmol each of the synthetic oligo DNA sense strand and antisense strand was dissolved in 10 ⁇ L of annealing buffer [10 mM Tris (pH 7.5) -50 mM NaCl-lmM EDTA] and incubated at 98 ° C. for 2 minutes. Then, it was cooled down and cooled to room temperature over about 3 hours. Thereafter, the annealed synthetic oligo DNA was diluted 100 times with sterile water.
  • annealing buffer 10 mM Tris (pH 7.5) -50 mM NaCl-lmM EDTA
  • each plasmid DNA obtained by cleaving the short DNA fragment of the short hairpin RNA loop by digestion with BamHI was used as a primer.
  • PAGE249-seq FW (SEQ ID NO: 9) and pAGE249- Using seq RV (SEQ ID NO: 10) it was confirmed that there was no mistake in the sequence of the inserted synthetic oligo DNA and the ligation part.
  • the plasmid inserted with the double-stranded DNA composed of the synthetic oligo DNAs siaAl and siaA2 is pSiaA
  • the plasmid inserted with the double-stranded DNA composed of the synthetic oligo DNAs siaBl and siaB2 is composed of pSiaB
  • PSiaC is the plasmid with the double-stranded DNA inserted
  • pSiaD is the plasmid with the double-stranded DNA composed of the synthetic oligo DNAs siaDl and siaD2
  • a plasmid with a synthetic oligo DNA siaFl and siaF2 also has a double-stranded DNA inserted into pSiaF
  • a synthetic oligo DNA sialFW and pSial is a plasmid in which double-stranded DNA consisting of sialRV is inserted
  • pSia2 a plasmid inserted with double-stranded DNA consisting of synthetic oligo DNAs sia3FW and sia3RV
  • pSia3 a plasmid inserted with double-stranded DNA consisting of synthetic oligo DNAs sia4FW and sia4RV
  • synthetic oligo DNA sia5FW and sia5RV PSia5 is the plasmid with the double-stranded DNA inserted
  • pSia6 is the plasmid with the double-stranded DNA composed of the synthetic oligo DNAs sia6FW and sia6RV
  • the plasmid is the double-stranded DNA composed of the synthetic oligo DNA sia7FW and sia7RV PSia7
  • pSia8 a plasmid inserted with a double-
  • the plasmid is pSiall, the plasmid inserted with the double-stranded DNA composed of the synthetic oligo DNA sial2FW and sial2RV is pSial2, the plasmid with the double-stranded DNA composed of the synthetic oligo DNA sial3FW and sial3RV is pSial3, and the synthetic oligo DNA pSial4 is a plasmid in which double-stranded DNA consisting of sial 4FW and sial4RV is inserted, pSial5 is a plasmid in which double-stranded DNA consisting of synthetic oligo DNA sial5FW and sial5RV is inserted, and double-stranded DNA consisting of synthetic oligo DNA sial6FW and sial6RV PSial6 is the plasmid with the inserted DNA, pSial7 is the plasmid with the double-stranded DNA composed of the synthetic oligo DNA sial7FW and sial
  • PSia29 is the inserted plasmid
  • pSia30 is the plasmid with the double-stranded DNA composed of the synthetic oligo DNAs sia30FW and sia30 RV
  • pSia31 is the plasmid with the double-stranded DNA composed of the synthetic oligo DNA sia31FW and sia31RV.
  • PSia32 is a plasmid in which double-stranded DNA consisting of oligo DNA si32FW and sia32RV is inserted
  • pSia33 is a plasmid in which double-stranded DNA consisting of synthetic oligo DNAs sia33FW and sia33RV is inserted
  • two plasmids are composed of synthetic oligo DNA sia34FW and sia34RV.
  • a plasmid with a double-stranded DNA consisting of pSia34 and a synthetic oligo DNA sia35FW and sia35RV is inserted into the plasmid with the double-stranded DNA inserted.
