WO2007108464A1 - Anticorps dirigé contre la sialidase cytoplasmique de mammifère - Google Patents

Anticorps dirigé contre la sialidase cytoplasmique de mammifère Download PDF

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Publication number
WO2007108464A1
WO2007108464A1 PCT/JP2007/055633 JP2007055633W WO2007108464A1 WO 2007108464 A1 WO2007108464 A1 WO 2007108464A1 JP 2007055633 W JP2007055633 W JP 2007055633W WO 2007108464 A1 WO2007108464 A1 WO 2007108464A1
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Prior art keywords
antibody
sialidase
amino acid
protein
seq
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PCT/JP2007/055633
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English (en)
Japanese (ja)
Inventor
Akiko Furuya
Mitsuo Satoh
Yutaka Kanda
Katsuhiro Mori
Reiko Kamochi
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Kyowa Hakko Kogyo Co., Ltd.
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Publication of WO2007108464A1 publication Critical patent/WO2007108464A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725—Proteoglycans, e.g. aggreccan
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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

Definitions

  • the present invention relates to an antibody that binds to a mammal-derived cytoplasmic sialidase and recognizes the three-dimensional structure of a mammal-derived cytoplasmic sialidase, a method for producing a glycoprotein using the antibody, and a saccharide obtained by the method It relates to protein.
  • 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.
  • the amino acid sequence is conserved.
  • 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 (Non-patent Document 10) is similar to the crystal structure of human NEU2 sialidase (Non-patent Document 11).
  • NEU2 sialidase gene encodes a single-chain polypeptide having 1679 base strength and 379 amino acid residues.
  • NEU2 sialidase has two aspartic acid box sequences, is rich in cysteine residues and
  • This enzyme is present in the cytoplasm, works near neutral pH, and uses oligosaccharides, gandariosides, and glycoproteins as substrates. In particular, it has been reported that it is highly expressed in testis and skeletal muscle and plays an important role in the differentiation of skeletal muscle cells! Speak (Non-Patent Document 12).
  • Non-patent document 13 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.
  • CHO cells derived from Chinese nomster ovary tissue and mouse myeloma.
  • a sugar chain that can exhibit optimal pharmacological activity is not always added.
  • it is important to control sialic acid modification in the production of glycoprotein drugs because the addition of non-reducing terminal sialic acid on the sugar chain can have a significant impact on the pharmacological activity of the administered glycoprotein. is there.
  • 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).
  • Glycoprotein drugs are secreted and produced by culturing transformants that have been established by stably incorporating cDNAs encoding these glycoproteins into host cells under appropriate culture conditions.
  • Production cell strength Glycoproteins secreted into the culture medium are added with a variety of sugar chains based on the sugar chain-modifying mechanism inherent to the cell. In many cases, the non-reducing terminal side of the mature sugar chain It is modified with a negatively charged sialic acid residue.
  • the rate of addition 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. In the production of this glycoprotein, one of the major causes of decreasing the rate of calorie with sialic acid is the involvement of sialidase in 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.
  • it is considered that there is a problem that the economic burden related to the preparation of the culture medium and waste liquid treatment is large. ing.
  • a new non-naturally occurring compound such as Neu5Ac2en for pharmaceutical production it is necessary to consider the safety of the substance itself or a derivative produced by the change of this substance during culture. Must not.
  • 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.
  • Patent Document 3 As an antibody that reacts with sialidase, an antibody obtained by immunizing a partial peptide of NEU2 (Patent Document 3) has been reported. However, it is a monoclonal antibody that binds to cytoplasmic sialidase and recognizes the three-dimensional structure of mammalian cytoplasmic sialidase! None before has it been known! /.
  • 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 Literature 7 Journal of Biological Chemistry vol.268,26435- 26440 (1993)
  • Non-Patent Literature 8 Genes and Development vol.10,3156- 3169 (1996)
  • Non-Patent Literature 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 Document 11 Journal of Biological Chemistry vol.280, 469-475 (2005)
  • Non-Patent Document 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 Literature 18 European Journal of Biochemistry 194, 457 (1990)
  • Non-Patent Literature 19 Journal of Biological Chemistry 265, 12127 (1990)
  • Non-Patent Literature 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
  • An object of the present invention is to recognize an anti-cytoplasmic sialidase monoclonal antibody that recognizes the three-dimensional structure of a mammal-derived cytoplasmic sialidase and neutralizes a mammal-derived cytoplasmic sialidase activity, and a glycoprotein using the antibody And a glycoprotein composition obtained by the production method.
  • the present invention relates to the following (1) to (11).
  • An anti-cytoplasmic sialidase monoclonal antibody that recognizes the three-dimensional structure of a mammal-derived cytoplasmic sialidase and neutralizes the mammal-derived cytoplasmic sialidase activity.
  • the monoclonal antibody according to (1) above which is a monoclonal antibody that binds to at least two types of mammalian cytoplasmic sialidases.
  • Cytoplasmic sialidase force The monoclonal antibody according to the above item (1) or (2), which is a protein encoded by a DNA selected from the following group forces (a) to (h).
  • cytoplasmic sialidase is a protein selected from the following group forces (a) to (1): (a) a protein having an amino acid sequence ability represented by SEQ ID NO: 5;
  • 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.
  • Monoclonal antibody force The antibody according to any one of (1) to (5) above, wherein the following (a) to (c) group force is also selected. (a) an antibody produced from Hypridoma;
  • a fusion antibody comprising (a) or (b).
  • Monoclonal antibody power The antibody according to any one of (1) to (6) above, which is a monoclonal antibody in which the following group powers (a) to (d) are selected.
  • a method for producing a glycoprotein composition by culturing cells characterized in that the monoclonal antibody described in (1) to (7) above, wherein the shear force is 1 is added to the culture solution
  • a method for producing a glycoprotein composition A method for producing a glycoprotein composition.
  • glycoprotein composition produced is compared with the glycoprotein composition obtained in the case where the monoclonal antibody according to any one of (1) to (7) above is not added to the culture solution.
  • the present invention provides an anti-cytoplasmic sialidase monoclonal antibody that recognizes the three-dimensional structure of a cytoplasmic sialidase derived from a mammal and neutralizes the cytoplasmic sialidase activity derived from a mammal.
  • sialic acid can be added to neutralize the activity of mammalian cytoplasmic sialidase.
  • a glycoprotein composition having a sugar chain with a high addition rate can be provided.
  • FIG. 1 Polyacrylamide gel electrophoresis image of E. coli recombinant GST-sialidase
  • FIG. 2 shows the measurement results of GST-sialidase sialidase activity.
  • GST shearidase decomposed the fluorescent substrate in a concentration-dependent manner and showed the shearidase activity with the value shown on the vertical axis. These activities were abolished by the sialidase inhibitor Neu5AC2en.
  • the reactivity in A is shown.
  • the solid line shows the reactivity to GST-sialidase, and the dotted line shows the reactivity to GST.
  • the horizontal axis of the graph represents the antiserum dilution rate, and the vertical axis represents the antibody binding activity.
  • FIG. 4 shows the reactivity of immunized rat serum obtained by immunization with E. coli expressed protein in a nodding ELISA.
  • the solid line shows the reactivity to GST-sialidase, and the dotted line shows the reactivity to GST.
  • the horizontal axis of the graph represents antiserum dilution, and the vertical axis represents antibody binding activity.
  • FIG. 5 shows the reactivity of monoclonal antibody binding ELISA.
  • the horizontal axis of the graph indicates the concentration of the purified antibody, and the vertical axis indicates the antibody binding activity.
  • FIG. 6 shows the neutralizing activity of monoclonal antibodies against GST-sialidase.
  • 1 to 3 indicate 1. Fluorescence intensity of fluorescent substrate only 2. Fluorescence intensity of GST-sialidase + fluorescent substrate 3. Fluorescence intensity of GST-sialidase + fluorescent substrate + sialidase inhibitor.
  • the horizontal axis of the right line graph shows the antibody / sialidase molar ratio, and the vertical axis shows the fluorescence intensity (sialidase activity).
  • FIG. 7 shows neutralizing activity of monoclonal antibodies against CHO-producing sialidase.
  • the results when using Ms705 pKAN-ATIII 27 culture supernatant lot 1 as CHO-producing sialidase are shown.
  • 1 to 3 are: 1. Ms705 pKAN-ATIII strain 27 culture supernatant 1 only fluorescence intensity, 2. Ms705 pKAN-ATIII strain 27 culture supernatant lot 1 + fluorescence substrate fluorescence intensity, 3. Ms705 The fluorescence intensity of pKAN-ATIII 27 strain culture supernatant lot 1 + fluorescent substrate + shearidase inhibitor is shown.
  • the horizontal axis of the line graph indicates the antibody concentration, and the vertical axis indicates the fluorescence intensity.
  • FIG. 8 shows neutralizing activity of monoclonal antibodies against CHO-producing sialidase.
  • the results when using Ms705 pKAN-ATIII 27 culture supernatant lot 2 as CHO-producing sialidase are shown. 1 to 3 in the left bar graph 1.
  • Ms705 pKAN-ATIII 27 culture supernatant 2 only fluorescence intensity
  • 3. Ms705 The fluorescence intensity of pKAN-ATIII strain 27 culture supernatant lot 2 + fluorescent substrate + shearidase inhibitor is shown.
  • the horizontal axis of the line graph indicates the antibody concentration, and the vertical axis indicates the fluorescence intensity.
  • FIG. 9 shows the neutralizing effect of monoclonal antibodies on sialidase derived from rat Myoblast cell line (A-10 strain).
  • the horizontal axis of the graph indicates the concentration of the added monoclonal antibody, and the vertical axis indicates the fluorescence intensity derived from the sialidase activity.
  • ⁇ indicates the fluorescence intensity when Neu5 Ac, a sialidase inhibitor, is added at a final concentration of 5 mM
  • ⁇ indicates the fluorescence intensity when mouse plasma-derived immunoglobulin G is added as a negative control
  • ⁇ indicates KM3627.
  • FIG. 10 shows changes in viable cell density during serum-free fed-batch culture of Ms705 ⁇ - ⁇ 27 strain, which is a cocoon-producing cell line.
  • the horizontal axis shows the number of culture days, and the vertical axis shows the viable cell density.
  • X indicates Ms705 pKAN-ATIII 27 strain
  • ⁇ indicates Ms705 ⁇ - ⁇ 27 strain with 10 ⁇ 3629 added.
  • FIG. 11 shows changes in the survival rate of Ms705 ⁇ - ⁇ 27 strain, a sputum producing cell line, during serum-free fed-batch culture.
  • the horizontal axis represents the number of culture days, and the vertical axis represents the viable cell density, viability, and the amount of soot accumulated in the culture supernatant.
  • X indicates Ms705 pKAN-ATIII 27 strain
  • ⁇ indicates Ms705 pKAN-ATIII 27 strain supplemented with 10 M monoclonal antibody KM3629.
  • FIG. 12 shows changes in the amount of sputum accumulated in the culture supernatant during serum-free fed-batch culture of the sputum-producing cell line Ms705 ⁇ ⁇ - ⁇ 27.
  • the horizontal axis represents the number of culture days, and the vertical axis represents the viable cell density, viability, and the amount of soot accumulated in the culture supernatant.
  • X is Ms705 pKAN-ATIII 27 strain
  • ⁇ is Ms705 pKAN- ⁇ 27 strain
  • 10 ⁇ The cases where the null antibody KM3629 was added are shown.
  • FIG. 13 shows changes in sialidase activity during serum-free fed-batch culture of Ms705 ⁇ - ⁇ 27 strain, a ⁇ -producing cell line.
  • a culture supernatant to which no sialidase fluorescent substrate was added was used as the knock ground.
  • the horizontal axis represents the number of days of culture, and the vertical axis represents the viable cell density, viability, and the amount of soot accumulated in the culture supernatant.
  • X represents Ms705 pKAN-ATIII 27 strain
  • ⁇ represents Ms705 pKAN-ATIII 27 strain added with 10 M monoclonal antibody KM3629.
  • FIG. 14 shows the sialic acid content of sputum in the supernatant on day 9 and day 14 of serum-free fed-batch culture of 27 strain Ms705 ⁇ M- ⁇ , which is a sputum producing cell line.
  • FIG. 15 shows changes in the density of viable cells during serum-free fed-batch culture using the spinner bioreactor of Ms705 ⁇ - ⁇ 27 strain, a sputum-producing cell line.
  • the horizontal axis indicates the number of days of culture, and the vertical axis indicates the viable cell density.
  • FIG. 16 Changes in the survival rate of Ms705 ⁇ - ⁇ 27, a ⁇ -producing cell line, during serum-free fed-batch culture using a spinner bioreactor.
  • the horizontal axis represents the number of culture days, and the vertical axis represents the survival rate.
  • FIG. 17 shows changes in the concentration of sputum accumulated in the culture supernatant during serum-free fed-batch culture using the spinner bioreactor of Ms705 ⁇ - ⁇ 27, a sputum-producing cell line.
