JP2005224240A - Immortalized cell line derived from non-human knockout animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide cells capable of carrying out a functional analysis of a sugar chain with a sugar chain structure without fucose modification and capable of producing a sugar protein having sugar chain structure without fucose modification and having a high physiological activity. <P>SOLUTION: The invention relates to the immortalized cell line derived from non-human knockout animal, which is a knockout genome gene of enzyme associated with sugar chain modification bonding to form an α-bonding on 1st position of fucose to 6th position of N-acetylglucosamine on the N-glycoside linkage complex type sugar chain reducing end. The invention relates to the transformant of the immortalized cell line, use of the immortalized cell line and method for preparing thereof. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention was established from a non-human animal in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar reducing end is knocked out. The immortalized cell line and a transformant of the cell line. The present invention also relates to the use and production method of the immortalized cell line.

Sugar chains modify membrane proteins and secreted proteins and control their functions. That is, the function of extracellular proteins involved in signal transduction in vivo in mammals is controlled by modification of sugar chains, and sugar chains play a physiologically important role.
A sugar chain is synthesized by an enzyme that changes the structure of a sugar chain added to a glycoprotein. Examples of enzymes that change the structure of sugar chains added to glycoproteins include β1,4-N-acetylglucosaminyltransferase III (hereinafter also referred to as GnTIII), α1,6-fucosyltransferase (hereinafter also referred to as FUT8), etc. These glycosyltransferase genes are introduced or deleted into cells to produce glycoproteins with altered sugar chain structures of the produced glycoproteins. The effect is analyzed (Non-Patent Document 1). Specifically, an enzyme that changes the structure of a sugar chain added to a glycoprotein is known to affect cell growth. In PC12 cells, a rat adrenal medulla-derived brown cell with enhanced expression of GnTIII that catalyzes the reaction of adding N-acetylglucosamine to the trimannose core β-mannose of the N-glycoside-linked glycan, growth factor receptor By reducing the function of (TrkA), the cell proliferation activity derived from nerve growth factor (NGF) decreases (Non-patent Document 2).

  In addition, as a technique for changing the structure of a sugar chain, there is a technique for culturing cells by adding an inhibitor of an enzyme that affects the structure of a sugar chain added to a glycoprotein, and analyzing the synthesized sugar chain. It is known that in cells cultured with tunicamycin, an inhibitor of sugar chain modification, the signal transduction system of epidermal growth factor receptor (EGFR) becomes abnormal (Non-patent Document 1). Furthermore, it is known that abnormalities in the structure of sugar chains that are also modified in the insulin receptor affect cell proliferation (Non-patent Document 3). As described above, since many of the growth factor receptors involved in cell proliferation are affected by the modified sugar chain structure, the expression change of the enzyme group that changes the structure of the sugar chain added to the glycoprotein is Affects the growth of living organisms.

In addition, glycosyltransferases may have multiple enzymes that catalyze the same transfer reaction, and complex sugar chain modifications performed by multiple glycosyltransferases delicately regulate protein functions.
In mammals, FUT8 is known as the only enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal. The enzyme is thought to play an important role in the living body, but its function is still unclear.

The structure of the FUT8 gene (Non-patent Document 4) involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycosidic complex reducing terminal N-acetylglucosamine was revealed in 1996 (Non-patent document 5, Non-patent document 6, Patent document 1). Although the enzyme activity of FUT8 has been confirmed in many organs, it is relatively high in the brain and small intestine (Non-patent Documents 7 and 8). As a physiological function, it has been pointed out that a fucose-modified sugar chain plays an important role in retinal formation, and its relationship with the expression regulation of FUT8 has attracted attention (Non-Patent Document 9). The role of FUT8 derived from platelets is also drawing attention in blood coagulation (Non-patent Document 10). It has also been reported that the modification of fucose to the sugar chain structure of immunoglobulin IgG1 affects the binding to FcγRIIIa, and the antibody-dependent cytotoxic activity of the antibody itself is changed (Non-Patent Document 11, Non-Patent Document 11). Reference 12). In relation to the pathological condition, an increase in FUT8 activity and an increase in the ratio of the enzyme reaction product have been observed in several diseases such as liver cancer and cystic fibrosis (Non-Patent Document 13, Non-Patent Document 14). ). In FUT8 overexpressing transgenic mice, steatosis-like degeneration in the liver and kidney is observed (Non-patent Document 15). In addition, in mice lacking the gene encoding FUT8, most individuals grow poorly and die within 2 to 3 weeks after birth (Non-patent Documents 16 and 17). Therefore, the individual of a mouse deficient in the FUT8 gene has poor cell growth, and no cell line derived from the mouse has been established so far.
WO92 / 27303 Molecular Medicine, 40, 1034 (2003) J. Biol. Chem., 272, 9629 (1997) J. Biol. Chem., 267, 17415 (1992) Biochem. Biophys. Res. Commun., 72, 909 (1976) J. Biol. Chem., 271, 27817 (1996) J. Biochem., 121, 626 (1997) Int. J. Cancer, 72, 1117 (1997) Biochim. Biophys. Acta., 1473, 9 (1999) Glycobiology, 9, 1171 (1999) Biochem. Soc. Trans., 15, 603 (1987) J. Biol. Chem., 277, 26733 (2002) J. Biol. Chem., 278, 3466 (2003) Hepatology, 13, 683 (1991) Hepatology, 28, 944 (1998) Glycobiology, 11, 165, (2001) GlycoT2002, Third Internatioal Symposiumon Glycosyltransferases, Djuronaset, Stockholm, September 19-22, 2002, A MarcusWallenberg Symposium Mysterious Life beyond Genome Information, Glycan, 2003

  An object of the present invention is to provide a non-human animal in which a genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. It is to provide a more established immortal cell line and a transformant of the cell line. Another object of the present invention is to provide a use and production method of the immortalized cell line.

The present invention relates to the following (1) to (25).
(1) Established from a non-human animal in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-binding complex-type sugar chain reducing end is knocked out. Immortalized cell line.
(2) The immortalized cell line according to (1), wherein the non-human animal is a mouse.
(3) All the alleles on the genome of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-binding complex sugar chain reducing end were knocked out. The immortalized cell line according to (1) or (2).

(4) N-glycoside-linked complex type sugar chain N-acetylglucosamine at the 6-position of N-acetylglucosamine is located in the exon region including the start codon of the genomic gene of the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked. The immortalized cell line according to any one of (1) to (3), which is deleted.
(5) The enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is α1,6-fucosyltransferase. The immortalized cell line according to any one of (4) to (4).
(6) The immortalized cell line according to (5), wherein α1,6-fucosyltransferase is a protein encoded by a DNA selected from the following (a) or (b):
(a) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity.

(7) The immortalized cell line according to (5), wherein α1,6-fucosyltransferase is a protein selected from the group consisting of the following (a) to (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 6;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity.
(8) A transformant in which a gene encoding a glycoprotein is introduced into the immortalized cell line of any one of (1) to (7).
(9) The transformant according to (8), wherein the glycoprotein is an antibody.

(10) The transformant according to (9), wherein the antibody is selected from the group consisting of the following (a) to (d).
(a) a human antibody;
(b) a humanized antibody;
(c) an antibody fragment comprising the Fc region of (a) or (b);
(d) A fusion protein having the Fc region of (a) or (b).
(11) The transformant according to (9) or (10), wherein the antibody class is IgG.
(12) A method for producing a glycoprotein, comprising the steps of: culturing the transformant according to (8) in a medium; producing and accumulating glycoprotein in the culture; collecting the glycoprotein from the culture; .
(13) The transformant according to any one of (9) to (11) is cultured in a medium, the antibody composition is produced and accumulated in the culture, the antibody composition is collected from the culture, The manufacturing method of an antibody composition including the process to refine | purify.

(14) A glycoprotein or antibody composition in which the N-glycoside-linked complex sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain is produced. The method according to 12) or (13).
(15) The method according to (14), wherein the N-glycoside-linked complex type sugar chain is a sugar chain in which the 1-position of fucose is not α-bonded to the 6-position of N-acetylglucosamine at the reducing end of the sugar chain. .
(16) A glycoprotein produced using the method according to any one of (12), (14) or (15).
(17) An antibody composition produced using the method according to any one of (13) to (15).

(18) A medicament comprising the glycoprotein according to (16) as an active ingredient.
(19) A medicament comprising the antibody composition according to (17) as an active ingredient.
(20) The drug is a diagnostic, prophylactic or therapeutic agent for diseases involving tumors, diseases involving allergies, diseases involving inflammation, cardiovascular diseases, autoimmune diseases, diseases involving viral infections or diseases involving bacterial infections. The pharmaceutical according to (18) or (19).
(21) Use of the glycoprotein according to (16) for producing the medicament according to (18) or (20).
(22) Use of the antibody composition according to (17) for producing the medicament according to (19) or (20).

(23) A method for establishing an immortalized cell line according to any one of (1) to (7), comprising the following steps (a) to (c):
(a) An organ from a non-human animal in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is knocked out Extraction step;
(b) making the extracted organ a non-aggregated cell population;
(c) A step of culturing a non-aggregated cell population.

(24) A method for establishing an immortalized cell line according to any one of (1) to (7), comprising the following steps (a) to (d):
(a) An organ from a non-human animal in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is knocked out Extraction step;
(b) making the extracted organ a non-aggregated cell population;
(c) introducing an oncogene into a non-aggregated cell population;
(d) A step of culturing a cell population into which an oncogene has been introduced.

(25) The method for establishing an immortalized cell line according to (23) or (24), wherein the organ is an organ selected from the group consisting of the following (a) to (d).
(a) liver;
(b) kidney;
(c) bone marrow;
(d) Fetus.

  Glycoprotein that is subjected to fucose modification by an enzyme involved in sugar chain modification in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end by the immortalized cell line of the present invention An unmodified form of fucose can be obtained. The obtained glycoprotein itself is useful as a drug or as a target molecule of a drug.

In the immortalized cell line of the present invention, the genomic gene of the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. Any immortalized cell line or a sub-line of the immortalized cell line is included as long as it is an immortalized cell line established from a non-human animal.
In the present invention, examples of non-human animals include non-human animals that can use genetic recombination techniques, such as mice, rats, cows, goats, and sheep, but mice are preferably used.
In the present invention, the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is defined as N-glycoside-linked complex sugar chain reduction. Any enzyme is included as long as it is an enzyme involved in the α-bonding reaction between the terminal 6-position of N-acetylglucosamine and the 1-position of fucose. Specific examples of enzymes involved in the α-bonding reaction between N-acetylglucosamine 6-position and N-acetylglucosamine 1-position of fucose at the N-glycosidic complex type sugar chain reducing terminal include α1,6-fucosyltransferase (FUT8) Can be given.
In the present invention, the α1,6-fucosyltransferase includes the protein encoded by the following DNA (a) or (b) or the following protein (c), (d) or (e).
(a) DNA comprising the base sequence represented by SEQ ID NO: 5
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity
Or
(c) a protein comprising the amino acid sequence represented by SEQ ID NO: 6
(d) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6, and having α1,6-fucosyltransferase activity
(e) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity

  Further, DNA encoding the amino acid sequence of α1,6-fucosyltransferase includes DNA having the base sequence represented by SEQ ID NO: 5 and DNA having the base sequence represented by SEQ ID NO: 5 under stringent conditions. Examples thereof include DNA that encodes the amino acid sequence of a protein that hybridizes and has α1,6-fucosyltransferase activity.