  • pSia35 a plasmid inserted with a double-stranded DNA composed of synthetic oligo DNAs sia36FW and sia36RV
  • pSia36 a plasmid inserted with a double-stranded DNA composed of synthetic oligo DNAs sia37FW and sia37RV
  • pSia37 a synthetic oligo DNA composed of sia38FW and sia38RV
  • the plasmids with double-stranded DNA inserted are called pSia38 (Fig. 3).
  • Sia! 2 is a sequence complementary to nucleotides 177 to 196 of the Chinese hamster NEU2 sialidase gene sequence shown in SEQ ID NO: 1, and siarv2 is a sequence complementary to nucleotides 1311 to 1333.
  • the PCR reaction was 25 ⁇ L of 5 ⁇ L CHO / DG44 cell-derived single-stranded cDNA [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP, s, 0.5 units of EX Prepare Taq polymerase (Takara Shuzo), 0.5 ⁇ of the above primers (SEQ ID NO: 146 and SEQ ID NO: 147)], and use GeneAmp PCR system 9700 (Perkin Elma) for 5 minutes at 94 ° C. After heating, 30 cycles of 94 ° C for 20 seconds, 65 ° C for 30 seconds, and 72 ° C for 30 seconds were performed.
  • a PCR amplified fragment of about 1 .1 Kbp was recovered using Qiaex II gel extraction kit (Qiagen).
  • the DNA fragment was ligated to the pT7 blue vector (Novagen) using Ligation high (Toyobo), and the resulting recombinant plasmid DNA was used to transform E. coli DH5a strain (Toyobo).
  • Recombinant plasmid DNA was isolated from the resulting ampicillin metacolonies using QIAprep Spin Miniprep Kit (Qiagen).
  • the base sequence of the PCR fragment contained in the plasmid was determined using DNA Sequencer ABI PRISM 377 (manufactured by PerkinElmer Co., Ltd.) according to a conventional method, and it was confirmed that there was no base mutation associated with PCR.
  • the resulting plasmid containing the NEU2 sialidase cDNA derived from Chinese hamster ovary cells (CHO) is called pT7 blue sialidase ( Figure 4).
  • firefly luciferase gene 1 ⁇ g of the reporter plasmid pG5Luc (Clontech) that encodes lysate is dissolved in 30 ⁇ L of Kashiwa Buffer 1 (Takara Shuzo), 10 units of HindllKNew England BioLab) and 10 units of XbaI (New England BioLab)
  • the reaction was carried out at 37 ° C for 2 hours.
  • the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment encoding about 1.7 Kbp of firefly luciferase was recovered using RECOCHIP (manufactured by Takara Bio Inc.).
  • plasmid pAGE249 is dissolved in 30 ⁇ L of koji buffer (Takara Shuzo), and 10 units of Hindlll (Takara Shuzo) and 10 units of BamHI (New England BioLab) are added at 37 ° C. And digested for 2 hours.
  • the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 6.4 Kbp derived from pAGE249 was recovered using RECOCHIP (Takara Bio Inc.).
  • pAGE Lucia is a plasmid that expresses a chimeric reporter gene that also has firefly luciferase and Chinese nomstar NEU2 sialidase activity under the control of the mouse Moro-1 LTR promoter.
  • siRNA expression plasmids and chimeric reporter expression plasmids were co-introduced into CHO / DG44 cells, effective siRNA expression plasmids were identified by measuring luciferase activity.
  • What is expressed from the plasmid pKANTEX Lucia prepared in this Example 3 is a chimeric reporter gene that also has the power of a firefly luciferase gene in which a Chinese nomstar NEU2 sialidase gene is placed in the untranslated region.
  • the NEU2 sialidase region on the chimeric reporter gene is cleaved by siRNA, the intracellular luciferase activity decreases with the disappearance of the chimeric reporter.
  • the DNA-lipid mixture was prepared by the following procedure. 40 ng chimeric reporter expression plasmid pKANTEX Lucia per well, 160 ng siRNA expression plasmid and 20 ng internal standard plasmid pRL-TK were dissolved in 25 ⁇ L OPTI-MEM medium, and 0.8 ⁇ L Lipofectamin2000 The DNA solution was added to a lipid mixture mixed with 25 ⁇ L of ⁇ -MEM medium and allowed to stand at room temperature for 20 minutes to prepare a DNA-lipid mixture.