  • the horizontal axis represents the number of days of culture, and the vertical axis represents the concentration of sputum accumulated in the culture supernatant.
  • FIG. 18 Changes in the sialidase activity in the culture supernatant of the Ms705 ⁇ M- ⁇ 27 strain, a ⁇ -producing cell line, during serum-free fed-batch culture using a spinner bioreactor.
  • As the knock ground a culture supernatant to which no sialidase fluorescent substrate was added was used.
  • the horizontal axis represents the number of days of culture, and the vertical axis represents the fluorescence intensity at an excitation wavelength of 340 and a detection wavelength of 460 nm.
  • FIG. 19 shows the number of sputum sialic acid in the culture supernatant of a sputum-producing cell line, Ms705 ⁇ - ⁇ 27, in a serum-free fed-batch culture using a spinner bioreactor.
  • the horizontal axis represents the number of culture days, and the vertical axis represents the number of sialic acids.
  • FIG. 20 shows the results of measuring the binding mode of cytosolic sialidase neutralizing antibody to GST-sialidase by the surface plasmon resonance method. 20, 10, 5, 2.5, 1.25, 0.625 g / mL KM36 The sensorgram obtained when 27 or KM3629 is added to the sensor chip holding GST-sialidase is shown.
  • FIG. 21 Surface plasmon resonance method for the binding of sialidase neutralizing antibodies to GST-sialidase in the presence of peptides corresponding to the three types of loops protruding from the substrate recognition surface of cytoplasmic sialidase. The measurement results are shown.
  • FIG. 22 shows the results of surface plasmon resonance analysis of the binding of KM3629 to GST-sialidase bound to KM3627 and the binding of KM3627 to GST-sialidase bound to KM3629.
  • the broken line indicates the result when the other antibody is added in advance, and the solid line indicates the result when the other antibody is added in advance.
  • the present invention relates to an anti-cytoplasmic sialidase monoclonal antibody that recognizes the three-dimensional structure of a mammal-derived cytoplasmic sialidase and neutralizes the cytoplasmic sialidase activity derived from a mammal.
  • mammals include humans, mice, rats, hamsters and the like.
  • the cytoplasmic sialidase derived from a mammal may be a cytoplasmic sialidase derived from one type of mammal, but is preferably a cytoplasmic sialidase derived from at least two types of mammals.
  • the cytoplasmic sialidase is an enzyme that exists in the cytoplasm and has an activity to catalyze the reaction of removing sialic acid from the non-reducing end of the sugar chain added to the glycoprotein! / Is included.
  • Non-reducing terminal force of sugar chain added to glycoprotein and present in the cytoplasm as an enzyme having an activity of catalyzing a reaction to remove sialic acid is, for example, producing a glycoprotein using the above-mentioned cells.
  • Examples include NEU2 sialidase.
  • NEU2 sialidase includes the following proteins (a), (b), (c), (d), (e), (1), (g) or a protein encoded by (h) DNA, Or (0, (j), (k), (1), (m), (n), (o), (p), (q) , (R), (s) or (t) protein.
  • (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;
  • 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 fragment thereof.
  • DNA obtained by using the Koguchi-1 'hybridization method or Southern blot hybridization method as a probe.
  • DNA derived from colonies or plaques is immobilized.
  • 0.1-2x concentrated SSC solution composition of 1x concentrated SSC solution is 150mM
  • hybridizable DNA examples include SEQ ID NOs: 1 and 2 DNA having at least 60% homology with the base sequence represented by 3 or 4, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more Particularly preferably 95 DNA having homology of at least%, most preferably at least 98% can be mentioned.
  • the amino acid sequence represented by SEQ ID NO: 5, 6, 7 or 8 has one or more amino acid deletions, substitutions, insertions and / or additions, and has a sialidase activity.
  • Proteins are molecular 'Crowung 2nd edition, current' profile Connorez 'in' molecular ⁇ ⁇ ⁇ Biology, Nucleic 'Acid' Nucleic Acids Research, 10, 6487 (1982), Proceedings 'National' Academic 'Science USA ( proc . Natl. Acad. Sci.) USA, 79, 6409 (1982), Gene, 34, 315 (1985), etc.
  • it means a protein which can be obtained by introducing a site-specific mutation into DNA encoding a protein having the amino acid sequence represented by SEQ ID NO: 5, 6, 7 or 8.
  • 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 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 a 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)]. For the amino acid sequence, 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.
  • G Cost to open gap
  • 11 for amino acid sequence
  • 2 for -E Cost to extend gap
  • -q penalty for nucleotide mismatch
  • 3 r eward for nucleotide match
  • 10 one W (wordsize) force base ⁇ ⁇ ⁇ 11 residues for lj, 3 residues for amino acid residues
  • X Dropoff (X) for blast extemsions in bits) is 20 for blastn, 25 for programs other than blastn (http: ⁇ www.ncbi.nlm.nih.gov/blastcgihelp.html)
  • 0 Software includes FASTA [Methods in Enzymology, 183, 63 (1990)].
  • the three-dimensional structure of cytoplasmic sialidase may be shifted as long as it has a three-dimensional structure equivalent to the three-dimensional structure that cytoplasmic sialidase can take in the natural state.
  • Examples of the monoclonal antibody in the present invention include the following (a) to (c).
  • a fusion antibody comprising (a) or (b).
  • a cell in which the cytoplasmic sialidase is expressed (2) a gene encoding a cytoplasmic sialidase is inserted into an expression vector, and then the expression vector is introduced into a host cell.
  • a cytoplasmic sialidase produced by a host cell is prepared as an antigen, a cell producing an antibody having antigen specificity is induced from an animal immunized with the antigen, and the cell and myeloma cell are fused.
  • Monoclonal antibodies can be obtained by culturing the hybridoma, or by administering the hybridoma to an animal, causing the animal to undergo ascites tumor, and separating and purifying the culture medium or ascites.
  • any animal to be immunized with an antigen any animal can be used as long as it can produce wild and hybridomas, but mouse, rat, wild, muster, rabbit, etc. are preferably used.
  • Antibodies produced by Hypridoma prepared by obtaining cells having antibody-producing ability from such animals, immunizing the cells with iQ vitro and then fusing with myeloma cells, and the like are also included in the antibodies of the present invention. Is included.
  • Hypridoma KM3627 is produced.
  • Hypridoma KM3627 and KM3629 were incorporated into the Patent Organism Depositary (National Institute of Advanced Industrial Science and Technology, Japan) on October 6, 2005, based on the Budapest Treaty. Deposited as FERM BP—10432 and FERM BP 10433!
  • Examples of the recombinant antibody include a human chimeric antibody, a humanized antibody, a human antibody, an antibody produced from a hybridoma or a recombinant antibody! /, An antibody fragment containing any variable region, and the like. Including antibodies produced by genetic recombination.
  • the human chimeric antibody comprises a heavy chain variable region (hereinafter referred to as VH) and a light chain variable region (hereinafter referred to as VL) of an antibody of a non-human animal and a heavy chain constant region (hereinafter referred to as CH) of a human antibody. And an antibody consisting of a light chain constant region (hereinafter referred to as CL).
  • VH heavy chain variable region
  • VL light chain variable region
  • CH heavy chain constant region
  • CL an antibody consisting of a light chain constant region
  • the human chimeric antibody of the present invention obtains cDNA encoding VH and VL from a hyperidoma that produces a monoclonal antibody that binds to cytoplasmic sialidase and recognizes the three-dimensional structure of cytoplasmic sialidase.
  • the CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hlg), but is preferably of the hlgG class, and more preferably hIgGl of the hlgG class, Any of the subclasses such as hIgG2, hIgG3, and hIgG4 can be used.
  • hlg human immunoglobulin
  • hIgG2 hIgG3
  • hIgG4 hIgG4
  • any ⁇ class can be used as long as it belongs to hlg! /, And those of the ⁇ class can be used.
  • a humanized antibody is an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to the appropriate positions of the human antibody VH and VL !,, and CDR-grafted antibody! .
  • the humanized antibody of the present invention produces a monoclonal antibody that binds to cytoplasmic sialidase and recognizes the three-dimensional structure of cytoplasmic sialidase, and the amino acid sequences of CDRs of VH and VL of antibodies from animals other than humans that also produce hybridoma force.
  • the cDNA encoding the V region grafted into the VH and VL FRs of any human antibody is constructed and inserted into the expression vector for animal cells having the genes encoding the CH and CL of the human antibody, respectively.
  • An antibody expression vector is constructed and introduced into animal cells for expression and production.
  • the VH and VL FR amino acid sequences of human antibodies may be any amino acid sequences as long as they are human antibody-derived VH and VL FR amino acid sequences.
  • the CH of the human rabbit antibody is preferably hlgG class if it belongs to hlg! /, But it is preferably of hlgG class, and hlgG1, hIgG2, hIgG3 belonging to hlgG class. HIgG4 and any subclass can be used.
  • the CL of human rabbit antibody may be any of those belonging to hlg, and those of ⁇ class or e class can be used.
  • a human antibody originally refers to an antibody that naturally exists in the human body, but a human antibody phage library prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from antibody-producing transgenic animal power.
  • the antibody naturally occurring in the human body can be cultured by isolating human peripheral blood lymphocytes, infecting and immortalizing EB virus, etc., and cloning the lymphocytes that produce the antibody.
  • the antibody can be purified from Seiyo.
  • One human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting an antibody gene prepared for human B cell force into the phage gene. From the library, phages expressing an antibody fragment having a desired antigen-binding activity on the surface can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule having two complete heavy chains and two complete light chain forces by genetic engineering techniques.
  • a human antibody transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a transgenic non-human animal producing a human antibody is produced by introducing a human antibody gene into a mouse embryonic stem cell, transplanting the embryonic stem cell to an early embryo of another mouse, and generating it. Can do. In addition, human antibody genes are introduced into animal fertilized eggs. It is also possible to produce a transgenic non-human animal that produces human antibodies by generating the fertilized egg. A method for producing a human antibody from a transgenic non-human animal producing a human antibody is carried out in a normal non-human mammal, and a human antibody hyperidoma is obtained and cultured by a method for producing a hyperidoma. Human antibodies can accumulate in the culture.
  • transgenic non-human animals examples include ushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit.
  • antibody fragment of the present invention examples include Fab, F (ab ′), Fab, and scFv.
  • Examples include peptides containing CDRs.
  • Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain), and about half of the N chain side of the H chain and the entire L chain are disulfide. It is an antibody fragment having an antigen binding activity with a molecular weight of about 50,000 bound by binding.
  • the Fab of the present invention can be obtained by treating a monoclonal antibody that binds to cytoplasmic sialidase and recognizes the three-dimensional structure of cytoplasmic sialidase with proteolytic enzyme papain.
  • the DNA encoding the antibody Fab can be inserted into a prokaryotic expression vector or eukaryotic expression vector, and the vector is introduced into prokaryotic or eukaryotic organisms to be expressed and produced. .
  • F (ab ') is the lower part of the two disulfide bonds in the IgG hinge region separated by the enzyme pepsin.
  • F (ab ′) of the present invention binds to cytoplasmic sialidase and is cytoplasmic sialidase.
  • Monoclonal antibodies recognizing the structure of a solid can be obtained by treating with the protease pepsin.
  • the following Fab ′ can be produced by thioether bond or disulfide bond.
  • & has a molecular weight of about 50,000, which is obtained by cleaving the disulfide bond in the hinge region of (&) above.
  • Fab ′ of the present invention can be obtained by treating F (ab ′), which binds to cytoplasmic sialidase and recognizes the three-dimensional structure of cytoplasmic sialidase, with a reducing agent dithiothreitol. Also Is produced by inserting a DNA encoding the Fab ′ fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism. Can do.
  • scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH and one VL are linked using an appropriate peptide linker (hereinafter referred to as P).
  • P an appropriate peptide linker
  • the scFv of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that binds to the cytoplasmic sialidase of the present invention and recognizes the three-dimensional structure of the cytoplasmic sialidase, constructs DNA encoding scFv, It can be produced by inserting DNA into a prokaryotic expression vector or eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • a diabody is an antibody fragment in which scFv is dimerized, and is an antibody fragment having a bivalent antigen-binding activity.
  • the bivalent antigen binding activity can be the same, or one can be a different antigen binding activity.
  • the diabody of the present invention obtains cDNA encoding VH and VL of a monoclonal antibody that binds to the cytoplasmic sialidase of the present invention and recognizes the three-dimensional structure of the cytoplasmic sialidase, and converts the scFv-encoding DNA into the amino acid sequence of P. Is constructed so that its length is 8 residues or less, the DNA is inserted into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector is prokaryotic! Spider can be expressed and produced by introduction into eukaryotes.
  • dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, and bonded via a disulfide bond between the cysteine residues.
  • the amino acid residue to be substituted for the cysteine residue can be selected based on the three-dimensional structure prediction of the antibody according to the method shown by Reiter et al. [Protein Engineering, 7, 697 (1994)].
  • the dsFv of the present invention binds to the cytoplasmic sialidase of the present invention and obtains cDNA encoding the VH and VL of a monoclonal antibody that recognizes the three-dimensional structure of the cytoplasmic sialidase, and constructs a DNA encoding dsFv.