  In the present invention, the DNA that hybridizes under stringent conditions is, for example, a colony hybridization method, a plaque using DNA such as DNA having the base sequence represented by SEQ ID NO: 5 or a fragment thereof as a probe. -This means DNA obtained by using the hybridization method or Southern blot hybridization method, specifically, in the presence of 0.7-1M sodium chloride using a filter on which colony or plaque-derived DNA is immobilized. After hybridization at 65 ° C, the filter is used at a temperature of 65 ° C using a 0.1 to 2-fold SSC solution (composition of 1-fold SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). DNAs that can be identified by washing can be listed. Hybridization is described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning Second Edition), Current Protocols in Molecular Biology, John Wiley & Sons, 1987-1997 (hereinafter referred to as Current). (Abbreviated as Protocols in Molecular Biology), DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) and the like. Specifically, the DNA capable of hybridizing is DNA having at least 60% homology with the base sequence represented by SEQ ID NO: 5, preferably 70% or more, more preferably 80% or more, and still more preferably 90%. As mentioned above, DNA having homology of 95% or more, most preferably 98% or more is particularly preferable.

  In the present invention, a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity is: Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci USA, 82, 488 (1985), etc. The protein which can be acquired by introduce | transducing a site-specific mutation into DNA which codes the protein which has the amino acid sequence represented by 6. 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, and insertion can be performed by a known technique such as the above-described site-directed mutagenesis. And / or a number that can be added, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.

  In the present invention, a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity is BLAST [J. Mol. Biol., 215 , 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], etc., and preferably at least 80% or more with the protein having the amino acid sequence set forth in SEQ ID NO: 6 Means 85% or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.

The immortalized cell line of the present invention includes an immortalized cell line established from a mouse in which the above-described enzyme genomic gene is knocked out or a sub-strain of the immortalized cell line.
In the present invention, the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. It means introducing a mutation into the expression regulatory region of the gene so as to delete the expression, or introducing a mutation into the amino acid sequence of the gene so as to delete the function of the enzyme. Introducing a mutation means modifying the base sequence such as deletion, substitution, insertion and / or addition to the base sequence on the genome. A specific example in which a genomic gene is knocked out is an example in which all or part of the target gene is deleted from the genome.

The non-human animal in which the genomic gene of the above enzyme is knocked out includes any non-human animal produced by any method as long as the genomic gene of the target enzyme is knocked out. .
In the immortalized cell line of the present invention, the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. Any cell line that is immortalized from a non-human animal is included. Establishment of an immortalized cell line requires a collective cell population, and in order to obtain a collective cell population, it is preferable to use a non-human animal organ or the like for the establishment of an immortalized cell line.

  As the organ of the non-human animal, any organ or tissue containing cells capable of establishing an immortalized cell line is included. Specifically, epithelial tissue, connective tissue, adipose tissue, cartilage tissue, bone tissue, muscle tissue, oral tissue, blood tissue, bone marrow, circulatory organ, lymphoid organ, tooth, digestive tract, liver, gallbladder, pancreas, breathing Organs, urinary organs, genital organs, endocrine organs or sensory organs are used, preferably liver, kidney or genital organs, more preferably blood tissue, bone marrow, etc., most preferably bone marrow. As genital organs, ovaries and testis are usually used. In addition, fetuses that are in the process of development can also be used to establish the immortalized cell line of the present invention.

The immortalized cell line of the present invention can be used for in vitro functional analysis of a sugar chain having a sugar chain structure without fucose modification, or for producing a highly bioactive glycoprotein having a sugar chain structure without fucose modification. it can.
Glycoproteins with high bioactivity by having a sugar chain structure without fucose modification include glycoproteins that have a sugar chain structure without fucose modification and improved affinity with receptors, and have a half-life in blood. Examples include glycoproteins that become longer, glycoproteins whose tissue distribution changes after administration in blood, or glycoproteins that improve interaction with proteins necessary for the expression of pharmacological activity.

  As a glycoprotein in the present invention, the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is not knocked out. When produced in a cell line, a glycoprotein having a fucose modification in the sugar chain structure of the produced protein is preferred. Specifically, antibodies, erythropoietin, thrombopoietin, tissue type plasminogen activator, prourokinase, thrombomodulin, antithrombin III, protein C, blood coagulation factor VII, blood coagulation factor VIII, blood coagulation factor IX, blood Coagulation factor X, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, bone morphogenetic factor, stem cell factor (SCF), interferon α, interferon β, interferon γ, interleukin 2, interleukin 6, interleukin 10, interleukin 11, soluble interleukin 4 receptor, tumor necrosis factor α, DNAseI, galactosidase, α-glucosidase, glucocerebrosidase and the like.

Examples of glycoproteins whose physiological activity is increased by having a sugar chain structure without fucose modification include antibodies. The physiological activity of an antibody includes an activity that binds to an antigen specific to the antibody, an antibody-dependent cytotoxic activity that removes cells expressing the antigen through the Fc region of the antibody bound to the antigen (hereinafter, “ ADCC activity ”) or an activity of binding to an antigen and damaging cells via complement (hereinafter referred to as“ CDC activity ”).
For example, in the immortalized cell line of the present invention, the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. Antibody compositions with higher ADCC activity can be produced than antibody compositions produced by untreated cell lines.
The antibody composition refers to a composition comprising an antibody molecule having an N-glycoside bond complex type sugar chain in the Fc region.

The antibody molecule is a tetramer in which two kinds of polypeptide chains of a heavy chain and a light chain are associated with each other. About one-quarter of the N-terminal side of the heavy chain and about one-half of the N-terminal side of the light chain (each about 100 amino acids) are called variable regions and are rich in diversity and directly involved in antigen binding To do. Most parts other than the variable region are called constant regions. Antibody molecules are classified into IgG, IgM, IgA, IgD, and IgE classes based on the homology of the constant region.
In the case of human antibodies, the IgG class is further classified into IgG1-IgG4 subclasses based on the homology of the constant region.

  The heavy chain is divided into four immunoglobulin domains, VH, CH1, CH2, and CH3, from the N-terminal side. Between CH1 and CH2, there is a highly flexible peptide region called a hinge region, which separates CH1 and CH2. . The structural unit consisting of CH2 and CH3 after the hinge region is called an Fc region, and an N-glycoside-linked sugar chain is bound to it. In addition, this region is a region where Fc receptors, complements, etc. bind (Immunology Illustrated Original Book 5th Edition, issued February 10, 2000, Nanedo Edition, Introduction to Antibody Engineering, January 25, 1994) First edition, Jinjinshokan).

  Since the Fc region of an antibody molecule has a region where N-glycoside-linked sugar chains are bound one by one, two sugar chains are bound per antibody molecule. The N-glucoside-linked sugar chain that binds to the antibody molecule includes any sugar chain that includes a core structure represented by the following chemical formula (1). Therefore, the two N-glucoside-linked sugar chains that bind to the antibody include There will be many combinations of sugar chains.

  Therefore, an antibody composition produced using the immortalized cell line of the present invention may be composed of antibody molecules having a single sugar chain structure as long as the effects of the present invention are obtained. The antibody molecule may have a plurality of different sugar chain structures. The antibody composition produced using the immortalized cell line of the present invention is preferably an antibody composition comprising an antibody molecule having an N-glycoside-linked complex type sugar chain in the Fc region, the N-glycoside-bound complex An antibody composition in which the type sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain.

A sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of N-glycoside-bonded complex sugar chain means that fucose is α-bonded to N-acetylglucosamine at the reducing end of N-glycoside-bound complex type sugar chain. No sugar chain. Specifically, a sugar chain in which the 1-position of fucose is not α-bonded to the 6-position of N-acetylglucosamine of the N-glycoside-bonded complex sugar chain can be mentioned.
An antibody composition comprising an antibody molecule having the above-mentioned N-glycoside-bonded complex sugar chain in the Fc region, wherein the N-glycoside-bonded complex sugar chain binds N-acetylglucosamine at the reducing end of the sugar chain An antibody composition that is an untreated sugar chain refers to a case where fucose is substantially undetectable in the sugar chain analysis described in 6. below. The level that cannot be substantially detected means that the level is below the detection limit of measurement.

  ADCC activity means that an antibody that binds to a cell surface antigen such as a tumor cell in vivo activates an effector cell through the binding of the Fc region of the antibody to the Fc receptor present on the effector cell surface. It refers to the activity of damaging cells and the like [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Capter 2.1 (1995)]. Examples of effector cells include killer cells, natural killer cells, activated macrophages, and the like.

  The ratio of the sugar chain in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end contained in the antibody composition comprising an antibody molecule having an N-glycoside-linked complex sugar chain in the Fc region is the antibody composition Glycans released and released using known methods such as hydrazine degradation and enzymatic digestion [Biochemical Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center) Takahashi Eiko (1989)] Can be determined by fluorescence labeling or isotope labeling and separating the labeled sugar chain by a chromatographic method. It can also be determined by analyzing the released sugar chain by the HPAED-PAD method [J. Liq. Chromatogr., 6, 1577 (1983)].

In the present invention, the antibody recognizes a tumor-related antigen, an antibody that recognizes an antigen related to allergy or inflammation, an antibody that recognizes an antigen related to cardiovascular disease, and an antigen related to an autoimmune disease. Or an antibody that recognizes an antigen associated with a virus or bacterial infection, and an IgG class antibody is preferably used.
The antibody molecule includes any molecule that contains the Fc region of an antibody. Specific examples include antibodies, antibody fragments, and fusion proteins containing the Fc region of antibodies.

Any antibody may be used as long as it is a protein produced in vivo by an immune reaction as a result of stimulation with a foreign antigen, and has an activity that specifically binds to the antigen. In addition to antibodies secreted by hybridomas produced from animal spleen cells, antibodies produced by gene recombination techniques, that is, antibodies obtained by introducing antibody expression vectors into which antibody genes have been inserted into host cells, etc. can give. Specific examples include antibodies produced by hybridomas, humanized antibodies, human antibodies, and the like.
A hybridoma is a monoclonal antibody having a desired antigen specificity obtained by fusing antibody-producing cells obtained by immunizing mammals other than humans with myeloma cells derived from mice, rats, etc. Refers to cells that produce

Examples of humanized antibodies include human chimeric antibodies and human CDR-grafted antibodies.
The human chimeric antibody is composed of an antibody heavy chain variable region (hereinafter, the variable region is also referred to as HV or VH as a V region) and an antibody light chain variable region (hereinafter, a light chain is referred to as LV or VL as an L chain). And a heavy chain constant region of a human antibody (hereinafter also referred to as CH) and a light chain constant region of a human antibody (hereinafter also referred to as CL). Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, hamsters, rabbits and the like.

Human chimeric antibodies are obtained by obtaining cDNAs encoding VH and VL from hybridomas producing monoclonal antibodies and inserting them into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively. A chimeric antibody expression vector can be constructed and expressed by introducing it into a host cell.
The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hIg). Any of the subclasses such as hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.

The human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL.
The human CDR-grafted antibody constructs a cDNA encoding a V region in which the CDR sequences of the VH and VL of the non-human animal antibody are grafted onto the CDR sequences of any human antibody, and the human antibody CH and A human CDR-grafted antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding CL of human antibody, and the human CDR-grafted antibody is expressed by introducing the expression vector into the host cell. Can be manufactured.

The CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but the hIgG class is preferable, and any of the subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class may be used. it can. The CL of the human CDR-grafted antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
A human antibody originally refers to an antibody that naturally exists in the human body. However, a human antibody phage library and a human antibody production transgene prepared by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Also included are antibodies obtained from transgenic animals or transgenic plants producing human antibodies.