  • PRL-TK is a Renilla luciferase expression plasmid added to correct the gene transfer efficiency for each well. Add 50 L of the DNA-lipid mixture and add it to the well containing the transfected cells replaced with 0 PTI-MEM medium, and incubate for 6 hours at 37 ° C and 5% CO. It was. After culturing for 6 hours, add 100% basic medium containing 20% serum.
  • the amount of firefly luciferase luminescence from each well was corrected using the amount of luminescence from renilla luciferase, and for each siRNA expression plasmid, the relative value of the firefly luciferase activity observed when co-introduced with the chimeric reporter plasmid was calculated. Indicated.
  • psiFLuc manufactured by GeneScript
  • psiFLuc as a positive control is a plasmid that expresses siRNA targeting a part of the firefly luciferase gene. By co-introducing this plasmid, the amount of firefly luciferase luminescence from the chimeric reporter is It was suppressed by 80% (Fig. 7).
  • siRNA expression plasmids targeting the sialidase gene 4 molecules of pSi5, pSil7, pSi28, and pSi34 clearly have the ability to suppress the activity of the co-introduced reporter by 80% or more, as in the positive control. became.
  • sialidase target siRNA expression plasmids pSi5, pSil7, pSi28, and pSi34 obtained in this section 2 into MS705 pKAN- ⁇ 27 strain described in WO2005 / 035563 (Department No. 13 ⁇ 41? ⁇ 1 BP-0 8472) Then, a cell line in which the cellular sialidase activity was stably suppressed was obtained. This strain is a CHO / MS705-derived strain that stably produces antithrombin.
  • the plasmid was introduced into the cell line by the following method.
  • puromycin was replaced with 10 mL of basic medium containing 12 ⁇ g / mL, and further cultured for 7 days to obtain puromycin-resistant clones.
  • 96 pure-mouthymycin resistant clones were isolated and cultured in 96-well plates for cell culture (Grainer). A replicated plate was prepared immediately before the cell density of each well reached confluence. That is, after each well was washed with 100 L PBS (Invitrogen), 25 ⁇ L of 0.05% Tripsin (Invitrogen) 25 ⁇ L was added and placed in a 5% CO incubator.
  • each basic well is supplemented with a basic medium containing 125 L of puromycin (SIGMA) at a concentration of 12 ⁇ g / mL, and the cells are suspended. Inoculate three 96-well plates containing basal medium containing 12 ⁇ g / mL of puromycin at a concentration of 12 ⁇ g / mL and leave at 37 ° C for 3 days in a 5% CO incubator.
  • SIGMA puromycin
  • sialidase activity was measured by dispensing 5 ⁇ L / wenore of the sialidase fluorescent substrate 4-methylumbellifery ⁇ a-a-D-acetyln euraminic acid ammonium salt (manufactured by Nacalai Tesque) prepared at a concentration of 4 mM, and capping the plate. And allowed to react at 37 ° C for 2 hours in a carbon dioxide incubator (smoked).
  • Serum-free batch culture is performed under conditions of a temperature of 35 ° C and a stirring speed of 85 rpm, and supplements the consumption of amino acids, etc. on the 3rd, 5th, 7th, 9th, and 11th days after the start of the culture.
  • Antithrombin was quantified by first using ⁇ ELISA Capture Antibody (Antibody Set, Affinity Biologocs C / N ⁇ -EIA) with 50 mM Carbonate (1.59 g Na CO and 2.93 g NaHC).
  • Dissolve O in an appropriate amount of distilled water adjust to pH 9.6 with 5N NaOH aqueous solution, and then add distilled water.
  • the solution was diluted 100 times with 1 L of the solution, and dispensed at 100 L / well into a 96-well plate (Nunc). After standing at room temperature for 1-2 hours, the inside of the well was washed with 0.005% Tween20-PBS, and 1% BSA-PBS was dispensed at 100 ⁇ L / well and stored frozen.