  • the DNA is a prokaryotic expression vector Alternatively, it can be produced by inserting into an eukaryotic expression vector and introducing the expression vector into a prokaryotic or eukaryotic organism.
  • the peptide containing CDR is constituted by including at least one region of CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via an appropriate peptide linker.
  • the peptide containing CDR of the present invention binds to the cytoplasmic sialidase of the present invention and recognizes the three-dimensional structure of cytoplasmic sialidase.
  • a DNA encoding R is constructed, inserted into a prokaryotic expression vector or eukaryotic expression vector, and the expression vector is introduced into prokaryotic or eukaryotic organisms to be expressed from the bottom. can do.
  • the peptide containing CDR is obtained by Fmoc method (fluorylmethyloxycarbol method).
  • the monoclonal antibody of the present invention includes antibodies produced from the above-mentioned hyperidoma, and fusion antibodies in which peptides, proteins, etc. are chemically or genetically bound to recombinant antibodies.
  • the fusion antibody of the present invention binds to the cytoplasmic sialidase of the present invention and recognizes the three-dimensional structure of the cytoplasmic sialidase, the monoclonal antibody H chain or L chain N-terminal side or C-terminal side, monoclonal antibody Chemical methods such as radioisotopes, low molecular weight drugs, high molecular weight drugs, proteins, etc. on appropriate substituents or side chains in sugar chains, as well as sugar chains in monoclonal antibodies, etc. It can be manufactured from Tsujiko by combining with Jinshokan (1994)].
  • a DNA encoding a monoclonal antibody that binds to the cytoplasmic sialidase of the present invention and recognizes the three-dimensional structure of the cytoplasmic sialidase is linked to a DNA encoding the protein to be bound and inserted into the expression vector.
  • the expression vector can be produced by introducing the expression vector into an appropriate host cell for expression.
  • radioisotope examples include 131 ⁇ and 125 ⁇ , and can be bound to an antibody by, for example, the chloramine T method.
  • alkylating agents such as nitrogen mustard and cyclophosphamide, metabolic inhibitors such as 5-fluorouracil and methotrexate, daunomycin, bleomycin, mitomycin C, daunorubicin, doxorubicin and the like.
  • Anticancer agents such as antibiotics, plant anolekaloids such as vincristine, vinblastine, vindesine, hormonal agents such as tamoxifen, dexamethasone [Clinical Oncology, edited by Japanese Society of Clinical Oncology, Cancer and Chemotherapy (1996)], or Hyde mouth cortisone , Steroids such as prednisone, non-steroids such as aspirin and indomethacin, immunomodulators such as gold zomarate and persilamine, immunosuppressants such as cyclophosphamide and azathioprine, chlorfe- maleate Min, anti-inflammatory agents, such as antihistamines such as Kuremashichin etc.
  • daunomycin and antibody can be bound by binding between daunomycin and the amino group of the antibody via dartal aldehyde, or binding the amino group of daunomycin and the carboxyl group of the antibody via water-soluble carpositimide.
  • Examples of the polymer drug include polyethylene glycol (hereinafter referred to as PEG), albumin, dextran, polyoxyethylene, styrene maleic acid copolymer, polybutyropyrrolidone, pyran copolymer, and hydroxypropyl methacrylamide. It is done.
  • a method of conjugating PEG with an antibody includes a method of reacting with a PEGylation modifying reagent [Bioconjugate Pharmaceutical, Yodogawa Shoten (1993)].
  • PEGylation modifying reagents include lysine ⁇ -amino group modifiers (JP-A 61-178926), aspartic acid and glutamic acid carboxyl group modifiers (JP-A 56-23587), arginine gua- Examples thereof include a modifier for a dino group (JP-A-2-117920).
  • Examples of the protein include cyto force-in which activates immunocompetent cells, such as human interleukin 2, human granulocyte macrophage colony stimulating factor, human macrophage colony stimulating factor, and human interleukin 12.
  • toxins such as ricin and diphtheria toxin having an activity of directly damaging cancer cells can be used.
  • a fusion antibody with a protein cDNA encoding the protein is linked to cDNA encoding the antibody or antibody fragment, and DNA encoding the fusion antibody is constructed, and the DNA is used for prokaryotic or eukaryotic organisms. Inserted into an expression vector and the expression vector is either prokaryotic or eukaryotic The fusion antibody can be produced by expressing it in
  • the monoclonal antibody of the present invention include a monoclonal antibody produced by hybridoma KM3627 (FER M BP-10432) and a monoclonal antibody produced by Hypridoma KM3629 (FE RM BP-10433).
  • the present invention also relates to a method for producing a glycoprotein composition, characterized in that, in the method for producing glycoprotein by culturing cells, the above-mentioned monoclonal antibody is added to a culture solution.
  • the monoclonal antibody can be added to the culture medium as long as the host cells that produce the glycoprotein composition are cultured and grown, but the cell viability is high and the state is maintained. It is particularly preferable to add at the beginning of the culture, which is preferably added to.
  • the state of high cell viability is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and most preferably 100%.
  • Adding to the culture medium at the start of culture means adding the above-mentioned monoclonal antibody to the basal medium in advance, or adding the above-mentioned monoclonal antibody to the culture medium containing the seeded cells.
  • the amount of the monoclonal antibody described above added to the medium is such that the final concentration is 0.1 ⁇ g / mL to 100 mg / mL, preferably 1 ⁇ g / mL to 10 mg / mL, more preferably 10 ⁇ g / mL. ⁇ lmg / mL, more preferably 10 ⁇ g / mL to 100 ⁇ g / mL.
  • the cell culture method includes any culture method as long as it is a method capable of producing the target glycoprotein without limitation.
  • methods that can produce the target glycoprotein include batch culture, fed-batch culture, reflux culture, high-density maintenance culture, microcarrier culture, and soft agar culture.
  • a glycoprotein composition according to the present invention wherein the antibody of the present invention is added to a culture solution
  • V production methods are also included.
  • a production method using a host cell into which a gene encoding a glycoprotein has been introduced an embryonic stem cell or a fertilized egg cell of a non-human animal into which a gene encoding a glycoprotein has been introduced into an early stage of a non-human animal
  • Examples include a method of producing using cells of a transgenic non-human animal generated after transplantation into an embryo.
  • a mammal-derived cell is preferably used as long as it can be any cell that can produce a glycoprotein composition.
  • mammalian cells include Chinese nomstar ovarian tissue-derived cells (CHO cells), rat myeloma cell line YB2Z0 cells, mouse myeloma cell line NSO cells, mouse myeloma cell line SP2Z0—Agl4 cells, and Syrian cells.
  • Hamster kidney tissue-derived BHK cells (ATCC CCL-10), MDCK (ATCC CCL-34), PER-C6 TM, human NM-F9 cells, human HEK293 cells, hyperpridoma cells, human leukemia cell line Namaluba cells, embryos Examples include sex stem cells and fertilized egg cells.
  • CHO cells include CHO-K1 strain (ATCC CCL-61), CHOZdhfr- strain (ATCCCRL-9096), Pro5 strain (ATCC CRL-1781), and commercially available CHO-S strain (Invitrigen Cat # 11619). Alternatively, sub-strains obtained by acclimating these cells to a serum-free medium can be used.
  • Examples of rat myeloma cells include Y3 Agl.2.3. (ATCC CRL-1631), Y0 (ECACC N .: 85110501), YB2Z0 (ATCC CRL 1662), and mouse cells such as NS 0 (ATCC CRL-1827). , Sp2 / 0 (ATCC CRL-1581), etc., and myeloma cells or myeloma cell line hybrid cells, or substrains (ATCC CRL-1581.1) in which these strains are conditioned to serum-free medium, etc. .
  • Examples of human cells include PER. C6 (ECACC 96022940), or substrains obtained by acclimating these strains to serum-free medium.
  • cells having properties equivalent to these cells obtained by subjecting these cells to mutation treatment or cell fusion with B cells obtained by immunizing a non-human mammal with an antigen are also available. It is contained in cells derived from mammals.
  • a host cell into which a gene encoding a glycoprotein is introduced includes an N-glycoside-linked complex type sugar chain reducing terminal at the 6th position of N-acetylyldarcosamine.
  • a host cell that is resistant to a lectin that recognizes a sugar chain structure to which the first position of the amino acid is bonded for example, a composition that also has glycoprotein molecular strength having an N-glycoside-linked complex sugar chain, Of all N-glycoside-bonded complex sugar chains contained in glycan, the glycoprotein thread having a sugar chain ratio of 20% or more, wherein fucose is bound to N-acetyl darcosamine at the sugar chain reducing end.
  • Examples of the host cell having the ability to produce an adult compound include cells in which the activity of at least one protein listed below is reduced or inactivated.
  • a host cell in which a gene encoding a1,6-fucosyltransferase in the host cell is knocked out is mentioned (WO02 / 31140, WO03 / 85107, WO03 / 85102).
  • the enzyme protein involved in the synthesis of intracellular sugar nucleotide GDP-fucose includes any enzyme that is involved in the synthesis of sugar nucleotide GDP-fucose, which is the source of fucose to sugar chains in the cell.
  • the Examples of enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose 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 nucleotides 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 is an enzyme protein involved in the sugar chain modification in which the 1-position of fucose is ex-linked to the 6-position of N-acetylyldarcosamine at the reducing end. 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.
  • N-glycosidic bond As an enzyme that affects the reaction in which the 6-position of N-glycidylcolcamine at the N-glycoside-linked complex type sugar chain reducing terminal and the 1-position of fucose are a-bonded, the above-mentioned N-glycosidic bond is used. It affects the activity of the enzyme involved in the reaction of the 1-position of fucose to the 6-position of N-acetyl darcosamine at the reducing end of the complex-type sugar chain, and the structure of the substance that is the substrate of the enzyme Enzymes that provide are also included.
  • 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.
  • a protein that affects the reaction of transporting intracellular sugar nucleotides GDP-fucose into the Golgi apparatus it affects the activity of the proteins involved in the transport of intracellular sugar nucleotides GDP-fucose to the Golgi apparatus. Proteins that give or affect expression are also included.
  • 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.
  • any lectin that recognizes a sugar chain structure in which the 6-position of N-glycidylcolcamine at the reducing end of N-glycoside-linked sugar chain and the 1-position of fucose are a-linked any lectin that can recognize the sugar chain structure is used. Any lectin can be used.
  • Lentil lectin LCA Lientil Agglutinin derived from Lens Culinaris
  • Endumen lectin PS A Porous lectin derived from Lens Culinaris
  • Endumen lectin PS A Porous lectin derived from Lens Culinaris
  • Endumen lectin PS A Porous lectin derived from Lens Culinaris
  • Endumen lectin PS A Porous lectin derived from Lens Culinaris
  • Endumen lectin PS A Pureasum sativum-derived Pea Lectin
  • Broad bean lectin VFA Adgglutin in derived from Vicia faba
  • Hiratiyawantake Examples include lectin AAL (Lectin derived from Aleuria aurantia).
  • 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.
  • the method for producing a glycoprotein composition using cells includes a method of producing using a host cell into which a gene encoding a glycoprotein has been introduced, and a method other than a human into which a gene encoding a glycoprotein has been introduced.
  • a method of producing using a host cell into which a gene encoding a glycoprotein has been introduced includes a method of producing using a host cell into which a gene encoding a glycoprotein has been introduced, and a method other than a human into which a gene encoding a glycoprotein has been introduced.
  • 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 that bind to asparagine N-glycoside-linked sugar chains
  • N-glycoside-linked sugar chains have a variety of structures, such as [Biochemical Experimental Method 23—Glycoprotein Sugar Chain Research Method (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 called the reducing end, and the opposite side is called 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.
  • N-acetylylgalatatosamine is bonded to the hydroxyl group of serine or threonine, and further, galactose, N-acetylyldarcosamine, N-acetylgalato Examples include samine, sialic acid, or a sugar chain to which sialic acid is bonded, a sugar chain in which xylose is bonded to a hydroxyl group of serine and j8, a sugar chain in which galactose is bonded to a hydroxyl group of hydroxylysine and j8.
  • a sugar chain in which xylose is bonded to the hydroxyl group of serine in a / 3 bond usually has a plurality of sugars bonded to the 4-position of the xylose, and a linear polysaccharide consisting of disaccharides is bonded to the end of the bonded sugar. ing.
  • 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 glycoprotein composition obtained by the production method of the present invention has a glycoprotein composition as compared with the glycoprotein composition obtained when the above-mentioned monoclonal antibody is not added to the culture solution.
  • a glycoprotein composition having an increased amount of sialic acid per molecule is compared with the glycoprotein composition obtained when the above-mentioned monoclonal antibody is not added to the culture solution.
  • Physiological activity includes affinity between glycoprotein and receptor, half-life time of glycoprotein in the body, change in tissue distribution after administration in blood, interaction with protein necessary for expression of pharmacological activity, etc. Can be given.