The antibody present in the human body can be cultured, for example, by isolating human peripheral blood lymphocytes, infecting an EB virus or the like, immortalizing and inserting the lymphocytes that produce the antibody. Can be purified.
The human antibody phage library is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by inserting antibody genes prepared from human antibody-producing cells into the phage genes. From the library, phages expressing antibody fragments having a desired antigen-binding activity 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 comprising two complete H chains and two complete L chains or a molecule having the shape of a desired antibody fragment by protein engineering techniques.

A human antibody-producing transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody-producing transgenic animal can be 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. Moreover, a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. The production method of human antibodies from human antibody-producing transgenic animals is obtained by culturing human antibodies-producing hybridomas by the conventional method of producing hybridomas in mammals other than humans, and producing human antibodies in the culture. Can be accumulated.
Examples of human antibody-producing transgenic non-human animals include cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, rabbits, and the like.

In the present invention, the antibody fragment refers to a fragment containing at least a part or all of the Fc region of the antibody. The Fc region means a region on the C-terminal side of the H chain of an antibody, a CH2 region and a CH3 region, and includes a natural type and a variant thereof. At least a part of the Fc region preferably refers to a fragment containing the CH2 region, more preferably a region containing the first aspartic acid present in the CH2 region. Fc region of IgG class, Kabat (Kabat) et al. EUIndex [Shikenshizu of Proteins of Immunological Interest (Sequences of Proteins of ImmunologicalInterest), 5 th Ed., Public Health Service, National Institutes ofHealth, Bethesda , MD. (1991)], from 226th cysteine to C-terminal, or 230th proline to C-terminal. Specific examples of the antibody fragment include a monomer or dimer of a polypeptide chain containing a part of the Fc region.

As the fusion protein containing the Fc region, any substance (hereinafter referred to as Fc fusion protein) obtained by fusing an antibody or antibody fragment containing an antibody Fc region with a protein may be used.
The protein to be fused with the Fc region contained in the Fc fusion protein includes any protein as long as it can be expressed by being fused with the Fc region. Specifically, antibodies, antibody-binding fragments, receptor ligands, soluble receptors, nucleic acid binding proteins, cell membrane binding proteins, lipid binding proteins, fatty acid binding proteins, sugar or sugar chain binding proteins, enzyme substrates Examples include dominant negative and enzyme dominant negative.

Examples of the binding fragment of an antibody include Fab, Fab ′, F (ab ′) 2, scFv, Diabody, dsFv, or a peptide containing CDR.
Fab is a fragment obtained by treating IgG with the proteolytic enzyme papain (cleaved at the 224th amino acid residue of the H chain). It is an antibody fragment having an antigen binding activity of about 50,000 molecular weight bound by SS binding.
Fab can be obtained by treating an antibody with the proteolytic enzyme papain. Alternatively, the Fab can be produced by inserting a DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic or eukaryotic organism to express the antibody. it can.

F (ab ') 2 is a fragment obtained by treating IgG with proteolytic enzyme pepsin (cleaved at the 234th amino acid residue of the H chain), and Fab binds via an SS bond in the hinge region. It is an antibody fragment having an antigen-binding activity with a molecular weight of about 100,000, which is slightly larger than the above.
F (ab ′) 2 can be obtained by treating an antibody with the proteolytic enzyme pepsin. Alternatively, Fab ′ described below can be produced by thioether bond or SS bond.

Fab ′ is an antibody fragment having an antigen-binding activity having a molecular weight of about 50,000, which is obtained by cleaving the SS bond in the hinge region of F (ab ′) 2.
Fab ′ can be obtained by treating F (ab ′) 2 with a reducing agent dithiothreitol. Alternatively, it can be produced by inserting a DNA encoding Fab ′ into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism.

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 (P) of 12 residues or more. It is an antibody fragment having
scFv obtains cDNA encoding the antibody VH and VL, constructs DNA encoding scFv, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and inserts the expression vector into a prokaryotic organism. Alternatively, it can be expressed and produced by introduction into a eukaryote.

Diabody is an antibody fragment in which scFv having the same or different antigen binding specificity forms a dimer, and is an antibody fragment having a bivalent antigen-binding activity for the same antigen or a bispecific antigen-binding activity for different antigens.
Diabody obtains cDNA encoding antibody VH and VL, constructs scFv-encoding DNA having a 3-10 residue polypeptide linker, and uses the DNA as a prokaryotic expression vector or eukaryotic expression vector Diabody can be expressed and produced by inserting the expression vector into a prokaryotic or eukaryotic organism.

dsFv refers to a polypeptide in which one amino acid residue in each of VH and VL is substituted with a cysteine residue, which are linked via an SS bond between the cysteine residues. The amino acid residue 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)).
dsFv obtains cDNA encoding antibody VH and VL, constructs DNA encoding dsFv, inserts the DNA into a prokaryotic expression vector or eukaryotic expression vector, and inserts the expression vector into a prokaryotic organism Alternatively, it can be expressed and produced by introduction into a eukaryote.

The peptide containing CDR is composed of at least one region of CDR of VH or VL. A peptide containing a plurality of CDRs can be produced by binding directly or via an appropriate peptide linker.
For peptides containing CDRs, construct cDNAs encoding antibody VH and VL CDRs, insert the cDNA into a prokaryotic expression vector or eukaryotic expression vector, and insert the expression vector into a prokaryotic or eukaryotic organism. It can be expressed and produced by introduction into the system. A peptide containing CDR can also be produced by a chemical synthesis method such as Fmoc method (fluorenylmethyloxycarbonyl method) or tBoc method (t-butyloxycarbonyl method).
Hereinafter, the method for producing an immortalized cell line of the present invention when a mouse is used as a non-human animal will be specifically described.

1. Method for producing a mouse in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-binding complex sugar reducing end is knocked out (1) The gene disruption technique targeting the gene The knockout mouse used in the present invention is involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. It can be prepared by targeting a gene of an enzyme (hereinafter referred to as “α1,6-fucose modifying enzyme”) and using a gene disruption method. Specific examples of the α1,6-fucose modifying enzyme include α1,6-fucosyltransferase.

  As a method for gene disruption, any method can be used as long as it can destroy a gene of a target enzyme. Examples thereof include a homologous recombination method, an RDO method, a method using a retrovirus, a method using a transposon, and the like. These will be specifically described below.

(A) Production of knockout mouse used in the present invention by homologous recombination method The knockout mouse used in the present invention targets the gene of α1,6-fucose modifying enzyme and uses the target gene on the chromosome using the homologous recombination method. It can be produced by modifying.
Modification of target genes on chromosomes, Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) , A Practical Approach, IRL Press at Oxford University Press (1993), BioManual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995) (hereinafter referred to as “mutant mice using ES cells” For example, it can be carried out as follows using the chromosome engineering technique described in “Production”.
Obtain cDNA of α1,6-fucose modifying enzyme.

Based on the obtained cDNA base sequence, genomic DNA of α1,6-fucose modifying enzyme is prepared.
Based on the base sequence of the genomic DNA, a target vector for homologous recombination of a target gene to be modified (for example, a structural gene of α1,6-fucose modifying enzyme or a promoter gene) is prepared.
The prepared target vector is introduced into embryonic stem cells, and cells that have undergone homologous recombination between the target gene and the target vector are selected.
The selected embryonic stem cells are introduced into fertilized eggs according to a known injection chimera method or assembly chimera method, and the introduced fertilized eggs are transplanted into the oviduct or uterus of pseudopregnant female mice to select germline chimeras.

The selected germline chimera is crossed and an individual having a chromosome into which the introduced target vector has been inserted by homologous recombination with the gene region on the genome encoding the α1,6-fucose modifying enzyme from the offspring born. select.
The selected target was bred and the introduced target vector was inserted into the homologous chromosome in both homologous chromosomes by causing homologous recombination with the gene region on the genome encoding α1,6-fucose modifying enzyme. A homozygote having a chromosome is selected.
By knocking the obtained homozygotes together and obtaining their progeny, the knockout mouse used in the present invention can be produced.
Examples of a method for obtaining cDNA and genomic DNA of α1,6-fucose modifying enzyme include the methods described below.

Preparation method of cDNA Total RNA or mRNA is prepared from mouse cells to be modified.
A cDNA library is prepared from the prepared total RNA or mRNA.

Based on the known amino acid sequence of α1,6-fucose modifying enzyme, for example, human amino acid sequence, a degenerative primer was prepared, and the α1,6-fucose modifying enzyme was obtained by PCR using the prepared cDNA library as a template. A gene fragment encoding is obtained.
Using the obtained gene fragment as a probe, a cDNA library can be screened to obtain a cDNA encoding an α1,6-fucose modifying enzyme.

Mouse cell mRNA may be commercially available (eg, Clontech) or may be prepared from total RNA prepared from cells as follows. Methods for preparing total RNA from cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154 , 3 (1987)], acidic guanidine thiocyanate, phenol, chloroform. (AGPC) method [Analytical Biochemistry, 162 , 156 (1987); Experimental Medicine, 9 , 1937 (1991)].

Examples of a method for preparing mRNA from total RNA as poly (A) ⁺ RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, Second Edition).
In addition, Fast Track mRNA Isolation Kit (Invitrogen), Quick Prep mRNA
MRNA can be prepared by using a kit such as Purification Kit (Pharmacia).

  A cDNA library is prepared from the prepared mouse cell mRNA. Methods for preparing a cDNA library include the methods described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or commercially available kits such as Examples include SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and ZAP-cDNA Synthesis Kit (STRATAGENE).

As a cloning vector for preparing a cDNA library, any phage vector or plasmid vector can be used as long as it can replicate autonomously in Escherichia coli K12. Specifically, ZAP Express [STRATAGENE, Strategies, 5 , 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)] , ΛZAP II (STRATAGENE), λgt10, λgt11 [DNA cloning, A Practical Approach, 1 , 49 (1985)], λTriplEx (Clontech), λExCell (Pharmacia) , PT7T318U (Pharmacia), pcD2 [Mol. Cell. Biol., 3 , 280 (1983)] and pUC18 [Gene, 33 , 103 (1985)], etc. Can do.

Any microorganism can be used as the host microorganism for preparing the cDNA library, but Escherichia coli is preferably used. Specifically, Escherichia coli XL1-Blue MRF '[STRATAGENE, Strategies, 5 , 81 (1992)], Escherichia coli C600 [Genetics, 39 , 440 (1954)], Escherichia coli Y1088 [Science, 222 , 778 (1983)], Escherichia coli Y1090 [Science, 222 , 778 (1983)], Escherichia coli NM522 [Journal of Molecular Biology (J. Mol. Biol ), 166 , 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16 , 118 (1966)] and Escherichia coli JM105 [Gene, 38 , 275 (1985)].

The cDNA library may be used for subsequent analysis as it is, but the oligo cap method [Gen Gene] developed by Kanno et al. Was developed to reduce the percentage of incomplete cDNA and to obtain full-length cDNA as efficiently as possible. (Gene), 138 , 171 (1994); Gene, 200 , 149 (1997); Protein Nucleic Acid Enzyme, 41 , 603 (1996); Experimental Medicine, 11 , 2491 (1993); cDNA Cloning (Yodosha) ) (1996); cDNA library prepared using gene library preparation method (Yodosha) (1994)] may be used for the following analysis.

  Based on the amino acid sequence of the α1,6-fucose modifying enzyme, a degenerative primer specific for the 5 ′ end and 3 ′ end base sequence predicted to encode the amino acid sequence is prepared, Gene fragments encoding α1,6-fucose-modifying enzyme by amplifying DNA using the prepared cDNA library as a template and PCR method [PCR Protocols, Academic Press (1990)] Can be obtained.