  • Samples and standards with known concentrations are diluted to an appropriate concentration with 1% BSA-PBS, dissolved at room temperature, and BSA-PBS in the wells removed, 50 ⁇ L / well And dispensed at room temperature for 1 hour. After washing the well with 0.05% Tween20-PBS, the sputum detection antibody solution was dispensed at 100 / z L / well and allowed to stand at room temperature for about 1 hour. After each well was washed with 350 L of 0.05% Tween20-PBS, ABTS solution containing 0.1% H0 was added.
  • the color development was stopped by adding 5% SDS solution with L / well. Using a microplate reader, the absorbance was measured at a wavelength of excitation 415 nm / absorption 490 nm, and the soot content was calculated.
  • the 28-17 strain suppressed the sialidase activity in the culture supernatant throughout the 14-day culture period.
  • the sialic acid number can maintain a high value.
  • the production method of the present invention can provide a glycoprotein composition in which the amount of sialic acid added is increased as compared with conventional glycoprotein compositions.

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Abstract

L'invention concerne la production d'une composition de glycoprotéine présentant une activité physiologique élevée en augmentant la quantité additionnelle d'acide sialique dans ladite composition. Selon l'invention, une cellule dans laquelle un ARNsi supprimant une activité de sialidase est introduit ou exprimé, un procédé pour la production d'une molécule de glycoprotéine à l'aide de la cellule, et une composition de glycoprotéine obtenue par le procédé de production sont fournis.
PCT/JP2007/054067 2006-03-02 2007-03-02 Procede pour la production d'une composition de glycoproteine WO2007102432A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049284A3 (fr) * 2007-10-12 2009-12-30 Sigma-Aldrich Co. Compositions et procédés pour une sialylation améliorée des glycoprotéines
JP2012524747A (ja) * 2009-04-23 2012-10-18 クルセル ホランド ベー ヴェー 組換えヒトアルファ１−アンチトリプシン
JPWO2013002330A1 (ja) * 2011-06-29 2015-02-23 協和発酵キリン株式会社 たん白質の精製方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FERRARI J. ET AL.: "Chinese hamster ovary cells with constitutively expressed sialidase antisense RNA produce recombinant DNase in batch culture with increased sialic acid", BIOTECHNOLOGY AND BIOENGINEERING, vol. 60, no. 5, 1998, pages 589 - 595, XP002981240 *
FERRARI J. ET AL.: "Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases", GLYCOBIOLOGY, vol. 4, no. 3, 1994, pages 367 - 373, XP002070047 *
MIYAGI T. ET AL.: "A crucial role of plasma membrane-associated sialidase (NEU3) in the survival of human cancer cells", GLYCOBIOLOGY, vol. 14, no. 11, 2004, pages 1176, XP002995283 *
PILATTE Y. ET AL.: "Sialic acids as important molecules in the regulation of the immune system: pathophysiological implications of sialidases in immunity", GLYCOBIOLOGY, vol. 3, 1993, pages 201 - 218, XP003017643 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009049284A3 (fr) * 2007-10-12 2009-12-30 Sigma-Aldrich Co. Compositions et procédés pour une sialylation améliorée des glycoprotéines
JP2011500032A (ja) * 2007-10-12 2011-01-06 シグマ−アルドリッチ・カンパニー 糖タンパク質のシアリル化を改善するための組成物および方法
US8273723B2 (en) 2007-10-12 2012-09-25 Sigma-Aldrich Co. Llc Compositions and methods for improved glycoprotein sialylation
JP2015057059A (ja) * 2007-10-12 2015-03-26 シグマ−アルドリッチ・カンパニー、エルエルシー 糖タンパク質のシアリル化を改善するための組成物および方法
EP2930245A1 (fr) * 2007-10-12 2015-10-14 Sigma Aldrich Co. LLC Lignée cellulaire et procédés pour une sialylation améliorée des glycoprotéines
JP2012524747A (ja) * 2009-04-23 2012-10-18 クルセル ホランド ベー ヴェー 組換えヒトアルファ１−アンチトリプシン
JPWO2013002330A1 (ja) * 2011-06-29 2015-02-23 協和発酵キリン株式会社 たん白質の精製方法

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