  • the composition is composed of glycoprotein molecules having ⁇ -glycoside-bonded complex type sugar chains, and all ⁇ -glycosides contained in the composition
  • a glycoprotein composition in which the proportion of sugar chains in which fucose is not bound to ⁇ -acetylcylcosamine at the sugar chain reducing end of the combined complex-type sugar chains is 20% or more is even better.
  • 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 N-acetyl at the reducing end of the sugar chain.
  • An antibody composition having a sugar chain in which fucose is bound to darcosamine has high ADCC activity.
  • fucose is bound, and as a sugar chain, fucose is not bound to N-acetyldarcosamine 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.
  • glycoprotein composition of the present invention examples include a glycoprotein composition described later, a glycoprotein fragment thereof, a fusion protein composition obtained by fusing two or more glycoproteins or glycoprotein fragments, and the like.
  • Glycoprotein compositions include antibodies, erythropoietin (EPO) [J. Biol. Chem., 252, 5 558 (1977)], thrombopoietin (TPO) [Nature, 369, 533 (1994)] tissue type Plasminogen activator, prolokinase, thrombomodulin, antithrombin III, a 1 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 clotting factor XII, blood clotting factor XIII, prothrombin complex, fibrinogen, albumin, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin , Bone morphogenetic factor, granulocyte colony stimulating factor (G-CSF
  • V may be an antibody having such 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 Cancer 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.
  • Antibodies that bind to antigens related to allergy or inflammation include anti-interleukin-6 antibody (Immunol. Rev., 127, 5-24, 1992), anti-interleukin-6 receptor antibody (Molecular 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
  • anti-chemokine antibody J. Immunol. Meth., 174, 249-257, 1994
  • anti-chemokine receptor antibody J. Exp.
  • Anti-IgE antibody anti-CD23 antibody, anti-CD11a antibody (Immunology Today, 21 (8), 403-410 (2000)), anti-CRTH2 antibody (J Immunol., 162, 1278-1286 (1999)), anti-CCR8 Examples include antibodies (W099 / 25734) and anti-CCR3 antibodies (US6207155).
  • Examples of antibodies that bind to an antigen associated with cardiovascular disease include anti-GpIIb / IIIa antibody (J. Immuno 1..152, 2968-2976, 1994), antiplatelet-derived growth factor antibody (Science, 253, 1129). -1132, 1991), anti-platelet-derived growth factor receptor antibody (J. Biol. Chem., 272, 17400-17404, 199 7) or anti-blood clotting factor antibody (Circulation, 101, 1158-1164, 2000) Can be mentioned.
  • Antibodies that bind to antigens associated with autoimmune diseases include anti-self DNA antibodies (Immunol. Letters, 72 , 61-68, 2000), anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin ⁇ 4 j8 7 antibody, anti-CD40L antibody, anti-IL-2 receptor And antibodies (Immunology Today, 21 (8), 403-410 (2000)).
  • anti-gpl20 antibody As an antibody that binds to an antigen associated with virus or bacterial infection, anti-gpl20 antibody
  • cytoplasmic sialidase leaked from cultured cells, produced by genetic engineering Cytoplasmic sialidases, and purified samples that retain the sialidase activity of these cytoplasmic sialidases.
  • the cytoplasmic sialidase leaked from the cultured cells is cultured by culturing the cells using a normal cell culture method, accumulating cytoplasmic sialidase in the culture supernatant, and collecting and purifying from the culture solution. Can be prepared.
  • the genetically modified cytoplasmic sialidase can be prepared, for example, as follows using genetic engineering techniques.
  • a cytoplasmic sialidase cDNA or genomic DNA is prepared.
  • a DNA fragment of an appropriate length containing a portion encoding cytoplasmic sialidase or a portion of the non-translated region is prepared.
  • a recombinant vector is produced by inserting the prepared DNA fragment or the 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.
  • Transformants are selected using the mRNA level or activity of the introduced cytoplasmic sialidase as an index.
  • the selected transformant is cultured using a normal cell culture method, cytoplasmic sialidase is accumulated in the culture supernatant, and collected and purified from the culture solution, thereby producing the antibody of the present invention.
  • Cytoplasmic sialidase used as an antigen can be prepared.
  • Any host cell can be used as long as it can express the target cytoplasmic sialidase, such as bacteria, yeast, animal cells, insect cells, and plant cells. Specific examples include host cells described in 4. below.
  • the above host cell can be replicated autonomously! /, Or can be integrated into a chromosome, and contains a promoter at a position where DNA encoding cytoplasmic sialidase can be transcribed. Used. Specifically, it is suitable for expression vectors of the type that are transcribed by DNA polymerase II or various host cells described in 4. below. Recombinant vectors can be used.
  • Examples of methods for obtaining cytoplasmic sialidase cDNA and genomic DNA 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 cytoplasmic sialidase is obtained by PCR using the prepared cDNA library as a cage.
  • a cDNA library can be screened to obtain DNA encoding cytoplasmic sialidase.
  • the human or non-human animal thread or tissue or cell mRNA may be a commercially available product (for example, manufactured by Clontech), and the tissue or cell strength of the human or non-human animal may also be as follows. May be prepared.
  • Examples of a method for preparing mRNA as total RNA poly (A) + RNA include an oligo (dT) -fixed cellulose column method (Molecular 'Cloung 2nd Edition).
  • mRNA can be prepared by using commercially available kits such as Fast Track mRNA Isolation Kit (Invitrogen) and Quick Prep mRNA Purification Kit (Pharmacia).
  • 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.
  • Superscript Plasmid Syste Examples include methods using m for cDNA Synthesis and Plasmid Cloning (manufactured by Life Technologies) and ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE).
  • any phage vector, plasmid vector, etc. can be used as long as it can autonomously replicate in Escherichia coli K12.
  • ZAP Express [Stratagenes, 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), ⁇ ExCell (Pharmacia), pT7 T318U (Pharmacia), pcD2 [Mole. Cell. Biol., 3, 280 (1983)] And 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 '[Stratagies, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [Jananore 'Ob' Molecular ⁇ Mol.
  • cytoplasmic sialidase Based on the amino acid sequence of cytoplasmic sialidase, encoding the amino acid sequence Create degenerative primers specific to the 5 'and 3' end of the predicted nucleotide sequence, and use the cDNA library as a saddle-shaped PCR method [PCR Protocols (PCR Protocols ), Academic Press (1990)], a gene fragment encoding cytoplasmic sialidase can be obtained by performing DNA amplification.
  • the obtained gene fragment is a DNA encoding cytoplasmic sialidase, for example, a commonly used nucleotide sequence analysis method such as Sanger et al.'S dideoxy method [Proceedings' Ob 'The National' Academia ⁇ By analyzing using a base sequence analyzer such as Abb PRISM377 DNA Sequencer (Applied Biosystems), such as ⁇ Ob'Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)] Can be confirmed.
  • a base sequence analyzer such as Abb PRISM377 DNA Sequencer (Applied Biosystems), such as ⁇ Ob'Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)
  • colony hybridization or plaque hybridization (molecular 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 cytoplasmic sialidase DNA.
  • PCR is performed using a cDNA or cDNA library that also synthesizes mRNA force contained in tissues or cells of human or non-human animals as a saddle.
  • the cytoplasmic sialidase cDNA can also be obtained by amplification using this method.
  • the nucleotide sequence of the DNA encoding the obtained cytoplasmic sialidase can be determined by using a commonly used nucleotide sequence analysis method such as Sanger et al.'S dideoxy method [Procedidas' Ob The National Academia ⁇ ⁇ DNA (Proc. Natl. Acad. Sci. USA), 74, 5 463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), etc.
  • the nucleotide sequence of can be determined.
  • a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. Among them, it can be confirmed that the gene encodes cytoplasmic sialidase.
  • the nucleotide sequence of the gene encoding cytoplasmic sialidase obtained by the above method is as follows:
  • cytoplasmic sialidase is synthesized by using a DNA synthesizer such as DNA synthesizer model 392 (manufactured by Perkin Elmer) using the phosphoramidite method. CDNA can also be obtained.
  • Examples of the method for preparing cytoplasmic sialidase genomic DNA include the methods described below.
  • genomic DNA of cytoplasmic 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 cytoplasmic sialidase activity as an index include the following methods.
  • a polyclonal antibody can be prepared by administering the antigen prepared in (1) above to an animal.
  • an antigen having an activity of catalyzing the reaction of removing sialic acid from the non-reducing end of the sugar chain added to the glycoprotein is desirable to use an antigen having an activity of catalyzing the reaction of removing sialic acid from the non-reducing end of the sugar chain added to the glycoprotein.
  • Usagi, goat, rat, mouse, hamster, etc. can be used as an animal to be administered.
  • the dose of the antigen is preferably 50- per animal: LOO ⁇ g.
  • the antigen that is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin. It can be prepared using cell culture technology or gene recombination technology as an antigen.
  • the antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. On the 3rd to 7th day after each administration, blood is collected from the fundus venous plexus and the serum reacts with the antigen used for immunization.
  • the enzyme immunoassay (ELISA): published by the medical school (1976) ), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • a polyclonal antibody can be obtained by obtaining serum from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
  • Separation and purification methods include centrifugation, salting out with 40-50% saturated ammonium sulfate, precipitation of caprinoleic acid (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory (1988)), or DEAE. —Seprose column, anion exchange column, protein A or G column, chromatography using a genore filtration column, etc. can be used alone or in combination.
  • Rats whose serum showed a sufficient antibody titer against the antigen prepared in (1) above used for immunization are used as a source of antibody-producing cells.
  • the spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer. [0109] The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
  • the spleen cells of the obtained precipitate fraction were treated with Tris-salt-ammonium buffer (PH 7.65) for 1-2 minutes to remove red blood cells, then washed with MEM medium 3 times. Spleen cells are used as antibody-producing cells.
  • myeloma cells cell lines obtained from mice or rats are used.
  • 8-azaguanine resistant mouse BALB / c-derived myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol, 81, 1 (1978), Europe J. Immunol., 6, 511 (1976)], SP2 / 0-Agl4 (SP-2) [Nature, 276, 269 (1978)], P3—X63—Ag8653 (65 3) [J.
  • MEM medium or PBS diisodium phosphate 1.83 g, monopotassium phosphate 0.21 g, salt 7.65 g, distilled water 1 Wash well with 1 liter, pH 7.2
  • Cytoplasmic sialidase a genetically modified recombinant that has an activity to catalyze the reaction of removing sialic acid from the non-reducing end of the sugar chain added to glycoprotein Cytoplasmic sialidase or a purified preparation that retains sialidase activity is specific for antigen Select a hyperidoma that responds to.
  • enzyme immunoassay include the following methods.
  • the purified sample while retaining the activity is coated on an appropriate plate, and the hyperpridoma culture supernatant or the purified antibody obtained in (d) described below is reacted as the first antibody.
  • an anti-rat or anti-mouse immunoglobulin antibody labeled with a chemiluminescent substance or a radiation compound is reacted and then reacted according to the labeling substance to react specifically with the antigen used for immunization. Selected as a high-pridoma that produces monoclonal antibodies.
  • cloning was repeated twice by limiting dilution (first time using HT medium (medium excluding HAT medium and aminopterin), and the second time using normal medium. ].
  • HT medium medium excluding HAT medium and aminopterin
  • a strain having a strong and strong antibody titer is selected as a hyperidoma strain producing the monoclonal antibody of the present invention.
  • pristane 2, 6, 10, 14-tetramethylpentadecane (Pristane) O. 5ml intraperitoneally and bred for 2 weeks] 8 ⁇ : To L0 week old mice or nude mice, (c) The obtained 5 monoclonal antibody-producing hybridoma cells 5-20 ⁇ 10 b cells of the present invention are injected intraperitoneally. Hypridoma becomes ascites tumor in 10-21 days.
  • Ascites fluid is collected from the ascites-carcinized mouse and centrifuged at 3000 rpm for 5 minutes to remove solids.
  • the monoclonal antibody can be purified and obtained by the same method as used for polyclonal.
  • the antibody subclass is determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit.
  • the protein mass is calculated from the Raleigh method or absorbance at 280 nm.
  • Recombinant antibody expression vector is an expression vector for animal cells in which DNA encoding human antibody CH and CL is incorporated. DNA encoding human antibody CH and CL in the expression vector for animal cells Can be constructed by crawling each.
  • the C region of a human antibody can be CH and CL of any human antibody, and includes, for example, CH of the ⁇ 1 subclass of human antibody and CL of the ⁇ class.
  • DNA encoding CH and CL of human antibodies it is preferable to use chromosomal DNA consisting of exons and introns, or cDNA which can use cDNA.
  • Any animal cell expression vector can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 (Cytotechnol., 3, 133 (1990)), pAGE103 (i. Biochem., 101, 1307 (1987), pHSG274 (Ge ne, 27, 223 (1984)), pKCR (Proc.
  • the promoter and enhancer used in the expression vector include SV40 early promoter i. Biochem., 1 01, 1307 (1987)], Morro-1 mouse leukemia virus LTR [: Biochem. Biophys. Res. Commun., 149, 960 (1987)], immunoglobulin heavy chain promoter [Cell, 41, 479 (1985) ] And Enhancer 611, 33, 717 (1983)].