The obtained gene fragment is a DNA encoding an α1,6-fucose modifying enzyme, which means that a commonly used nucleotide sequence analysis method, such as the dideoxy method of Sanger et al. [Proceedings of the National Academy, This can be confirmed by analysis using a base sequence analyzer such as of Science (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or ABIPRISM377 DNA Sequencer (manufactured by PE Biosystems). .

By using the gene fragment as a DNA probe, colony hybridization or plaque hybridization (Molecular Cloning 2nd edition) is performed on cDNA or cDNA library synthesized from mRNA contained in mouse cells to be modified. Therefore, DNA of 6-fucose modifying enzyme can be obtained.
In addition, using the primers used to obtain the gene fragment encoding the α1,6-fucose modifying enzyme, using a cDNA or cDNA library synthesized from mRNA contained in mouse cells to be modified as a template, PCR method The α1,6-fucose modifying enzyme DNA can also be obtained by screening using

The base sequence of the obtained DNA encoding the α1,6-fucose modifying enzyme is used as a base sequence analysis method, such as Sanger et al. [Proceedings of the National Academy of Sciences]. Science (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or by analyzing using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (PE Biosystems), the base sequence of the DNA decide.

Based on the determined cDNA base sequence, a homology search program such as BLAST is used to search a base sequence database such as GenBank, EMBL, and DDBJ. It is also possible to search for and use a gene encoding a modifying enzyme.
Examples of the base sequence of the gene encoding the α1,6-fucose modifying enzyme obtained by the above method include the base sequence described in SEQ ID NO: 5.

Based on the determined DNA base sequence, the cDNA of α1,6-fucose modifying enzyme can be synthesized by chemical synthesis using a DNA synthesizer such as Perkin Elmer's DNA synthesizer model 392 using the phosphoramidite method. It can also be acquired.
Examples of the method for preparing genomic DNA of α1,6-fucose modifying enzyme include the methods described below.

Methods for preparing genomic DNA Examples of methods for preparing genomic DNA include known methods described in Molecular Cloning Second Edition, Current Protocols in Molecular Biology, and the like. In addition, genomic DNA of α1,6-fucose modifying enzyme can be isolated by using a genomic DNA library screening system (Genome Systems), Universal GenomeWalker ^™ Kits (CLONTECH), or the like.
The base sequence of the genomic DNA of the α1,6-fucose modifying enzyme obtained by the above method can be confirmed from the fact that the base sequence in the cDNA of the α1,6-fucose modifying enzyme obtained by the above method is included.

  Target vectors for homologous recombination of target genes are Gene Targeting, A Practical Approach, IRL Pressat Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of mutant mice using ES cells (Yodosha) (1995) and the like. The target vector may be any of a replacement type, an insertion type, or a gene trap type.

As a method for introducing a target vector into embryonic stem cells, any method can be used as long as it introduces DNA into animal cells. For example, electroporation [Cytotechnology, 3 , 133 (1990) ], Calcium phosphate method [JP-A-2-27075], lipofection method [Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. USA), 84 , 7413 (1987)], injection Method [Manipulating Mouse Embryo 2nd edition], Method using particle gun (Gene gun) [Patent No. 2606856, Patent No. 2517813], DEAE-Dextran method [Bio-manual series 4-Gene transfer and expression / analysis method ( Yodosha) Takashi Yokota, Kenichi Arai (1994)], virus vector method [Manipulating Mouse Embryo 2nd Edition], etc. It is possible.

  Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, APractical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells The method such as positive selection, promoter selection, negative selection, poly A selection described in (1995) and the like can be used. Specifically, in the case of a target vector containing the hprt gene, after introduction into an embryonic stem cell lacking the hprt gene, the embryonic stem cell is cultured in a medium containing aminopterin, hypoxanthine and thymidine, and an aminopterin resistant strain is obtained. By selecting, positive selection for selecting homologous recombinants containing the hprt gene can be performed. In the case of a target vector containing a neomycin resistance gene, the embryonic stem cell into which the vector has been introduced is cultured in a medium containing G418, and a G418 resistant strain is selected to select a homologous recombinant containing the neomycin resistance gene. A selection can be made. In the case of a target vector containing the DT gene, the embryonic stem cell into which the vector has been introduced is cultured, and the growing strain is selected (recombinants randomly inserted into the chromosome other than homologous recombination have the DT gene Therefore, it is possible to carry out negative selection to select homologous recombinants not containing the DT gene. Methods for selecting the desired homologous recombinants from the selected cell lines include Southern hybridization to genomic DNA (Molecular Cloning 2nd Edition) and PCR [PCR Protocols, Academic Press (1990)].

When embryonic stem cells are incorporated into a fertilized egg using the assembly chimera method, it is generally preferable to use a fertilized egg at a developmental stage prior to the 8-cell stage. When embryonic stem cells are incorporated into a fertilized egg using an injection chimera method, it is generally preferable to use a fertilized egg from the 8 cell stage to the blastocyst development stage.
When a fertilized egg is transplanted into a female mouse, the fertilized egg obtained in a pseudopregnant female mouse in which fertilization ability is induced is artificially transplanted and implanted by mating with a non-human mammal ligated with a vas deferens. Pseudopregnant female mice can be obtained by natural mating, but fertilizing ability can be achieved by mating with male mice after administration of luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) or its analogs. Induced pseudopregnant female mice can also be obtained. Examples of LHRH analogs include [3,5-Dil-Tyr5] -LHRH, [Gln8] -LHRH, [D-Ala6] -LHRH, des-Gly10- [D-His (Bzl) 6] -LHRHethylamide, etc. Is given.

(B) Production of knockout mouse used in the present invention by RDO method The knockout mouse used in the present invention targets the gene of α1,6-fucose modifying enzyme and uses the RDO (RNA-DNA oligonucleotide) method. Can be produced as follows.

Prepare cDNA of α1,6-fucose modifying enzyme or genomic DNA.
Determine the nucleotide sequence of the prepared cDNA or genomic DNA.
Based on the determined DNA sequence, an RDO construct of an appropriate length including a portion encoding α1,6-fucose modifying enzyme, a portion of an untranslated region or an intron portion is designed and synthesized.

The synthesized RDO is introduced into embryonic stem cells, and embryonic stem cells in which the target enzyme, ie, α1,6-fucose modifying enzyme is mutated, are selected.
The selected embryonic stem cells are introduced into a fertilized egg according to an injection chimera method or an assembly chimera method, and the introduced fertilized egg is transplanted into the oviduct or uterus of a pseudopregnant female mouse to select a germline chimera.

The selected germline chimera is crossed and an individual having a chromosome into which the introduced target vector has been inserted by homologous recombination with the gene region on the genome encoding the α1,6-fucose modifying enzyme from the offspring born. select.
The selected target was bred and the introduced target vector was inserted into the homologous chromosome in both homologous chromosomes by causing homologous recombination with the gene region on the genome encoding α1,6-fucose modifying enzyme. A homozygote having a chromosome is selected.
By knocking the obtained homozygotes together and obtaining their progeny, the knockout mouse used in the present invention can be produced.

For introduction of RDO into embryonic stem cells, the method for introducing a target vector described in (1) (a) above can be used.
Examples of the method for preparing cDNA of α1,6-fucose modifying enzyme include “cDNA preparation method” described in (1) (a) of 1 above.
Examples of a method for preparing genomic DNA of α1,6-fucose modifying enzyme include the “method for preparing genomic DNA” described in (1) (a) of 1 above.

The DNA base sequence is cleaved with an appropriate restriction enzyme and inserted into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used base sequence analysis method such as the dideoxy method [Sanger et al. Procedures of the National Academy of Science (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (PE Biosystems) This can be confirmed by analyzing them. RDO can be prepared by a conventional method or using a DNA synthesizer.
As a method of introducing RDO into embryonic stem cells and selecting cells with mutations in the target enzyme α1,6-fucose modifying enzyme gene, Molecular Cloning 2nd Edition, Current Protocols In -Methods for directly detecting gene mutations on chromosomes described in molecular biology and the like.

RDO constructs are described in Science, 273 , 1386, (1996); Nature Medicine, 4 , 285, (1998); Hepatology, 25 , 1462, (1997); Gene Therapy, 5 , 1960, (1999); Gene Therapy, 5 , 1960, (1999); Journal of Molecular Medicine (J. Mol. Med.), 75 , 829, (1997); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 8774, (1999); Proceedings of the National Academy of Sciences Science (Proc. Natl. Acad. Sci. USA), 96 , 8768, (1999); Nuc. Acids. Res., 27 , 1323, (1999); Investment of Damato Logie, 111, 1172, (1998) (Invest Dematol..); (. Nature Biotech) Nature biotechnology, 16, 1343, (1998); Nature -It can design according to description of Nature Biotech., 18 , 43, (2000); Nature Biotech. ( 18 ), 555, (2000) etc.

(C) Preparation of a knockout mouse used in the present invention by a method using a transposon The knockout mouse used in the present invention has the transposon system described in Nature Genet., 25 , 35, (2000), etc. It can be prepared by selecting a mutant of α1,6-fucose modifying enzyme.

A transposon system is a system that induces mutations by randomly inserting foreign genes onto a chromosome. Usually, a foreign gene inserted into a transposon is used as a vector to induce mutations, and this gene is used as a chromosome. A transposase expression vector for random insertion is simultaneously introduced into the cell.
Any transposase may be used as long as it is suitable for the transposon sequence to be used.

As the foreign gene, any gene can be used as long as it induces a mutation in cell DNA.
For introduction of a gene into a cell, the method for introducing a target vector described in (1) (a) of 1 above can be used.

(2) Technique for Introducing Mutation for Enzyme The knockout mouse used in the present invention introduces a mutation for the gene of α1,6-fucose modifying enzyme, and selects a desired mouse having the mutation in the enzyme. It can be produced by using a technique.
Specific examples of the α1,6-fucose modifying enzyme include α1,6-fucosyltransferase.

Specifically, a desired mouse with a mutation in the α1,6-fucose-modifying enzyme gene is selected from mutants produced from germ cells treated with mutagenesis or spontaneously generated mutants. The method of doing is mentioned.
Examples of germ cells include cells having the ability to form individuals such as sperm, eggs, or embryonic stem cells.

As the mutagenesis treatment, any treatment can be used as long as it induces point mutation, deletion, or frameshift mutation in cell DNA. Specific examples include treatment with ethyl nitrosourea, nitrosoguanidine, benzopyrene, acridine dye, irradiation with radiation, and the like. Various alkylating agents and carcinogens can also be used as mutagens. Examples of methods for causing mutagens to act on cells include, for example, tissue culture technology 3rd edition (Asakura Shoten) edited by Japanese Society for Tissue Culture (1996), Nature Genet., 24 , 314, ( 2000) and the like.
Examples of spontaneously generated mutants include mutants that occur spontaneously by continuing normal breeding without any special mutagenesis treatment.

(3) A method for producing a clone individual using the nucleus of a cell subjected to the targeted genetic modification The knockout mouse used in the present invention is a cloned mouse described in the literature [Nature, 394 , 369 (1998), Nature Genetics, 22 , 127 (1999)], for example, can be produced as follows.
Above 1. Using the method described in (1) or (2) above, a mutation is introduced into the α1,6-fucose modifying enzyme gene on the chromosome of any mouse cell.
Next, the nuclei of the obtained cells are initialized (an operation for returning the nuclei to a state where they can be repeated again).
Development is initiated by injecting reprogrammed cell nuclei into enucleated mouse unfertilized eggs.