  • Recombinant antibody expression vectors can be used in the type in which the antibody H chain and L chain are on separate vectors, or in the same vector (tandem type). However, it is easy to construct a recombinant antibody expression vector, to be introduced into animal cells, and to balance the expression level of antibody H and L chains in animal cells. Recombinant antibody expression vectors are preferred (CF. Immunol. Methods, 167, 271 (1994)). The recombinant antibody expression vector of the tandem type, p KANTEX93 (WO97Zl0354), pEE18 [Hybridoma, 17, 559 (1998)] and the like.
  • the constructed recombinant antibody expression vector can be used to express human chimeric antibodies and humanized antibodies in animal cells.
  • Non-human animal antibodies for example, cDNAs encoding mouse antibody VH and VL can be obtained as follows.
  • mRNA is extracted from a hybridoma cell producing a mouse antibody or the like to synthesize cDNA.
  • the synthesized cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library.
  • a DNA encoding the C region portion or V region portion of the mouse antibody is used as a probe, and a recombinant phage or cDNA containing cDNA encoding VH or VL is isolated.
  • any animal can be used as long as it can produce a high-pridoma cell such as a mouse, rat, mouse, muster, or rabbit.
  • guanidine thiocyanate can also be used.
  • Methods for preparing mRNA from total RNA include oligo (dT) immobilized cellulose cell column method [Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989)].
  • kits for preparing mRNA from high predoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
  • any vector can be used as long as it is a vector into which the cDNA synthesized by using the mRNA extracted from the hyperpridoma cell force as a cocoon can be incorporated.
  • ZAP Express [Strategies, 5, 5 8 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1 989)]
  • ⁇ ZAPII (Stratagene)
  • gtlO gtlO
  • l gtl l DNA Cloning: A Practical Approach, I, 49 (1985)]
  • Lambda BlueMid (Clontech)
  • X ExCell pT7T3 18U (Pharmacia)
  • pcD2 [Mol. Cell. Biol., 3, 280 (1983) )]
  • pUC18 Gene, 33, 103 (1985)].
  • any one can be used as long as it can introduce, express and maintain the cDNA library.
  • XL 1-Blue MRF [Strategies, 5, 81 (1 992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778 (1983)], NM522 Q1. Mol Biol., 166, 1 (1983)], K802 Q [. Mol. Biol., 16, 118 (1966)] 3 ⁇ 4U iM105 [Gene, 38, 275 (1985)]
  • cDN encoding VH or VL of non-human animal antibody from cDNA library A clone can be selected by using a collodium hybridization method or a plaque hybridization method using an isotope or fluorescently labeled probe [Molecula r lonmg, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989)]. In addition, a primer is prepared and a cDNA or cDNA library synthesized from mRNA is used as a cage.
  • PCR method Polym erase chain reaction
  • the cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger. (Sanger, F.) et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] and the like, and a base sequence automatic analyzer such as an ALF DNA sequencer (Pharmacia).
  • a plasmid such as pBluescript SK (-) (Stratagene)
  • a commonly used nucleotide sequence analysis method such as Sanger. (Sanger, F.) et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] and the like
  • a base sequence automatic analyzer such as an ALF DNA sequencer (Pharmacia
  • amino acid sequences of CDRs of VH and VL are compared with those of known antibodies VH and VL [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Can be found. [0129] Furthermore, any database using the complete amino acid sequences of VH and VL, for example, BLAST method for SW ISS-PROT, PIR-Protein, etc. ⁇ . Mol. Biol., 215, 403 (1990)] etc. The novelty of the sequence can be examined by performing a homology search of the sequences.
  • each of the cDNAs can be cloned to construct a human chimeric antibody expression vector.
  • each cDNA encoding VH or VL of a non-human animal antibody may be obtained by using the nucleotide sequence of VH or VL of a non-human animal antibody and the 5 'end of CH or CL of a human antibody.
  • a recombinant antibody expression vector according to (1) of this section 2 which is ligated to a synthetic DNA comprising a base sequence on the side and having an appropriate restriction enzyme recognition sequence at both ends, respectively.
  • a human chimeric antibody expression vector can be constructed by cloning each of the genes encoding CH or CL upstream so that they are expressed in an appropriate form.
  • cDNAs encoding the non-human animal antibodies VH or VL were amplified by PCR using synthetic DNAs with appropriate restriction enzyme recognition sequences at both ends, respectively. It can also be cloned into the recombinant antibody expression vector described in 1.
  • a cDNA encoding the humanized antibody VH or VL can be constructed as follows. First, the amino acid sequence of the VH or VL framework region (hereinafter referred to as FR) of the human antibody to be grafted with the VH or VL CDR amino acid sequence of the target non-human animal antibody is selected. Any amino acid sequence derived from a human antibody can be used as the amino acid sequence of VH of human antibody or FR of VL. For example, human amino acid VH or VL FR amino acid sequences registered in databases such as Protein Data Bank, human antibody VH or VL FR subgroup consensus amino acid sequences [sequences of Proteins of Immunological Interest, US Dept.
  • the designed amino acid sequence is converted into a DNA sequence in consideration of the frequency of use of cotton in the nucleotide sequence of the antibody gene [sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].
  • Based on the designed DNA sequence several synthetic DNAs with a length of around 100 bases are synthesized, and PCR is performed using them. In this case, it is preferable to design six synthetic DNAs for both the H and L chains, based on the PCR reaction efficiency and the length of DNA that can be synthesized.
  • the recombinant antibody expression vector constructed in (1) of this section 2 can be easily humanized.
  • CDNA encoding the VH or VL of the conjugated antibody can be cloned.
  • the amplified product is cloned into a plasmid such as pBluescript SK (-) (Stratagene), the nucleotide sequence is determined by the method described in (2) of this section 2, and the desired DNA sequence is determined.
  • a plasmid having a DNA sequence encoding the amino acid sequence of the VH or VL of the antibody is obtained.
  • Humanized antibodies can be obtained by transplanting only the VH and VL CDRs of the target non-human animal antibody into the VH and VL FRs of the human antibody, and their antigen-binding activity is higher than that of the original non-human animal antibody. [BIOZTECHNOLOGY, 9, 266 (1991)]. This is because, in the VH and VL of the original non-human animal antibody, not only CDR but also some amino acid residues of FR are directly or indirectly involved in antigen binding activity. Residual force It is considered that VH and VL of human antibodies change to different amino acid residues with the SCDR transplantation. In order to solve this problem, humanized antibodies have anti-antibodies directly within the FR amino acid sequences of VH and VL of human antibodies.
  • Modification of FR amino acid residues of human antibody VH and VL can be achieved by performing the PCR method described in (4) of this section 2 using the synthetic DNA for modification. Determine the nucleotide sequence of the amplified product after PCR by the method described in (2) of this section 2 and confirm that the target modification has been made.
  • the cDNA encoding the humanized antibody VH or VL constructed upstream of each gene encoding CH or CL of the human antibody of the recombinant antibody expression vector described in (1) of this section 2 Each can be cloned to construct a humanized antibody expression vector.
  • the recombinant antibody expression vector described in (3) and (6) of this section 2 or a modified expression vector thereof can be used for transient expression of the recombinant antibody.
  • the host cell into which the expression vector is introduced can be used in any cell that can express a recombinant antibody. Because of its high expression level, COS-7 cells (ATCC CRL 1651) Commonly used [Methods in Nucleic Acids Res., CRC press, 283 (1991)].
  • Methods for introducing expression vectors into COS-7 cells include the DEAE-dextran method [Methods in Nucleic Acids Res., CRC press, 283 (1991)], the lipofuxion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • ELIS A method Monoclonal Antibodies—Principles and practice, Third edition , Academic Press, 996 996, Antibodies——A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] and the like.
  • Examples of a method for introducing an expression vector into a host cell include the electopore position method [Japanese Patent Laid-Open No. 2-257891, Cytotechnology, 3, 133 (1990)].
  • any host cell capable of expressing the recombinant antibody can be used.
  • Host cells with reduced or deleted activity of proteins such as enzymes involved in sugar chain modification in which position 1 of oc binds, or proteins involved in transport of intracellular sugar nucleotides GDP-fucose to the Golgi apparatus (WO02 / 31140, WO03 / 85107, WO03 / 8510 2) and the like can also be used.
  • G418 sulfate (hereinafter referred to as G418: SIG MA) according to the method disclosed in JP-A-2-57891.
  • the transformed strain can increase the expression level of the humanized antibody using a dhfr amplification system or the like according to the method disclosed in JP-A-2-257891.
  • Recombinant antibodies can be purified from the culture supernatant of transformants using a protein A column.
  • other purification methods usually used for protein purification can be used. For example, it can be purified by combining gel filtration, ion exchange chromatography and ultrafiltration.
  • the molecular weight of the purified recombinant antibody H chain, L chain, or whole antibody molecule is expressed by polyacrylamide gel electrophoresis [hereinafter SDS-PAGE: Nature, 227, 680 (1970)] or Western blotting. [Monoclonal Antibodies ⁇ Principles and prac tic e, Third edition, Academic Press (1996), Antibodies ⁇ A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
  • the binding activity of the purified antibody or antibody fragment of the present invention with the antigen is determined by ELISA or fluorescent antibody method [Cancer Immunol. Immunother., 36, 373 (1993)] or surface plasmon resonance using BIAcore TM. It can be measured.
  • the enhancement of fluorescence generated when sialidase decomposes sialidase light ⁇ ' ⁇ 4-methylumbelliferyl-N--D-acetylneuraminic acid ammonium salt is used as an index.
  • a method of measuring the rate of inhibition by addition of an antibody or antibody fragment [Glycobiology vol. 3, 455-463 (1993), BIO / TECH NOLOGY vol. 13, 692 (1995)] can be used.
  • the medium supplemented with the antibody of the present invention can be prepared using a medium used for cell culture as a basal medium.
  • BME medium Proc. Soc. Exp. Biol. Med., 89, 362, 1965
  • BGJb medium Exp. Cell Res., 25, 41, 1961
  • CMRL 1066 medium NY Academy of) Sciences, 5, 303, 1957
  • Glasgow MEM medium Virology, 16, 147, 1962
  • Improved MEM Zinc Opt ion medium J. National Cancer Inst., 49, 1705, 1972
  • IMDM medium In Vitro, 9 , 6, 1970
  • Medium 199 medium Proc. Soc. Exp. Biol.
  • a medium added with serum a medium added with various growth factors as serum substitutes, a medium added with factors produced by stromal cells, or a protein-free medium may be added to these media. If the cells can be cultured, embryos can be used.
  • serum-free medium supplemented with commercially available KNOCKOUT TM SR (MD Goldsborough et al .; Focus, 20, 8, 1998)
  • serum-free medium supplemented with insulin and transferrin eg, CHO-S-SFM II (GIBCOBRL), Hybridoma- SFM (GIBCO BRL), eRDF Dry Powdered Media (GIBCOBRL), UltraCULTURE TM (Bio Whittaker), UltraDOMA TM (BioWhittaker), UltraCHO TM (BioWhittaker) ), UltraMDCK TM (BioWhittaker), ITPSG medium (S. Hosoi et al .; Cytotechnology, 5, S17, 1991), ITSFn medium (A. Rizzino and C.
  • mN3 medium S. Okabe et al .; Mech. Dev., 59, 89, 1996) etc.
  • medium supplemented with cell-derived factors eg, pluripotent teratocarcinoma cell culture supernatant of PSA1 Added medium (GR Martin; Proc. Natl. Acad. Sci. USA, 78, 7634, 1981) or protein-free medium (eg CD-CHO (GIBCOBRL), PFHM-II (GIBCOBRL) UltraDOM A-PF TM (manufactured by BioWhittaker) and the like.
  • cell-derived factors eg, pluripotent teratocarcinoma cell culture supernatant of PSA1 Added medium (GR Martin; Proc. Natl. Acad. Sci. USA, 78, 7634, 1981
  • protein-free medium eg CD-CHO (GIBCOBRL), PFHM-II (GIBCOBRL) UltraDOM A-PF TM (man
  • basal media include EXCELL302 (manufactured by JRH Biosciences) and CHO-S-SFM IKGIBCOBRL).
  • concentration of each additive contained in the IMDM medium may be changed to 100 times to 1/100, preferably 10 times to 1/10 of the above concentration. ! /
  • the antibody of the present invention is added to these basal media at a concentration of 0.1 ngZml to 100 ⁇ g / mU, preferably lng ⁇ 1 to 10 / ⁇ gZml, more preferably 10 ngZml to 1 ⁇ gZml.
  • An added medium can be prepared.
  • an antibody that neutralizes the activity of the cytoplasmic sialidase of the present invention can be added as a 10- to 100-fold concentrated solution during cell culture without directly adding it to the basal medium.
  • a feed medium in which the antibody of the present invention is dissolved may be used, or a solution in which the antibody of the present invention is dissolved in an appropriate buffer may be used.
  • the glycoprotein composition of the present invention is produced by adding the monoclonal antibody of the present invention prepared in 1 and 2 above to a culture solution and culturing cells that produce the target glycoprotein composition. Can do.
  • glycoprotein composition is obtained from Molecular 'Crowing 2nd Edition, Current' Protocols 'in' Molecular 1 ⁇ Nylon 1 ⁇ , Antibodies, A Laboratory manual, old Spring Harbor.
  • antibody engineering Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press, 1993 (hereinafter abbreviated as “monoclonal nanoreantibodies”), Antibody Engineering, A Practical Approach, IRL Press Using the method described in at Oxford University Press, 1996 (hereinafter abbreviated as “antibody engineering”), for example, it can be obtained by expressing in a host cell as follows.
  • 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.
  • Examples of host cells include cells derived from mammals.
  • cells derived from mammals into which an enzyme related to sugar chain modification has been introduced using genetic engineering techniques can also be used as host cells.
  • 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.
  • cDNA can be prepared from a tissue or cell of a human or non-human animal using a probe or primer specific to the target glycoprotein. it can.
  • examples of expression vectors include pcDNAU pcD M8 (sold by Funakoshi), pAGE107 [JP 3-22979; Cytotechnology, 3, 133, (1990)] , PAS3-3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 32 9, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGElO 3 [Journal ' C. Biochemistry, 101, 1307 (1987)], pAGE210, and the like.
  • 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 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 [JP 2-227075]
  • lipofussion Law Proceedings ⁇ The ⁇ National ⁇ Academic ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ Mouse Embryo Laboratories Manual]
  • Method Using Particle Gun (Gene Gun) [Patent No. 2606856, Patent No.
  • the transformant obtained as described above is cultured, the target glycoprotein composition is produced in the culture supplemented with the monoclonal antibody of the present invention, and the isoprotein composition is collected from the culture. By doing so, the intended glycoprotein composition can be produced.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing host cells.
  • the RPMI 1640 medium [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-Transgenic • How to make a mouse (Kodansha) Motoya Katsaki (1987) Hota can use media such as fetal calf serum added to these media.
  • the culture is usually carried out for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C, and 5% CO.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
  • a method for producing a glycoprotein composition a method of producing a glycoprotein composition in a host cell, Secreted or produced on the outer membrane of the host cell.
  • a more appropriate method can be selected by changing the host cells used and the structure of the glycoprotein molecule to be produced.
  • 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.
  • a glycoprotein composition produced by a transformant into which a gene encoding a glycoprotein molecule has been introduced is, 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 Using methods such as electrophoresis such as electrophoresis alone or in combination A purified preparation of the glycoprotein composition can be obtained.
  • a solvent extraction method e.g., a salting-out method using ammonium sulfate, a desalting method, a precipitation method
  • 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. After the solubilized solution is diluted or dialyzed to return the glycoprotein composition to a normal three-dimensional structure, a purified preparation of the glycoprotein composition can be obtained by the same isolation and purification method as described above. I'll do it.
  • 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 a glycoprotein molecule, the cell is cultured and the desired glycoprotein composition is purified from the culture. Thus, a glycoprotein composition can be produced.
  • the sugar chain structure of a glycoprotein molecule created using various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein. For example, neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetylyldarcosamine, and acidic sugars such as sialic acid are analyzed for sugar structure using the sugar composition analysis and two-dimensional sugar chain mapping method. Etc. can be used.
  • 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.
  • composition analysis of the glycan of a glycoprotein is performed by acid hydrolysis of the glycan with hydrochloric acid or sulfuric acid to release the acidic saccharide and label the released acidic saccharide to analyze the composition ratio.
  • the monosaccharides that make up the sugar chains of glycoproteins can be broadly divided into sialic acid, neutral sugars, and amino sugars.
  • the stability of released monosaccharides against acids is the order of amino acids, neutral sugars, and sialic acids. Since sialic acid is completely decomposed under conditions that cleave the glycosidic bond of amino sugar and neutral sugar, it is usually analyzed separately.
  • a sample hydrolyzed according to a known method [Chem. Pharm. Bull. 35, 687 (1987); Anal. Biochem. 179, 162-166 (1989)], for example, is commercially available.
  • L 2-diamino-4,5-methylenedioxybenzene (DMB) fluorescently labeled using a reagent kit for fluorescent labeling of sialic acid (manufactured by Takara Shuzo Co., Ltd.) and the composition ratio can be calculated by HPLC analysis it can.
  • DMB 2-diamino-4,5-methylenedioxybenzene
  • the glycoprotein composition is hydrazine-degraded to release the sugar chain from the glycoprotein molecule, and fluorescent labeling of the sugar chain 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. Then sorted Normal phase chromatography is performed for each peak of the sugar chain.
  • ⁇ 2-aminoviridine
  • 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 mapping method.
  • the purified glycoprotein is labeled with a compound such as a radioisotope, and the strength of the binding reaction with the labeled glycoprotein receptor or interacting protein is quantitatively measured. How to do. It is also possible to measure protein-protein interactions using various devices such as Biacore's BIAcore series (J. Immnunol. Methods, 145, 229 (1991), Biomedical Biomedical Supplement, UP Series Protein Intermolecular). Interaction experiment method, Yodosha (1996)).
  • the half-life in blood or the distribution to the tissue after blood administration can be known.
  • a method for detecting the labeled substance is used.
  • a system that combines the antibody-antigen reaction specific to the glycoprotein to be detected is preferred.
  • glycoprotein composition of the present invention has a sugar chain structure with high sialic acid modification, for example, improved affinity with receptors, improved blood half-life, improved tissue distribution after administration in blood In addition, an effect such as improvement of interaction with a protein necessary for expression of pharmacological activity can be expected, and high physiological activity is exhibited.
  • the glycoprotein composition of the present invention has a longer blood half-life than the glycoprotein composition obtained when the antibody of the present invention is not added to the culture broth and cultured in sputum. .
  • These highly bioactive glycoprotein compositions are useful in the prevention and treatment of various diseases including cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, or viral or bacterial infections. It is.
  • an anticancer agent comprising a conventional glycoprotein composition as an active ingredient is characterized by suppressing the growth of cancer cells.
  • an anticancer agent comprising a glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long blood half-life and can treat cancer with a sustained cell killing effect. It is more effective as a therapeutic agent than an anticancer agent comprising the glycoprotein composition.
  • glycoprotein compositions that are effective components of anticancer agents include interferon, tumor necrosis factor, and interoral chickins.
  • an immunological disease therapeutic agent comprising the glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long half-life in blood and can treat an immune disease due to a sustained mediator molecule inhibitory effect. so Therefore, it is more effective than conventional therapeutic agents for immune diseases containing glycoprotein as an active ingredient.
  • the glycoprotein composition that is an active ingredient of a therapeutic drug for immune diseases include TNF receptor p75IgGlFc fusion protein and antithrombin III.
  • Cardiovascular diseases include diseases associated with thrombosis, diseases associated with bleeding such as hemophilia, and diseases associated with decreased renal function.
  • a conventional antithrombotic drug comprising a glycoprotein composition as an active ingredient is characterized by inhibiting the blood coagulation system or activating the fibrinolysis system.
  • an antithrombotic therapeutic agent comprising the glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long blood half-life and can treat hemorrhage by a continuous anticoagulant action. Therefore, it is more effective than anti-thrombotic therapeutic drugs that use conventional glycoprotein as an active ingredient.
  • glycoprotein compositions that are active ingredients of antithrombotic drugs include antithrombin III, active protein C, and thrombomodulin.
  • a therapeutic agent for hemophilia comprising a conventional glycoprotein composition as an active ingredient is characterized by activating the blood coagulation system or inhibiting the fibrinolytic system.
  • a therapeutic agent for hemophilia comprising the glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long blood half-life and treats hemophilia by a continuous blood coagulation action. Therefore, it is more effective than the conventional hemophilia drug containing glycoprotein as an active ingredient.
  • the glycoprotein composition that is an active ingredient of a therapeutic agent for hemophilia include various blood coagulation factors.
  • Examples of the disease accompanied by a decrease in renal function include renal anemia.
  • a conventional therapeutic agent for renal anemia comprising a glycoprotein composition as an active ingredient is characterized by having an erythrocyte increasing action.
  • a therapeutic agent for renal anemia comprising the glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long blood half-life and can treat renal anemia by a continuous erythrocyte increasing action. Therefore, it is more effective than the conventional therapeutic drugs for renal anemia containing glycoprotein as an active ingredient.
  • Erythropoietin is an example of a glycoprotein composition that is an active ingredient of a therapeutic agent for renal anemia.
  • An anti-infective therapeutic agent comprising a conventional glycoprotein composition as an active ingredient has a function of suppressing the growth of cells infected with bacteria or viruses.
  • an anti-infective drug comprising a glycoprotein composition obtained by the production method of the present invention as an active ingredient has a long blood half-life and is a persistent cell infected with bacteria or viruses.
  • the growth of Infectious diseases can be treated by its inhibitory action, so it is more effective than conventional infectious agents for treating infectious diseases containing glycoproteins as effective components.
  • glycoprotein yarns that are active ingredients of infectious disease therapeutic agents include interferons and interleukins.
  • the medicament containing the glycoprotein composition of the present invention can be administered alone as a prophylactic or therapeutic agent.
  • one or more pharmacologically acceptable carriers and one or more carriers are used. It is desirable to mix them together and provide them as pharmaceutical preparations produced by any method well known in the pharmaceutical arts!
  • the route of administration can be oral administration where it is desirable to use the most effective treatment, or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • intravenous administration can be preferably mentioned.
  • dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes, and the like.
  • Suitable formulations 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 a 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 a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • preparations suitable for parenteral administration include injections, suppositories, and sprays.
  • 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, body 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 examining the biological activity of glycoprotein compositions include, in vitro experiments, methods for measuring glycoprotein receptor binding activity, methods for measuring enzyme activity of glycoprotein compositions, and glycoprotein receptor expression. 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.
  • PCR reaction CHO / DG44 cell-derived-25 ⁇ L reaction solution containing 5 i uL of single-stranded cDNA [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP, s, 0.5 units EX Taq polymerase (Manufactured by Takara Shuzo Co., Ltd.), 0.5 ⁇ of the above primers (SEQ ID NO: 12 and SEQ ID NO: 13)] were prepared and heated at 94 ° C.
  • coli DH5a strain (Toyobo Co., Ltd.).
  • Recombinant plasmid DNA was isolated from the obtained ampicillin resistant colonies using QIAprep Spin Miniprep Kit (Qiagen).
  • the nucleotide sequence of the PCR fragment contained in the plasmid was determined according to a conventional method using DNA Sequencer ABI PRISM 377 (manufactured by Perkin Elma Co., Ltd.), and it was confirmed that there was no base mutation associated with PCR.
  • the obtained plasmid containing NEU2 sialidase cDNA derived from Chinese nomstar ovary cells (CHO) is called pT7 blue sialidase.
  • a vector pGEX-Sialidase for expressing a dartathione S transferase (GST) fusion sialidase protein was prepared by the following procedure. Primer specific to the 5'-terminal untranslated region of cytoplasmic sialidase using the pT7 blue sialidase prepared in (1) above as a saddle type BamHIsiaFW (SEQ ID NO: 14) and a primer specific to the 3'-terminal untranslated region siaRVSall Perform polymerase chain reaction (PCR) using (SEQ ID NO: 15) No, NEU2 sialidase cDNA was amplified.
  • GST dartathione S transferase
  • BamHIsiaFW encodes the start codon in the Chinese and Muster cytoplasmic sialidase gene sequences shown in SEQ ID NO: 1.
  • the 187th force also corresponds to the 210th base, while siaRVSall encodes the stop codon from the 1303 position.
  • the sequence is complementary to the 1326th base.
  • PCR using this primer amplifies the entire cytoplasmic sialidase cDNA that can be inserted into the vector in a state where the codon frame matches that of GST.
  • PCR reaction is 50 ⁇ l reaction solution containing pT7 blue sialidase lng [1 X KOD Buffer (Toyobo), 0.2 mM dNTP, s, ImM MgCl, 2.5 units
  • the DNA fragment was ligated to the pGEX-KG vector using Ligation high (Toyobo Co., Ltd.), and the resulting recombinant plasmid DNA was used to transform E. coli DH5a strain (Toyobo Co., Ltd.).
  • pGEX-KG is a GST fusion protein expression vector modified from the multicloning site of pGEX-2T (manufactured by Pharmacia) (Analytical Biochem. (1991) 192, 262-267).
  • Recombinant plasmid DNA was isolated from the resulting ampicillin metacolonies using QIAprep Spin Miniprep Kit (Qiagen).
  • the nucleotide sequence of the PCR fragment contained in the plasmid was determined according to a conventional method using DNA Sequencer ABI PRISM 377 (manufactured by Perkin Elma Co.), and it was confirmed that there was no base mutation associated with PCR.
  • the resulting plasmid is called pGEX-Sialidase.
  • the gene sequence of GST-sialidase encoded by this plasmid is shown in SEQ ID NO: 16.