A heterozygote having a mutation introduced into the α1,6-fucose modifying enzyme gene is obtained by artificially transplanting and implanting an embryo that has started development into a female mouse.
A homozygote is obtained by crossing the heterozygotes obtained.
By knocking the homozygotes obtained and obtaining their progeny, the knockout mouse used in the present invention can be produced.
It is known that the method of initializing the cell nucleus differs depending on the type of non-human mammal, but in the case of mice, etc., exogenous genes can be added to enucleated unfertilized eggs of the same non-human mammal. It is preferable to initialize by injecting the nuclei of the cells into which is introduced and culturing for several hours, preferably about 1 to 6 hours.

It is known that the method of initiating development in an unnucleated unfertilized egg is different depending on the type of non-human mammal, but in the case of mice, etc. Stimulate an unfertilized egg into which the nucleus of the cell into which the cell has been introduced is stimulated with an egg activator (eg, strontium) and treat with a cell division inhibitor (eg, cytochalasin B) to release the second polar body. Generation | occurrence | production is preferably started by suppressing.
As a method of artificially transplanting and implanting an egg that has started to develop into a female mouse, the above-mentioned 1. And the method described in (1) (a).

2. Production of Immortalized Cell Line The immortalized cell line of the present invention can be obtained by using a mouse in which the genomic gene of α1,6-fucose modifying enzyme produced by the method described in 1 above is knocked out, for example, as follows. Can be produced.

Organs are removed from mice in which the genomic gene of α1,6-fucose modifying enzyme has been knocked out.
The removed organ is made into a non-aggregated cell population by physicochemical treatment.
The immortalized cell line of the present invention can be obtained by aseptically continuously culturing the prepared non-aggregated cell population for at least 2 months.

  At this time, an oncogene may be introduced to promote immortalization of the prepared non-aggregated cell population. Examples of the oncogene include known oncogenes that are already known to promote immortalization by introduction into primary cultured cells such as SV40 large T antigen, Ras, Myc, or Src. The immortalized cell line of the present invention can be efficiently obtained by incorporating these oncogenes into an appropriate animal cell expression vector described in 3 below and introducing them into the prepared non-aggregated cell population.

  Methods for excising organs from mice include animal tissue culture, Modern Biology Series 23, Kyoritsu Shuppan, (1974), Cell Culture Manual, Kodansha Scientific, (1982), Useof Isolated Liver Cells and Kidney Tubules in Metabolic Studies, Elaevier / North-Holland Publishing Co., New York / Amsterdam / Oxford, (1976), Illustrated techniques for animal experiments, protein nucleic acid enzyme, separate volume No. 24, (1981), mouse laboratory manual, gene introduction and Focusing on analysis, Department of Experimental Animal Research, Tokyo Metropolitan Institute of Medical Science, Springer Fairlark Tokyo Co., Ltd. (1998), Mouse Lab Manual [2nd edition], Experimental methods in the post-genomic era, ( Well-known methods described in the Tokyo Metropolitan Institute of Medical Science, Laboratory Animal Research Division, Springer Fairlark Tokyo Co., Ltd. (2003), and the like.

  3rd edition, tissue culture technology [basic edition], edited by Japanese Society for Tissue Culture, Asakura Shoten, (1996), 3rd edition. And known techniques described in Tissue Culture Technology [Application], Japanese Tissue Culture Society, Asakura Shoten, (1996), and the like. Specifically, a method using enzymatic digestion or a physical separation method using a mesh or the like is preferably used.

  The non-aggregated cell population is a single cell state in which adhesion between cells is released. The single cell state refers to a state in which individual cells are separated from each other by performing treatment such as a physical separation method using enzyme digestion, mesh, or the like, so that the cells do not adhere to each other. In order to obtain such a single cell population, for example, the excised organ is shredded with a scalpel as much as possible, and a phosphate buffered saline solution (hereinafter abbreviated as “PBS”) is used. Wash several times, preferably 2-3 times, add an enzyme digestion solution suitable for digestion of the removed organ (for example, PBS containing 1 mM EDTA and 0.25% trypsin), and at 37 ° C for several tens of minutes, preferably Incubate for 5 to 20 minutes, suspend the digested organ in the culture medium, perform centrifugation (eg, 4 ° C., 200 × g for 5 minutes), and resuspend the precipitated cells in the culture medium. Thus, a non-aggregated cell population can be obtained.

  As a method for continuously culturing a non-aggregated cell population aseptically for at least 2 months, any cell culture method may be used as long as the culture can be continued without killing the cells. Specifically, 3rd edition, Tissue culture technology [Basics], Japan Tissue Culture Society, Asakura Shoten, (1996), 3rd edition, Tissue culture technology [Application], Japan Tissue Culture Society, A publicly known method described in Asakura Shoten, (1996) and the like can be mentioned. As a general method, there is a monolayer culture method using a flask or a dish, but a high-density maintenance culture method, a microcarrier culture method, a reflux culture method, a soft agar culture method, or the like can also be used.

In addition, depending on the type of immortal cell line to be established, it is effective for the efficient establishment of the immortal cell line for which the auxotrophic factor of the cell line is continuously supplied during the culture period. The co-culture method used or a culture method using a medium supplemented with cytokines can be used. Specific examples of co-culture methods using feeder cells include NIH3T3 cells (J. Virol., 4 , 549, 1969), OP9 cells (Science, 272 , 722, 1996), MC3T3-G2 / PA6 (J Cell. Physiol., 112 , 89, 1982), STO cells (Proc. Natl. Acad. Sci. USA, 78 , 7634, 1981; Nature, 292 , 154, 1981), mouse embryo primary fibroblasts (manipulating)・ The Mouse Embryo A Laboratory Manual; Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Examples include culture methods. Specific examples of culture methods in a medium supplemented with cytokines include erythropoietin, thrombopoietin, tissue-type plasminogen activator, prourokinase, thrombomodulin, gonadotropin, thyroid-stimulating hormone, epidermal growth factor ( EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, osteogenic factor, stem cell factor (SCF), fibroblast growth factor (FGF), nerve growth factor (NGF), insulin, interferon α, interferon β And a culture method using a medium supplemented with interferon γ, interleukin 1-16, soluble interleukin 4 receptor, tumor necrosis factor α, tumor necrosis factor β, chemokine, estrogen, testosterone and the like.

  As an incubator that can be used for aseptic continuous culture for at least 2 months, any incubator can be used as long as it can be used for culturing primary cultured cells taken out from a living tissue, but it is preferably used for cell culture. Incubators are preferred. Examples of incubators for cell culture include flasks, bacterial culture flasks, cell culture flasks, dishes, petri dishes, tissue culture dishes, tour dish, permanox dishes, multi dishes, micro plates, and micro well plates. Multiplate, Multiwell plate, Separate strip well, Terasaki plate, Tissue culture chamber slide, Petri dish, Cell culture petri dish, Tissue culture tube, Tray, Cell culture tray, Cell factory, Culture bag, Technopot, Roller Examples include bottles, spinners, and follow fibers. In order to control the adhesion between the incubator and the cells, the surface of the incubator on the side in contact with the cells can be artificially treated. Examples of artificially treating the surface of the incubator include collagen coat, gelatin coat, poly L-lysine coat, fibronectin coat, laminin coat, proteoglycan coat, glycoprotein coat, matrigel coat, silicon coat and the like. Moreover, it can also be processed so as to have a negative charge, such as primaria (manufactured by Becton Dickinson). The incubator subjected to these treatments can also be used for culture for establishing the immortalized cell line of the present invention.

As the basal medium used for the culture, BME medium (Proc. Soc. Exp. Biol. Med., 89 , 362, 1965), BGJb medium (Exp. Cell Res., 25 , 41, 1961), CMRL1066 medium (NY Academy) of Sciences, 5 , 303, 1957), Glasgow MEM medium (Virology, 16 , 147, 1962), Improved MEM Zinc Option medium (J. National Cancer Inst., 49 , 1705, 1972), IMDM medium (In Vitro, 9 , 6, 1970), Medium 199 medium (Proc. Soc. Exp. Biol. Med., 73 , 1, 1950), Eagle MEM medium (Science, 130 , 432, 1959), Alpha MEM medium (NatureNew Biology, 230 , 310, 1971), Dulbecco MEM medium (Virology, 8 , 396, 1959), Ham medium (Exp. Cell Res., 29 , 515, 1963; Proc. Natl. Acad. Sci. USA, 53 , 288, 1965), RPMI 1640 medium (JAMA, 199 , 519, 1967), Fischer's medium (Methods in Med. Res., 10, 1964), McCoy's medium (Proc. Soc. Exp. Biol. Med., 100 , 115, 1959), Williams E medium (Exp Cell Res, 69, 106 , 1971;.... Exp Cell Res, 89, 139, 1974) and mixed with a mixture of these basal medium Can be used, so long as the medium which can be used for culturing animal cells such as media.

In addition, the embryo culture described in Manipulating the Mouse Embryo A Laboratory Manual, Methodsin Enzymology volume 225, Guide to Techniques in Mouse Development, AcademicPress (1993), creation of mutant mice using ES cells, etc. Any medium that can be used for embryo culture, such as M2 medium, M16 medium, Whitten medium, and in vitro fertilization medium, can be used as the basal medium. These media are preferably used when establishing immortalized cell lines from fetuses.
By adding fetal bovine serum or the above-mentioned cytokines to these basal media, a medium used for culture can be prepared. The concentration of fetal bovine serum and cytokines to be added may be appropriately determined according to the immortalized cell line to be established. More preferably, it is 2 to 20%.

Furthermore, any medium can be used as long as it can culture animal cells and embryos even in a medium obtained by adding various growth factors as serum substitutes to the above-mentioned basal medium, or a protein-free medium. Specific examples thereof include serum-free medium (Focus, 20 , 8, 1998) supplemented with commercially available KNOCKOUT ^™ SR, serum-free medium supplemented with insulin and transferrin [for example, CHO-S-SFMII (manufactured by GIBCOBRL), Hybridoma-SFM (GIBCOBRL), eRDF Dry Powdered Media (GIBCOBRL), UltraCULTURE ^TM (BioWhittaker), UltraDOMA ^TM (BioWhittaker), UltraCHO ^TM (BioWhittaker), UltraMDCK ^TM (BioWhittaker) ITPSG medium (Cytotechnology, 5 , S17, 1991), ITSFn medium (Proc. Natl. Acad. Sci. USA, 77 , 457, 1980), mN3 medium (Mech. Dev., 59 , 89, 1996), etc.] Medium supplemented with cell-derived factors [eg, medium supplemented with culture supernatant of pluripotent teratocarcinoma cell PSA1 (Proc. Natl. Acad. Sci. USA, 78 , 7634, 1981), protein-free medium (eg, CD-CHO (manufactured by GIBCOBRL), PFHM-II (manufactured by GIBCOBRL), UltraDOMA-PF ^™ (manufactured by BioWhittaker) and the like. A medium obtained by further adding the above-described cytokines to these mediums can also be used.

In the process of establishing an immortalized cell line, the survival rate of the cell may temporarily decrease and the cell may die. For this reason, in order to return the cell viability or keep it high, the cell density is 1 × 10 ⁴ to 1 × 10 ⁶ cells / ml, preferably 5 × 10 ^{4 to} 5 × at the time of cell inoculation into the culture medium. It is preferable to inoculate at 10 ⁵ cells / ml. For example, cells are inoculated into a medium, cultured using a normal animal cell culture method such as batch culture in a 37 ° C., 5% CO ₂ incubator, and the cell saturation is 70 to 90% and the cell concentration is 5 When reaching 10 ⁴ to 1 × 10 ⁶ cells / ml, the cells may be inoculated in a fresh medium so that the cell concentration is diluted 2 to 20 times, and the culture may be repeated under the same conditions. If the cells are poorly proliferating, the medium is changed every 4 to 7 days until the proliferating properties are restored. Thus, an immortalized cell line can be obtained by continuing the culture continuously for at least 2 months.

The obtained immortalized cell line can be prepared as a cloned (single cell) cell line by using a 96-well plate limiting dilution method, colony forming method, or the like.
The following shows a method for preparing a cloned cell line using the limiting dilution method.
Dilute the cell suspension, inoculate so that there is less than one cell per well, and incubate for several weeks in a 30-40 ° C., 5% CO ₂ incubator. After completion of the culture, select cells in which cell growth has been observed.

The method of cloning using the colony formation method is as follows.
In the case of adherent cells, the cell suspension is diluted, the cells are inoculated into a petri dish and cultured, and then colony formation is confirmed. A colony is separated with a ring such as a penicillin cap, and after separating cells with an enzyme such as trypsin, it is transferred to an appropriate incubator and a desired cell line is selected.
In the case of suspension cells, dilute the cell suspension, inoculate the cells in soft agar and culture, pick up the resulting colonies under a microscope, return to static culture and select the desired cell line .
This makes it possible to obtain clones with better growth from the entire cell population.

3. Method for Producing Glycoprotein Using Antibody Composition as an Example A method for producing a glycoprotein composition using the immortalized cell line of the present invention is shown below by taking the production of an antibody composition as an example.
Antibody compositions include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Antibodies, A Laboratory manual, ColdSpring Harbor Laboratory, 1988 (hereinafter abbreviated as Antibodies), Monoclonal Antibodies: principlesand practice , Third Edition, Acad. Press, 1993 (hereinafter abbreviated as Monoclonal Antibodies), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter abbreviated as Antibody Engineering), etc. For example, a gene encoding an antibody molecule can be expressed and obtained using the immortalized cell line of the present invention as a host cell as follows.
Prepare cDNA of antibody molecule.
Based on the prepared full-length cDNA of the antibody molecule, if necessary, a cDNA fragment of an appropriate length containing a portion encoding the protein is prepared.

A recombinant vector is prepared by inserting the DNA fragment or full-length cDNA downstream of the promoter of an appropriate expression vector.
By introducing the recombinant vector into the immortalized cell line of the present invention, a transformant producing an antibody composition can be obtained.
cDNA is the same as in 1. above. According to the “cDNA preparation method” described in (a) of (1), it can be prepared from a tissue or cell of a human or non-human animal using a probe primer specific for the antibody molecule of interest.

Examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [JP 3-22979; Cytotechnology, 3 , 133, (1990)], pAS3-3 [JP 2-27075], pCDM8 [Nature, 329 , 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochemistry, 101 , 1307 (1987) ], PAGE210 and the like.

  Any promoter can be used as long as it can be expressed in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein Examples include promoters, heat shock promoters, SRα promoters and the like. In addition, an IE gene enhancer of human CMV may be used together with a promoter.

Examples of the host cell include an immortalized cell line established from the knockout non-human animal of the α1,6-fucose modifying enzyme of the present invention established according to the method described in 2 above. Specific examples include cell lines such as LIV86 strain, LIV86-SV strain, KID86 strain, KID86-SV strain, Bom86 strain, Bom86-SV strain, and Fut8-/-strain.
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation [Cytotechnology, 3 , 133 (1990)], calcium phosphate method [JP-A-2-27075], lipofection method [Procedure of the National Academy of Science (Proc. Natl. Acad. Sci. USA), 84 , 7413 (1987)], injection method [manipulating・ Mouse Embryo 2nd edition], Method using particle gun (Gene gun) [Patent No. 2606856, Patent No. 2517813], DEAE-Dextran method [Bio-manual series 4-Gene introduction and expression / analysis method (Yodosha) Takashi Yokota and Kenichi Arai (1994)], virus vector method [Manipulating Mouse Embryo 2nd Edition] and the like.

As an expression method of the antibody composition, in addition to direct expression, secretory production, expression of a fusion protein between the Fc region and another protein, etc. may be performed according to the method described in Molecular Cloning 2nd edition, etc. it can.
The antibody composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the antibody composition in the culture, and collecting the composition from the culture. The method of culturing the transformant in the medium can be performed according to the usual method used for culturing the immortalized cell line of the present invention.

As a medium for culturing the obtained transformant, generally used RPMI1640 medium [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 [Procedure of the Society]・ For the Biological Medicine (Proceeding of the Society for the Biological Medicine), 73 , 1 (1950)], Whitten Medium [Genetic Engineering Experiment Manual-How to Make Transgenic Mice (Kodansha) edited by Motoya Katsuki (1987) )] Or a medium obtained by adding insulin, insulin-like growth factor, transferrin, albumin, or the like to these media.

The culture is usually performed for 1 to 7 days under conditions such as pH 6 to 8, 30 to 40 ° C., and the presence of 5% CO ₂ . The culture can be performed for 1 day to several months using a culture method such as fed-batch culture or follow-fiber culture.
Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

As described above, a transformant derived from the immortalized cell line of the present invention having a recombinant vector incorporating a DNA encoding an antibody molecule is cultured according to a normal culture method, and an antibody composition is produced and accumulated. By collecting the antibody composition from the culture, the antibody composition can be produced.
As a method for producing the antibody composition, a method for producing the antibody composition in the immortalized cell line of the present invention, a method for producing it secreted outside the immortalized cell line of the present invention, or a method for producing it on the outer membrane of the immortalized cell line of the present invention. The method can be selected by changing the type of the immortalized cell line of the present invention to be used and the structure of the antibody molecule to be produced.

When the antibody composition is produced in the immortal cell line of the present invention or on the outer membrane of the immortal cell line of the present invention, the method of Paulson et al. [J. Biol. Chem. , 264 , 17619 (1989)], Law et al. [Proc. Natl. Acad. Sci. USA], 86 , 8227 (1989); Gene Development (Genes Develop.), 4 , 1288 (1990)], or by applying the method described in Japanese Patent Application Laid-Open No. 05-336963, Japanese Patent Application Laid-Open No. 06-823021, etc. Can be actively secreted.

That is, using genetic recombination techniques, DNA encoding an antibody molecule and DNA encoding a signal peptide appropriate for expression of the antibody molecule are inserted into an expression vector, and the expression vector is used as the immortalized cell of the present invention. By expressing the antibody molecule after introduction into the strain, the target antibody molecule can be actively secreted outside the immortalized cell line of the present invention.
Further, according to the method described in JP-A-2-27075, the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.

  An antibody composition produced by a transformant into which a gene encoding an antibody molecule has been introduced, for example, when the antibody composition is expressed in a dissolved state in the cell, the cell is recovered by centrifugation after completion of the culture. After suspending in an aqueous buffer solution, the cells are disrupted with an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, a dynomill, or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the 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, diethylamino Anion exchange chromatography using resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (Mitsubishi Chemical Corporation), cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resin such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing etc. By using alone or in combination, a purified preparation of the antibody composition can be obtained.

Further, when the antibody composition is expressed by forming an insoluble substance in the cell, the cell is similarly collected and then crushed and centrifuged to collect the insoluble substance of the antibody composition as a precipitate fraction. The recovered insoluble body of the antibody composition is solubilized with a protein denaturant. The antibody composition is returned to a normal three-dimensional structure by diluting or dialyzing the solubilized solution, and then a purified preparation of the antibody composition can be obtained by the same isolation and purification method as described above.
When the antibody composition is secreted extracellularly, the antibody composition can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture with a technique such as centrifugation as described above, and the antibody composition is purified from the soluble fraction by using the same isolation and purification method as described above. A standard can be obtained.

Examples of the antibody composition thus obtained include antibodies, antibody fragments, fusion proteins having the Fc region of antibodies, and the like.
In the following, as a more specific example of obtaining an antibody composition, a method for producing a humanized antibody composition and an Fc fusion protein composition will be described, but other glycoproteins are also described in accordance with the above method and the method. Can be acquired.

A. Manufacture of humanized antibody composition (1) Construction of humanized antibody expression vector Humanized antibody expression vector is an expression vector for animal cells into which genes encoding H chain and L chain C region of human antibody are incorporated. It can be constructed by inserting genes encoding the H chain and L chain C region of a human antibody into an expression vector for animal cells, respectively.

Examples of the C region of a human antibody include the H chain and L chain C region of any human antibody. Specifically, the C region of the IgG1 subclass of the H chain of the human antibody (hereinafter referred to as hCγ1) and human Examples include the C region of the κ class of the L chain of the antibody (hereinafter referred to as hCκ).
As a gene encoding the H chain and L chain C region of a human antibody, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used.

Any expression vector for animal cells can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 [Cytotechnology, 3 , 133 (1990)], pAGE103 [J. Biochem., 101 , 1307 (1987)], pHSG274 [Gene, 27 , 223 (1984)], pKCR [Procedures of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78 , 1527 (1981)], pSG1βd2-4 [Cytotechnology ), 4 , 173 (1990)]. Promoters and enhancers used in animal cell expression vectors include SV40 early promoter and enhancer [J. Biochem., 101 , 1307 (1987)], Moloney murine leukemia virus LTR [biochemical And Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 149 , 960 (1987)], immunoglobulin heavy chain promoter [Cell, 41 , 479 (1985)] and enhancers [ Cell, 33 , 717 (1983)] and the like.

The humanized antibody expression vector can be used as either a type in which the antibody H chain and L chain are on separate vectors or a type on the same vector (hereinafter referred to as tandem type), Tandem-type humanized antibody expression in terms of the ease of construction of humanized antibody expression vectors, ease of introduction into animal cells, and the balance of expression levels of antibody H and L chains in animal cells. Vectors are preferred [J. Immunol. Methods, 167 , 271 (1994)].
The constructed humanized antibody expression vector can be used to express human chimeric antibodies and human CDR-grafted antibodies in animal cells.

(2) Obtaining cDNA encoding V region of non-human animal antibody Obtain cDNA of non-human animal antibody, for example, cDNA encoding mouse antibody H chain and L chain V regions as follows. Can do.

  MRNA is extracted from the hybridoma producing the desired mouse antibody, and cDNA is synthesized. The synthesized cDNA is inserted into a vector such as a phage or a plasmid to prepare a cDNA library. From this library, a recombinant phage or recombinant plasmid having a cDNA encoding an H chain V region and a cDNA encoding an L chain V region are used using the C region portion or V region portion of an existing mouse antibody as a probe. Recombinant phage or recombinant plasmid is isolated, respectively. Determine the entire nucleotide sequence of the cDNA encoding the H chain and L chain V region of the desired mouse antibody on the recombinant phage or recombinant plasmid, and estimate the entire amino acid sequence of the H chain and L chain V region from the base sequence .

Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, hamsters, rabbits and the like.
Methods for preparing total RNA from hybridoma cells include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154 , 3 (1987)], and mRNA from total RNA. Examples of the preparation method include an oligo (dT) -immobilized cellulose column method [Molecular Cloning 2nd Edition]. Examples of kits for preparing mRNA from hybridomas include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.

For cDNA synthesis and cDNA library preparation, conventional methods [Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology] or commercially available kits such as Super Script ^™ Plasmid System for cDNA Synthesis and plasmid Cloning (GIBCO BRL) and ZAP-cDNA Synthesis Kit (Stratagene).