  • E. coli DH5a carrying the plasmid pGEX-Sialidase prepared in (2) above was inoculated into 2 mL of LB medium (Difco) and cultured at 37 ° C. The next day, 1 mL of the culture solution was inoculated into 500 mL of LB medium and swirl culture was performed at 37 ° C. When the absorbance at 600 nm reached 0.6, isopropyl- ⁇ -thiogalactopyranoside (IPTG) (manufactured by Takara Bio Inc.) was added at a final concentration of 0. ImM, followed by further overnight culture at room temperature. The next day, the cells are collected by centrifugation.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the suspension was suspended in 10 mL of B-PER Bacterial Protein Extraction Reagent (Pierce), and then disrupted 3 times for 10 minutes with an ultrasonic crusher (Sonics).
  • the E. coli lysate obtained by the above operation was centrifuged at 12,000 rpm for 20 minutes using an R-12A2 rotor (manufactured by Hitachi, Ltd.), and the supernatant was recovered and pre-treated with B-PER Bacterial Protein.
  • Glutathion Sepharose 4 Fast Flow manufactured by Falumasia
  • 1 mL washed with Extraction Reagent was added and mixed by inverting at 4 ° C for 8 hours. After 8 hours, the mixed solution of E.
  • coli lysate and Glutathion Sepharose 4 Fast Flow was transferred to a 0.8 cm diameter column, and then washed twice by passing 10 mL of PBS containing 0.1% TritonX-100 twice. After washing, 1 mL of elution buffer (50 mM Tris-HC1 pH 9.5, 10 mM reduced glutathione) was added, and GST-sialidase bound to Glutathion Sepharose 4 Fast Flow was eluted. The elution fraction was dialyzed by replacing the buffer with PBS, and the protein concentration was calculated by measuring the absorbance at 280 nm.
  • elution buffer 50 mM Tris-HC1 pH 9.5, 10 mM reduced glutathione
  • Fig. 1 shows an image of 2 g of the resulting GST-fused sialidase separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue (CBB).
  • CBB Coomassie brilliant blue
  • GST-sialidase O.lmg or O.Olmg obtained in (3) above was added to 0.4 mM sialidase light ⁇ ' ⁇ 4-methylumbelliferyl-N--D-acetylneuraminic acid ammonium salt (manufactured by Naito Lightesta). It was added to 60 ⁇ L of phosphate buffer ( ⁇ 7.0) containing 5 ⁇ L, and incubated at 37 ° C for 1 hour in a 96-well black ⁇ LISA plate (manufactured by Sumitomo Bakelite).
  • the value obtained by dividing the concentration of 4-methylumbelliferyl (4MU), which shows the same fluorescence value as the value obtained by subtracting the background fluorescence value from the fluorescence value obtained by this measurement, by the reaction time (h) reaction volume (mL) (nmol / h / ml) is shown in FIG. 2 as sialidase activity.
  • the concentration-dependent sialidase activity of GST-sialidase was confirmed. These activities disappeared by the addition of a casease inhibitor. Since the prepared GST fusion sialidase has sialidase activity, it was confirmed to be a recombinant protein retaining the structure necessary for activity.
  • E. coli-expressed GST-sialidase 50 ⁇ g obtained in Example 1 was added to each of aluminum hydroxide and aluminum 7-Nuunt Antibodies-A Laboratory Manual, old Spring Harbor Laboratories, p99, 1988] 2 mg and pertussis vaccine (Chiba prefectural serum Laboratory Ltd.) 1 X 10 9 cells together with the 6 Shureimesu Balb / c mice were administered by 4 Shureimesu SD rats each 3 rats. From 2 weeks after administration, 50 g of GST-sialidase was administered once a week for a total of 3 times.
  • Fig. 3 shows the binding ELISA results of the immunized mouse antiserum
  • Fig. 4 shows the binding ELISA results of the immunized rat antiserum.
  • the antisera of mouse No.2, mouse No.3, rat No.2, and rat No.3 immunized with GST-sialidase were more effective in the GST-sialidase solidified plate than in the GST solidified plate. A high reactivity was confirmed, and it was confirmed that a polyclonal antibody against cytoplasmic sialidase was produced.
  • Example 2 The mouse (or rat) spleen cell obtained in (1) and the myeloma cell obtained in Reference Example 1 (6) were mixed at a ratio of 10: 1, and described in Reference Example 1 (7) below. A high pre-dorman was prepared according to the above method.
  • mice Seven-week-old female nude mice (ICR) treated with pristane were injected intraperitoneally with 5-20 ⁇ 10 6 cells / mouse of the mouse hybrid obtained in Example 2 (2). Ten to 21 days later, ascites collected in the abdominal cavity was collected from mice in which the hyperidoma became ascites cancer. Ascitic fluid was collected from 1 to 8 mL per animal.
  • Binding activity (binding ELISA)
  • Example 2 It was carried out according to the method shown in Reference Example 1 (4). However, the purified antibodies (KM3627, KM3628, KM3629, KM3630, and KM3631) obtained in Example 2 (3) were diluted to 10, 1, 0.1, 0.01, and 0.001 / zg / mL as the primary antibody. A thing was used. As shown in FIG. 5, all purified antibodies showed specific binding activity to GST-sialidase.
  • Example 2 the purified antibody obtained in Example 2 (3) was used in place of the immunized animal antiserum and Hypridoma culture supernatant.
  • the purified antibody was diluted with GST-sialidase so that the molar ratio was 100, 10, 1, 0.1, 0.01.
  • neutralization activity against GST-sialidase was observed with all purified antibodies.
  • Example 2 ⁇ Production Using the culture supernatant of CHO cells, it was confirmed whether the purified antibody obtained in Example 2 (3) neutralized the cytoplasmic sialidase that also leaked the cell force.
  • a As Till-producing CHO cells the sputum-producing cell line Ms705 pKAN-ATIII 27 (FERM BP-10089) described in WO2005 / 035563 was used.
  • Ms705 pKAN-ATIII strain 27 was cultured in the medium, and the culture supernatant on day 14 of culture was used for the neutralizing activity against leaked sialidase in the method shown in Reference Example 1 (5) Measured according to
  • the culture supernatant on the 14th day of culture is the culture solution on the 14th day of the fed batch culture in which the above-mentioned sputum producing cells were subjected to a spinner reactor (manufactured by ABLE) having a culture tank volume of 1 L.
  • Fig. 7 (Lot 1) and Fig. 8 (Lot 2) show the results of measurement using the obtained culture supernatant in different lots. In all experiments, neutralizing activity against CHO-producing sialidase was observed with all purified antibodies.
  • the cells were cultured in a medium (Invitrogen) and grown to confluence. After the cells were detached from the incubator by trypsin treatment, the cells were washed with Dulbecco's PBS buffer (Invitrogen). A portion of the washed cell suspension was collected, and the viable cell density was measured using a cell auto-counter ViCELL (manufactured by Beckman Coulter) to confirm that the cell viability was 95% or more. .
  • Washed cells are uniformly suspended in 150 ⁇ L Dulbecco's PBS buffer with 1.25 x 10 6 viable cells, dispensed into microcentrifuge tubes (Eppendorffne earth), and frozen at -80 ° C overnight. I let you. The next day, the microcentrifuge tube was left at room temperature for 30 minutes to thaw the cell suspension. After the cell suspension was completely thawed, it was centrifuged at 15000 rpm at 4 ° C for 15 minutes using a microcentrifuge (manufactured by TOMY). The supernatant cell lysate was collected in a new microcentrifuge tube and stored on ice.
  • the final concentrations of the five monoclonal antibodies were 100 / zg / ml, 1 ⁇ g / ml, and O ⁇ g / m1.
  • 50 ⁇ L of cell lysate supplemented with 10 mM phosphate buffer (pH 7.0) was added to the sialidase inhibitor ⁇ -Acety ⁇ 2,3-dehydro- 2-deox y.
  • -Wells supplemented with neuraminic acid (Neu5AC2en) (Fluka) to a final concentration of 5 mM were prepared.
  • sialidase fluorescent substrate 4-methylumbelliferyl—N——D-acetylneuraminic acid ammonium salt prepared at a concentration of 4 mM was added to each well of the 96-well microwell plate at 5 L / well.
  • the solution was dispensed, a lid was placed on the plate, and the mixture was reacted at 37 ° C for 2 hours in a carbon dioxide incubator (manufactured by TABAI).
  • Serum-free fed-batch culture was carried out by using the Ms705 pKAN-ATIII strain 27 established in Example 3 by the following procedure.
  • MTX manufactured by SIGMA
  • L-glutamine manufactured by Invitrogen
  • SIGMA 3,3,5- Triiodo-L-thyronine
  • EX- CELL302 medium containing 100 nmol / L Pluronic F-68 (Invitrogen) at a concentration of 0.1%
  • D (+)-glucose Nacalai Testa
  • JRH serum-free fed-batch culture medium
  • Amino acids (L-alanin 0.177 g / L, L-arginine, prepared at a higher concentration than the usual addition concentration) Monohydrochloric acid 0.593g / L, L-asparagine monohydrate 0.177g / L, L-aspartic acid 0.212g / L, L-cystine dihydrochloride 0.646g / L, L-glutamic acid 0.530g / L, L-glutamine 5.84 g / L, glycine 0.212 g / L, L-histidine monohydrochloride dihydrate 0.297 g / L, L-isoleucine 0.742 g / L, L-leucine 0.742 g / L, L-lysine monosalt Acid 1.031g / L, L-methionine 0.212g / L, L-Feralan 0.466g / L, L-proline 0.283g / L, L-serine 0.297g /
  • the solution was diluted 100 times with 1 L of distilled water), and dispensed into a 96-well plate (Nunc) at 100 ⁇ L / well.
  • the mixture was allowed to stand at room temperature for 1 to 2 hours, and the inside of the well was washed with 0.05% Tween20-PBS, and then 1% BSA-PBS was dispensed with 100 / well and stored frozen.
  • Specimens and standards with known concentrations were diluted to an appropriate concentration using 1% BSA-PBS, dissolved at room temperature, and BSA-PBS in the tool was removed.
  • the solution was dispensed with L / well and allowed to stand at room temperature for 1-2 hours.
  • the sputum detection antibody solution was dispensed at 100 ⁇ L / well and allowed to stand at room temperature for about 1 hour.
  • 0.05% Tween20-PBS was injected several times at 350 L / well to wash the well. Dispense the ABTS solution containing 0.1% H 0 with 50 ⁇ L / well to develop the color.
  • Color development was stopped by dispensing 50% L / well of% SDS solution. Using a microplate reader, the absorbance was measured at a wavelength of excitation 415 nm / absorption 490 and the soot content was calculated.
  • Example 5 (1) 10 ⁇ g / mL of the monoclonal antibody ⁇ 3629 was added to the initial culture medium of the ⁇ -producing cell line and the Ms705 pKAN-ATII I 27 strain, and the Erlenmeyer flask was added. Serum free batch culture was performed. On day 0, 3, 6, 9, 12, and 14 after the start of culture, collect 2 to 4 mL of the culture solution, and the viable cell density (cells / mL), viability, ⁇ Concentration (mg / mL) and sialidase activity were measured. In addition, the number of sialic acids contained at the sugar chain end bound to the produced koji was measured using the culture solution on day 9 and day 14 of the culture.
  • the viable cell density and viability are the dye exclusion method using 0.4% trypan blue solution (manufactured by Invitrogen), and the soot concentration is the soot amount described in this section (2).
  • the sialidase activity was determined by the method for measuring the sialidase activity described in Reference Example 1 (5), and the sialic acid content was measured using anion exchange HPLC (Shimadzu Corporation).
  • Fig. 10 shows the viable cell density
  • Fig. 11 shows the survival rate
  • Fig. 12 shows the production volume
  • Fig. 13 shows the sialidase activity in the culture supernatant
  • Fig. 14 shows the sialic acid content.
  • the monoclonal antibody KM3629 has the effect of suppressing the elimination of sialic acid from sputum by neutralizing intracellular sialidase activity.
  • Serum-free fed-batch culture was performed using the Ms705 pKAN-ATIII strain 27 established in Example 3 in the same manner as described in Example 5 (1).
  • Ms705 pKAN-ATIII 27 strain was suspended in a serum-free Fuedbachi culture medium so that the viable cell density was 2.5 ⁇ 10 5 cells / mL, and the cell suspension was seeded in a 1 L spinner bioreactor.
  • Serum-free fed-batch culture was performed under conditions of a temperature of 35 ° C. and a stirring speed of 85 rpm, and KM3629 prepared in Example 2 was added at a final concentration of 1 mg / mL on the third day after the start of the culture. Performed on the 3rd, 5th, 7th, 9th, and 11th days after the start of the culture to reduce the consumption of amino acids, etc.
  • Fig. 15 shows the viable cell density
  • Fig. 16 shows the survival rate
  • Fig. 17 shows the production volume
  • Fig. 18 shows the sialidase activity in the culture supernatant
  • Fig. 19 shows the number of sialic acids.