When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from a hybridoma as a template can be incorporated. For example, ZAP Express [Strategies, 5 , 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)], λZAP II (Stratagene) ), Λgt10, λgt11 [DN Cloning: A Practical Approach, I , 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3 18U (Pharmacia) ), PcD2 [Mole. Cell. Biol., 3 , 280 (1983)], pUC18 [Gene, 33 , 103 (1985)] and the like.

Any Escherichia coli for introducing a cDNA library constructed by a phage or plasmid vector can be used as long as it can introduce, express and maintain the cDNA library. For example, XL1-Blue MRF '[Strategies, 5 , 81 (1992)], C600 [Genetics, 39 , 440 (1954)], Y1088, Y1090 [Science, 222 , 778 (1983)], NM522 [Journal of Molecular Biology (J. Mol. Biol.), 166 , 1 (1983)], K802 [Journal of Molecular Biology (J. Mol. Biol.)] , 16 , 118 (1966)] and JM105 [Gene, 38 , 275 (1985)] and the like are used.

  cDNA clones encoding H and L chain V regions of non-human animal antibodies from cDNA libraries can be obtained by colony hybridization or plaque hybridization using molecularly isolated or isotope or fluorescently labeled probes [molecular cloning. Second Edition] can be selected. Polymerase chain reaction (hereinafter referred to as PCR method; Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology) using primers and cDNA synthesized from mRNA or cDNA library as template ] Can also be used to prepare cDNAs encoding H chain and L chain V regions.

The cDNA selected by the above method is cleaved with an appropriate restriction enzyme or the like, and then inserted into a plasmid such as pBluescript SK (-) (Stratagene). Base sequence analysis of dideoxy method [Procedures of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or ABIPRISM377 DNA sequencer (PE Biosystems) It can confirm by analyzing using an apparatus.

  The entire amino acid sequences of the H chain and L chain V regions are deduced from the determined base sequences, and the entire amino acid sequences of the known antibody H chain and L chain V regions [Sequences of Proteins of Immunological Interest ( Sequences of Proteins of Immunological Interest), US Dept. Health and Human Services, 1991], the obtained cDNA encoded the complete amino acid sequence of the H chain and L chain V regions of the antibody including the secretory signal sequence. Can be confirmed.

(3) Analysis of V region amino acid sequence of non-human animal antibody Regarding the complete amino acid sequence of antibody H chain and L chain V region including secretory signal sequence, known antibody H chain and L chain V region The length and N-terminus of the secretory signal sequence by comparison with the full amino acid sequence of [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991] Amino acid sequences can be deduced, and the subgroup to which they belong can be known. In addition, the amino acid sequence of each CDR of the H chain and L chain V region is also the same as the amino acid sequence of the known antibody H chain and L chain V region [Sequences of Proteins of Immunological Interest (Sequences of Proteins ofImmunological Interest), US Dept. Health and Human Services, 1991].

(4) Construction of human chimeric antibody expression vector The humanized antibody expression vector according to (1) of A in (3) above is upstream of the gene encoding the H chain and L chain C regions of the human antibody. A human chimeric antibody expression vector can be constructed by inserting cDNAs encoding the H-chain and L-chain V regions of the animal antibody. For example, a cDNA encoding the H chain and L chain V region of a non-human animal antibody, the base sequence on the 3 ′ end side of the antibody H chain and L chain V region of a non-human animal, the H chain of the human antibody and The human DNA according to (1) of A in (3) above, which is ligated to a synthetic DNA consisting of a base sequence on the 5 ′ end side of the L chain C region and having an appropriate restriction enzyme recognition sequence at both ends. It is possible to construct a human chimeric antibody expression vector by inserting it into the upstream of the gene encoding the H chain and L chain C region of the human antibody of the vector for expression of the modified antibody so that they are expressed in an appropriate form.

(5) Construction of cDNA encoding V region of human CDR-grafted antibody A cDNA encoding H chain and L chain V region of human CDR-grafted antibody can be constructed as follows. First, select the amino acid sequence of the framework of the H chain and L chain V region (hereinafter referred to as FR) of the human antibody to which the CDR of the H chain and L chain V region of the target non-human animal antibody is grafted. . As the amino acid sequence of the FR of the H chain and L chain V region of a human antibody, any amino acid sequence can be used so long as it is derived from a human antibody. For example, human amino acid H chain and L chain V region FR amino acid sequences, human antibody H chain and L chain V region FR subgroup common amino acid sequences registered in databases such as Protein Data Bank [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991], among others, human CDR transplantation with sufficient activity In order to produce an antibody, it is desirable to select an amino acid sequence having as high a homology as possible (at least 60% or more) with the FR amino acid sequence of the H chain and L chain V region of the antibody of the target non-human animal. .

  Next, the CDR amino acid sequence of the H chain and L chain V region of the target non-human animal antibody is transplanted to the FR amino acid sequence of the H chain and L chain V region of the selected human antibody, and human CDR grafting The amino acid sequences of the H chain and L chain V regions of the antibody are designed. Frequency of codon usage in the amino acid sequence of the designed amino acid sequence [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991] Is converted to a DNA sequence, and a DNA sequence encoding the amino acid sequence of the H chain and L chain V regions of the human CDR-grafted antibody is designed. Based on the designed DNA sequence, the DNA is synthesized by dividing it into several synthetic DNAs having a length of about 100 bases, and the synthesized DNAs are ligated by PCR. In this case, it is preferable to design and link 6 synthetic DNAs for each of the H chain and L chain from the reaction efficiency in PCR and the length of DNA that can be synthesized.

  In addition, by inserting an appropriate restriction enzyme recognition sequence into the 5 ′ end of the synthetic DNA located at both ends, it can be easily inserted into the humanized antibody expression vector constructed in (1) of A in this section 3. Can do. After PCR, the amplified product is inserted into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the nucleotide sequence is determined by the method described in A, (2) of this section 3, and the desired human CDR grafted A plasmid having a DNA sequence encoding the amino acid sequence of the H chain and L chain V region of the antibody is obtained.

(6) Modification of V region amino acid sequence of human CDR-grafted antibody The human CDR-grafted antibody is obtained by converting only the CDR of the H chain and L chain V region of the target non-human animal antibody to the H chain and L of the human antibody. It is known that the antigen-binding activity is reduced only by grafting to the FR of the chain V region as compared with the antibody of the original non-human animal [BIO / TECHNOLOGY, 9 , 266 (1991)]. This is because, in the H chain and L chain V region of the antibody of the original non-human animal, not only CDR but also some amino acid residues of FR are directly or indirectly involved in antigen binding activity. These amino acid residues are considered to change to different amino acid residues in the FRs of the H and L chain V regions of human antibodies with CDR grafting. In order to solve this problem, in human CDR-grafted antibodies, amino acid residues that are directly involved in binding to the antigen or CDR amino acids in the FR amino acid sequences of the H and L chain V regions of human antibodies. Identify amino acid residues that interact with residues, maintain antibody conformation, and indirectly participate in antigen binding, and identify those amino acid residues found in the original non-human animal antibody. It has been carried out to increase the decreased antigen binding activity [Bio / Technology (BIO / TECHNOLOGY), 9 , 266 (1991)].

In the production of human CDR-grafted antibodies, the most important point is how to efficiently identify the amino acid residues of FRs involved in these antigen-binding activities. Therefore, X-ray crystallography [Journal of Molecular・ Construction and analysis of antibody three-dimensional structure by biology (J.Mol. Biol.), 112 , 535 (1977)] or computer modeling [Protein Engineering, 7 , 1501 (1994)] etc. ing. The information on the three-dimensional structure of these antibodies has provided a great deal of useful information for the production of human CDR-grafted antibodies. However, at present, various kinds of trial and error are required, such as preparing several types of variants for each antibody and examining the correlation with each antigen-binding activity.

  Modification of the FR amino acid residues of the H chain and L chain V region of the human antibody can be achieved by the method described in A (5) of this section 3 using synthetic DNA for modification. For the amplified product after PCR, the base sequence is determined by the method described in A, (2) of this section 3, and it is confirmed that the target modification has been performed.

(7) Construction of human CDR-grafted antibody expression vector In the upstream of the gene encoding the human antibody H chain and L chain C regions of the humanized antibody expression vector described in A of (1) of this section 3 A cDNA encoding the H chain and L chain V regions of the human CDR-grafted antibody constructed in (5) and (6) of A of Item 3 can be inserted to construct a human CDR-grafted antibody expression vector. For example, among the synthetic DNAs used for constructing the H chain and L chain V regions of the human CDR-grafted antibody in (5) and (6) of A in this section 3, the 5 ′ end of the synthetic DNA located at both ends By introducing a restriction enzyme recognition sequence into the upstream of the gene encoding the H chain and L chain C regions of the human antibody of the humanized antibody expression vector described in A of (1) of this section 3 They are inserted so that they are expressed in an appropriate form, and a human CDR-grafted antibody expression vector can be constructed.

(8) Stable production of humanized antibody By introducing the humanized antibody expression vector according to (4) and (7) of A in this section 3 into an appropriate host animal cell, a human chimeric antibody and a human CDR A transformant that stably produces a transplanted antibody (hereinafter also referred to as a humanized antibody) can be obtained.

Examples of methods for introducing humanized antibody expression vectors into animal cells include electroporation (Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3 , 133 (1990)).
As an animal cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it is an immortalized cell line of the present invention capable of producing a humanized antibody.

Specifically, cell lines such as the LIV86 strain, the LIV86-SV strain, the KID86 strain, the KID86-SV strain, the Bomb86 strain, the Bomb86-SV strain, the Fut8 − / − strain described in the examples of the present specification can be mentioned. .
A transformed strain that stably produces a humanized antibody after introduction of the humanized antibody expression vector is G418 sulfate (hereinafter referred to as G418; manufactured by SIGMA) according to the method disclosed in JP-A-2-257891. It can be selected by an animal cell culture medium containing a drug such as As an animal cell culture medium, RPMI1640 medium (manufactured by Nissui Pharmaceutical), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMDM medium (manufactured by GIBCO BRL), Hybridoma-SFM medium (Manufactured by GIBCO BRL) or a medium obtained by adding various additives such as insulin, insulin-like growth factor, transferrin, and albumin to these media. By culturing the obtained transformant in a medium, the humanized antibody can be produced and accumulated in the culture supernatant. The production amount and antigen binding activity of the humanized antibody in the culture supernatant are determined by enzyme-linked immunosorbent assay (hereinafter referred to as ELISA method; Antibodies: A Laboratory Manual), Cold Spring Harbor Laboratory, Chapter 14, 1998, Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996]. In addition, the transformed strain can increase the production amount of the humanized antibody using a DHFR gene amplification system or the like according to the method disclosed in JP-A-2-57891.

The humanized antibody can be purified from the culture supernatant of the transformant using a protein A column [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988. Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996]. In addition, 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 humanized antibody H chain, L chain or whole antibody molecule is determined by polyacrylamide gel electrophoresis (hereinafter referred to as SDS-PAGE; Nature, 227 , 680 (1970)) or Western blotting. [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 12, 1988, Monoclonal Antibodies: Principles and Practice, Academic Press Limited , 1996].

B. Production of Fc fusion protein (1) Construction of Fc fusion protein expression vector
An Fc fusion protein expression vector is an expression vector for an animal cell in which a gene encoding a protein to be fused with the Fc region of a human antibody is incorporated. It can be constructed by inserting a gene encoding.
The Fc region of a human antibody includes not only a region containing CH2 and CH3 regions, but also a region containing a hinge region and part of CH1. In addition, any amino acid may be used as long as at least one amino acid of CH2 or CH3 is deleted, substituted, added and / or inserted and has a binding activity to the Fcγ receptor substantially.