  • the sialidase activity in the culture supernatant was suppressed throughout the 14-day culture period.
  • Figure 19 It was confirmed that the number of sialic acids in the cocoons produced was high!
  • the above indicates that monoclonal antibody ⁇ 3629 suppresses sialic acid elimination from glycoproteins by neutralizing sialidase activity even in culture using spinner reactors that mimic actual industrial production methods. Show that it is effective.
  • the affinity of the anti-sialidase antibodies ⁇ 3627 and ⁇ 3629 obtained in Example 2 for sialidase as an antigen was measured using a surface plasmon resonance method (SPR method). Analysis by the SPR method was performed as follows using a BiacoreTlOO system (Biacore). First, an anti-GST antibody was immobilized on flow cell 1 and flow cell 2 of Biacore S series sensor chip CM5 so that the binding amount was 13,000 RU. GST Capture Kit (Biacore) was used for the solid phase.
  • the dissociation constant for the binding of each antibody to GST-sialidase was calculated from a curve obtained by plotting the binding equilibrium value (RU value) at each antibody concentration against the antibody concentration. The results are shown in FIG. Biacore T100 evaluation software (Biacore) was used for the calculation. Further, Ka (l / Ms) is KM3267 is 2.27 X 10 5, KM3629 is 2.08 X 10 5, Kd (l / s) is KM3267 is 2.45 X 10, KM3629 was 2.22 X 10- 6. Therefore, KM3627 and KM3629 have 1.1 X 10- 9 M Contact and 1.1 X 10- 11 M dissociation constant (KD), that are attached to Shiaridaze with very high affinity becomes bright et force It was.
  • KD dissociation constant
  • Asp L-aspartic acid
  • Asx L-aspartic acid or L-asparagine
  • Glx L-glutamic acid or L-glutamine
  • Trt Trityl
  • Fmoc-Ser (t-Bu) -OH Na-9-fluorenylmethyloxycarbonyl-O-t-butyl-L-serine
  • Fmoc-Thr (t-Bu) -OH N a—9-funoleolenyl methenoreoxycarbonyl—O—t—butyl—L-thread
  • Fmoc- Lys (Boc)-OH N -9-Fluoro-rumethyloxycarbonyl- N ⁇ -1-butinoreoxycarbonyl-L-lysine
  • Fmoc- Asn (Trt)-OH N a -9-Fluorenylmethyloxycarbonyl- N ⁇ -tritinole- L-asno lagin
  • Fmoc-Asp (0-t-Bu) -OH Na-9-fluorenylmethyloxycarbol-L-aspartic acid- ⁇ -t-butyl ester
  • Fmoc-Glu (0-t-Bu) -OH N ⁇ —9—Fluoreninoremethinoreoxycarbo-nore—L-gnoletamic acid- ⁇ -t-butyl ester
  • Fmoc- Arg (Pmc)-OH N a -9-Fluorenylmethyloxycarbonyl- Ng- 2, 2,5, 7,8- Pentamethylchroman-6-sulfol- L-arginine
  • Fmoc-Tyr (t-Bu) -OH N ⁇ —9—Fluorore-noremethinoreoxycanolepool—O—t—Butinore— L-tyrosine
  • reaction solvents reaction reagents, and the like.
  • the ⁇ 1 and ⁇ 2 regions are not fixed in the structure of apo-type Neu2 to which no substrate is bound, and are considered to have a relatively random and flexible structure.
  • the structure was confirmed in the body, suggesting that it is an important site for sialidase activity, which changes its structure by substrate binding. From this, it was considered that it is suitable as an antigen site aiming at neutralization activity of sialidase.
  • the structure of the third domain loop has been confirmed for both the apo type and the substrate complex type, and the force is clearly protruding to the protein surface force side of the substrate recognition surface. The possibility of sterically hindering the substrate from entering the active center was suggested.
  • SEQ ID NO: 9 is an amino acid sequence in which a Cys residue is added to the N-terminus of the amino acid sequence corresponding to amino acid sequence Nos. 37 to 52 of the cheese hamster Neu2 for the purpose of binding to a resin used for synthesis.
  • SEQ ID NO: 10 is the amino acid sequence corresponding to amino acid sequence numbers 106 to 125 of Chinese Noom Star Neu2.
  • SEQ ID NO: 11 is an amino acid sequence corresponding to amino acid sequence numbers 180 to 196 of Chineseno and Muster Neu2.
  • step (d) Fmoc- Asp (0-1-Bu) -OH, Fmoc-Ala-OH, Fmoc-His (Trt) -OH, Fmoc-Glu (0-t-Bu) -OH, Fmoc — Asp (0— t— Bu) — OH, Fmoc— Thr (t— Bu) — OH, Fm oc- Lys (Boc)-OH, Fmoc- Thr (t- Bu)-OH, Fmoc- Leu- OH, Fmoc- Arg (Pmc) -OH, Fmoc- Lys (Boc) -OH, Fmoc-Glu (0-1-Bu) -OH, Fmoc-Ala-OH, Fmoc-Phe-OH ), (E), (b), (c) were repeated in this order.
  • KLH Keyhole limpet hemo cianin; manufactured by Wako Pure Chemical Industries
  • PBS PBS
  • MBS N- (m-Maleimidobenzoyloxy) succinimide
  • Peptide lmg obtained by dissolving 2.5 mg of KLH-MBS in 0.1 M sodium phosphate buffer (PH7.0) after removing free MBS with a gel filtration column such as Sephadex G-25 column previously equilibrated with PBS And stirred for 3 hours at room temperature. After the reaction, dialyzed with PBS was used as an immunogen.
  • PH7.0 sodium phosphate buffer
  • the spleen was chopped in a MEM (Minimum Essential Medium) medium (manufactured by Nissui Pharmaceutical), loosened with a pin set, and centrifuged (1200 rpm, 5 minutes). The resulting precipitate fraction was supplemented with Tris monosalt diamine buffer (PH7.6) and treated for 1 to 2 minutes to remove erythrocytes. The resulting precipitate fraction (cell fraction) was washed 3 times with MEM medium and used for cell fusion.
  • MEM Minimum Essential Medium
  • PH7.6 Tris monosalt diamine buffer
  • the conjugates 1 to 3 obtained in Reference Example 1 (1) were each conjugated with thyroglobulin.
  • the preparation method is as described in Reference Example 1 (2), but SMCC [4- (N-Maleimidomethyl) -cyclyl is used as a cross-linking agent instead of MBS. ohexane-1-carboxylic acid N-hydroxysuccinimidoester; manufactured by Sigma Co.] was used.
  • the conjugate prepared as described above was dispensed into a 96-well ELISA plate (manufactured by Grainer) at 10 ⁇ / 1 ⁇ 2 50 L / well and allowed to stand at 4 ° C. for adsorption.
  • GST-sialidase is added to a 96-well ELISA plate (Grainer Co., Ltd.) at 5 ⁇ g / mL, 50 ⁇ L / It was dispensed in holes and left to stand overnight at 4 ° C for adsorption.
  • BSA bovine serum albumin
  • PBS Dulbecco phosphate buffer
  • Tween-PBS Peroxidase-labeled Usagi anti-mouse (or rat) immunoglobulin
  • ABTS 2.2-azinobis (3-ethylbenzothiazole-6-sulfonic acid) ammonium] substrate solution [lmmol / L ABTS — 0.1 mol / L citrate buffer (pH 4 .2), 0.1% H2O2]
  • the color was developed, and the absorbance at OD415 nm was measured using a plate reader (Emax; manufactured by Molecular Devices).
  • Sialidase 3 ⁇ 4: Photosubstrate 0.4 mM 4-methylumbelliferyl-N- — D—acetylneura minic acid ammonium salt (Nacalai Testa) 5 ⁇ L / well, incubated at 37 ° C for 1 hour, 0.2 M glycine (pH 10) was dispensed at 140 L / well to stop the reaction. Reaction stop After stopping, using a fluorescent plate reader ARVO (manufactured by Perkin Elmer), the intensity of the 460 nm fluorescence wavelength with an excitation wavelength of 340 nm was measured for 0.1 second. The value obtained by dividing the background fluorescence value from the fluorescence value obtained by this measurement was defined as the sialidase activity.
  • the mouse spleen cells obtained in Reference Example 1 (3) and the myeloma cells obtained in Reference Example 1 (6) were mixed at 10: 1 and centrifuged (1200 rpm, 5 minutes). After loosening the cells of the obtained precipitate fraction, mix 10 8 solutions of polyethylene glycol-1000 (PEG-lOOOO) lg, MEM medium lmL, and dimethyl sulfoxide 0.35 mL at 37 ° C with stirring. 0.5 mL per mouse spleen cell was added, and 1 mL of MEM medium was added several times to the suspension every 1 to 2 minutes, and then the MEM medium was added so that the total volume became 50 mL.
  • PEG-lOOOO polyethylene glycol-1000
  • an anticytoplasmic sialidase monoclonal antibody that recognizes the three-dimensional structure of a cytoplasmic sialidase derived from a mammal and neutralizes the cytoplasmic sialidase activity derived from a mammal, the antibody in a culture solution It is possible to provide a method for producing a glycoprotein composition characterized by being added. Sequence listing free text

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Abstract

L'invention concerne un anticorps monoclonal anti-sialidase cytoplasmique capable de reconnaître la conformation et de neutraliser l'activité d'une sialidase cytoplasmique de mammifère. Elle concerne également un procédé utilisant cet anticorps pour produire une composition glycoprotéinique comprenant une chaîne sucre à laquelle de l'acide sialique est rattaché.
PCT/JP2007/055633 2006-03-20 2007-03-20 Anticorps dirigé contre la sialidase cytoplasmique de mammifère WO2007108464A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008120801A1 (fr) 2007-04-02 2008-10-09 Kyowa Hakko Kirin Co., Ltd. Procédé de production de composition d'antithrombine
JP2013136530A (ja) * 2011-12-28 2013-07-11 National Center For Global Health & Medicine Ｉｌ−２８ｂの分析方法
JP2019530664A (ja) * 2016-09-08 2019-10-24 ザ テキサス エー アンド エム ユニバーシティ システム 抗線維症シアリダーゼ阻害剤化合物および使用の方法
EP3994275A4 (fr) * 2019-07-03 2023-11-29 Palleon Pharmaceuticals Inc. Sialidases humaines recombinantes, protéines de fusion de sialidases et leurs procédés d'utilisation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026908A1 (fr) * 1993-05-17 1994-11-24 Genentech, Inc. Sialidase de cellule de cho obtenue par technique de recombinaison d'adn
US6528286B1 (en) * 1998-05-29 2003-03-04 Genentech, Inc. Mammalian cell culture process for producing glycoproteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026908A1 (fr) * 1993-05-17 1994-11-24 Genentech, Inc. Sialidase de cellule de cho obtenue par technique de recombinaison d'adn
US6528286B1 (en) * 1998-05-29 2003-03-04 Genentech, Inc. Mammalian cell culture process for producing glycoproteins

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CROSS A.S. ET AL.: "Recruitment of Murine Neutrophils in Vivo through Endogenous Sialidase Activity", J. BIOL. CHEM., vol. 278, no. 6, 2003, pages 4112 - 4120, XP003018026 *
GRAMER M.J. ET AL.: "Removal of sialic acid from a glycoprotein in CHO cell culture supernatant by action of an extracellular CHO cell sialidase", BIO/TECHNOLOGY, vol. 13, no. 7, 1995, pages 692 - 698, XP002033147 *
GU X. ET AL.: "Site- and branch-specific sialylation of recombinant human interferon-gamma in Chinese hamster ovary cell culture", BIOTECHNOL. BIOENG., vol. 55, no. 2, 1997, pages 390 - 398, XP003018025 *
HASEGAWA T. ET AL.: "Molecular Cloning of Mouse Ganglioside Sialidase and Its Increased Expression in Neuro2a Cell Differentiation", J. BIOL. CHEM., vol. 275, no. 11, 2000, pages 8007 - 8015, XP002214721 *
MIYAGI T. ET AL.: "Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase", J. BIOL. CHEM., vol. 268, no. 35, 1993, pages 26435 - 26440, XP003018024 *
MONTI E. ET AL.: "Cloning and characterization of NEU2, a human gene homologous to rodent soluble sialidases", GENOMICS, vol. 57, no. 1, 1999, pages 137 - 143, XP004444960 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008120801A1 (fr) 2007-04-02 2008-10-09 Kyowa Hakko Kirin Co., Ltd. Procédé de production de composition d'antithrombine
JP2013136530A (ja) * 2011-12-28 2013-07-11 National Center For Global Health & Medicine Ｉｌ−２８ｂの分析方法
JP2019530664A (ja) * 2016-09-08 2019-10-24 ザ テキサス エー アンド エム ユニバーシティ システム 抗線維症シアリダーゼ阻害剤化合物および使用の方法
EP3994275A4 (fr) * 2019-07-03 2023-11-29 Palleon Pharmaceuticals Inc. Sialidases humaines recombinantes, protéines de fusion de sialidases et leurs procédés d'utilisation

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