  As a gene encoding a protein to be fused with the Fc region of a human antibody, chromosomal DNA consisting of exons and introns can be used, and cDNA can also be used. As a method of linking these genes and Fc regions, PCR can be performed using each gene sequence as a template (Regular Cloning 2nd Edition; Current Protocols in Molecular Biology, Supplement 1-34). can give.

Any expression vector for animal cells can be used as long as it can incorporate and express a gene encoding the C region of a human antibody. For example, pAGE107 [Cytotechnology, 3 , 133 (1990)], pAGE103 [Journal of Biochemistry (J. Biochem.), 101 , 1307 (1987)], pHSG274 [Gene, 27 , 223 (1984)], pKCR [Procedure of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78 , 1527 (1981)], pSG1βd2-4 [Cytotechnology ), 4 , 173 (1990)]. Promoters and enhancers used in animal cell expression vectors include SV40 early promoter and enhancer [J. Biochem., 101 , 1307 (1987)], Moloney murine leukemia virus LTR [biochemical And Biophysical Research Communications (Biochem. Biophys. Res. Commun.), 149 , 960 (1987)], immunoglobulin heavy chain promoter [Cell, 41 , 479 (1985)] and enhancer [ Cell, 33 , 717 (1983)] and the like.

(2) Obtaining DNA encoding a protein to be fused with the Fc region of a human antibody DNA encoding a protein to be fused with the Fc region of a human antibody can be obtained as follows.
MRNA is extracted from cells or tissues expressing a protein to be fused with the target Fc, and cDNA is synthesized. The synthesized cDNA is inserted into a vector such as a phage or a plasmid to prepare a cDNA library. A recombinant phage or recombinant plasmid having cDNA encoding the target protein is isolated from the library using the gene sequence portion of the target protein as a probe. The entire base sequence of the target protein on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence is deduced from the base sequence.
As animals other than humans, any mouse, rat, hamster, rabbit, etc. can be used as long as cells and tissues can be removed.

Methods for preparing total RNA from cells and tissues include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154 , 3 (1987)], and mRNA from total RNA. Examples of the method for preparing the above include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, Second Edition). Examples of kits for preparing mRNA from cells and tissues include FastTrack mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
For cDNA synthesis and cDNA library preparation, conventional methods (Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology) or commercially available kits such as SuperScript ^™ Plasmid System for cDNA Synthesis and Examples include methods using Plasmid Cloning (GIBCOBRL) and ZAP-cDNA Synthesis Kit (Stratagene).

When preparing a cDNA library, any vector can be used as a vector into which cDNA synthesized using mRNA extracted from cells or tissues as a template is incorporated. For example, ZAPExpress [Strategies, 5 , 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)], λZAPII (Stratagene), λgt10, λgt11 [DNA Cloning: A Practical Approach, I , 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3 18U (Pharmacia), pcD2 [Mole. Cell. Biol., 3 , 280 (1983)] and pUC18 [Gene, 33 , 103 (1985)] are used.

Any Escherichia coli for introducing a cDNA library constructed by a phage or plasmid vector can be used as long as it can introduce, express and maintain the cDNA library. For example, XL1-BlueMRF '[Strategies, 5 , 81 (1992)], C600 [Genetics, 39 , 440 (1954)], Y1088, Y1090 [Science, 222 , 778 ( 1983)], NM522 [Journal of Molecular Biology (J. Mol. Biol.), 166 , 1 (1983)], K802 [Journal of Molecular Biology (J. Mol. Biol.), 16 , 118 (1966)] and JM105 [Gene, 38 , 275 (1985)] and the like are used.

  Selection of cDNA clones encoding the protein of interest from the cDNA library is done by colony hybridization or plaque hybridization (Molecular Cloning Second Edition) using isotopes or fluorescently labeled probes. be able to. Alternatively, a cDNA encoding a target protein can be prepared by PCR using a primer prepared and a cDNA or cDNA library synthesized from mRNA as a template.

  Examples of the method for fusing the cDNA encoding the protein of interest with the cDNA encoding the Fc region of a human antibody include the PCR method. For example, an arbitrary synthetic oligo DNA (primer) is set on the 5 ′ side and 3 ′ side of the gene sequence of the target protein, and PCR is performed to obtain a PCR product. Similarly, an arbitrary primer is set for the gene sequence of the Fc region of the human antibody to be fused to obtain a PCR product. At this time, primers are set so that the same restriction enzyme recognition site or the same gene sequence exists on the 3 ′ side of the PCR product of the protein to be fused and the 5 ′ side of the PCR product of the Fc region. When amino acid modification around this linking portion is necessary, the mutation is introduced by using the primer into which the mutation has been introduced. Both genes are linked by further PCR using the two types of PCR fragments obtained. Alternatively, ligation can also be performed by performing a ligation reaction after the same restriction enzyme treatment.

The gene sequence linked by the above method is cleaved with an appropriate restriction enzyme and inserted into a plasmid such as pBluescriptSK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger et al. The dideoxy method [Procedures of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or ABI PRISM 377 DNA sequencer (PE Biosystems) and other bases By analyzing using a sequence analyzer, the base sequence of the DNA can be determined.
Estimate the entire amino acid sequence of the Fc fusion protein from the determined nucleotide sequence and compare it with the target amino acid sequence to confirm that the obtained cDNA encodes the complete amino acid sequence of the Fc fusion protein including the secretory signal sequence can do.

(3) Stable production of Fc fusion protein By introducing the Fc fusion protein expression vector described in (1) above into an appropriate host animal cell, a transformant that stably produces Fc fusion protein can be obtained. it can.
Examples of a method for introducing an Fc fusion protein expression vector into animal cells include electroporation (Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3 , 133 (1990)).
As an animal cell into which the Fc fusion protein expression vector is introduced, any cell can be used as long as it is an immortalized cell line of the present invention capable of producing an Fc fusion protein.
Specifically, cell lines such as the LIV86 strain, the LIV86-SV strain, the KID86 strain, the KID86-SV strain, the Bomb86 strain, the Bomb86-SV strain, the Fut8 − / − strain described in the examples of the present specification can be mentioned. .

  After the introduction of the Fc fusion protein expression vector, a transformant that stably produces the Fc fusion protein composition is selected by an animal cell culture medium containing a drug such as G418 according to the method disclosed in JP-A-2-257891. it can. As an animal cell culture medium, RPMI1640 medium (manufactured by Nissui Pharmaceutical), GIT medium (manufactured by Nippon Pharmaceutical), EX-CELL302 medium (manufactured by JRH), IMDM medium (GIBCO BRL), Hybridoma-SFM medium (Manufactured by GIBCO BRL) or a medium obtained by adding various additives such as insulin, insulin-like growth factor, transferrin, and albumin to these mediums can be used. By culturing the obtained transformant in a medium, the Fc fusion protein composition can be produced and accumulated in the culture supernatant. The production amount and antigen binding activity of the Fc fusion protein in the culture supernatant can be measured by ELISA or the like. The transformed strain can increase the production amount of Fc fusion protein using a dhfr gene amplification system or the like according to the method disclosed in JP-A-2-57891.

The Fc fusion protein composition can be purified from the culture supernatant of the transformant using a protein A column or protein G column (Antibodies, Chapter 8, Monoclonal Antibodies). In addition, 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 total molecular weight of the purified Fc fusion protein molecule should be measured by SDS-PAGE [Nature, 227 , 680 (1970)], Western blotting (Antibodies, Chapter 12, Monoclonal Antibodies), etc. Can do.

As described above, the antibody composition and the Fc fusion protein composition production method using the immortalized cell line of the present invention have been shown as examples. As described above, the immortalized cell line of the present invention is a glycoprotein other than these. It can also be used for the production of compositions.
In the case of an immortalized cell line that already has the ability to express a glycoprotein such as an antibody molecule, the cell line is cultured, and the desired antibody composition or glycoprotein composition is purified from the culture. By doing so, the antibody composition and glycoprotein composition of the present invention can be produced.

4). Evaluation of activity of glycoprotein composition Protein amount of purified glycoprotein composition, affinity with receptor, half-life in blood, distribution to tissue after blood administration, or protein interaction required for expression of pharmacological activity As a method to measure the change of the current, Current Protocols In Protein Science, John Wiley & Sons Inc., (1995), Japan Biochemical Society edited new life chemistry experiment lecture 19 animal experiment method, Tokyo Chemical Dojin (1991), Japan Biochemical Society new life Chemistry Experiment Course 8 Intracellular Information and Cell Response, Tokyo Chemical Doujin (1990), Japan Biochemical Society, New Chemistry Experiment Course 9 Hormone I Peptide Hormones, Tokyo Chemical Doujin (1991), Laboratory Biology Course 3 Isotope Experiment, Maruzen Stock Company (1982), Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., (1995), Enzyme Immunoassay 3rd Edition, Medical School (1987), Revised Enzyme Antibody Method, Interdisciplinary Planning (1985), etc. The known methods described can be used.

As a specific example, the purified glycoprotein composition is labeled with a compound such as a radioisotope, and the strength of the binding reaction with the receptor or interacting protein of the labeled glycoprotein composition is quantitatively determined. A method of measuring is given. In addition, protein-protein interactions can be measured using various devices such as Biacore's BIAcore series (J. Immnunol. Methods, 145 , 229 (1991)). Interaction experiment method, Yodosha (1996)).
By administering the labeled glycoprotein composition into the body, it is possible to know the half-life in blood or the distribution to the tissue after blood administration. A system in which a glycoprotein-specific antibody-antigen reaction to be detected is combined is preferable.

5). Evaluation of activity of antibody composition As a method for measuring the protein amount, antigen binding or effector function of the purified antibody composition, a known method described in Monoclonal Antibodies or Antibody Engineering may be used. it can.
As a specific example, when the antibody composition is a humanized antibody, the binding activity to the antigen and the binding activity to the antigen-positive cultured cell line are determined by ELISA method and fluorescent antibody method [Cancer Immunol. Immunother.), 36 , 373 (1993)]. The cytotoxic activity against an antigen-positive cultured cell line can be evaluated by measuring CDC activity, ADCC activity, etc. [Cancer Immunology. Immunother., 36 , 373 (1993)].
Moreover, the safety and therapeutic effect of the antibody composition in humans can be evaluated using an appropriate model of animal species relatively close to humans such as cynomolgus monkeys.

6). Analysis of sugar chains of glycoprotein composition and antibody composition The sugar chain structure of the glycoprotein composition and antibody composition expressed in the immortalized cell line of the present invention should be performed in accordance with the analysis of normal sugar chain structure. Can do. For example, sugar chains bound to IgG molecules are composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetylglucosamine, and acidic sugars such as sialic acid. It can be performed using a method such as a sugar chain structure analysis using a sugar chain map method or the like.

(1) Neutral sugar / amino sugar composition analysis The composition analysis of a sugar chain of a glycoprotein composition or an antibody composition is carried out by conducting acid hydrolysis of the sugar chain with trifluoroacetic acid or the like to obtain a neutral sugar or amino sugar. And the composition ratio can be analyzed.

As a specific method, there is a method using a sugar composition analyzer (BioLC) manufactured by Dionex. BioLC is a device for analyzing sugar composition by HPAEC-PAD (high performance anion-exchange chromatography-pulsed amperometric detection) [J. Liq. Chromatogr., 6 , 1577 (1983)]. is there.
The composition ratio can also be analyzed by fluorescence labeling with 2-aminopyridine. Specifically, a sample obtained by acid hydrolysis according to a known method [Agruc.Biol.Chem., 55 (1) , 283-284 (1991)] It is possible to calculate the composition ratio by labeling with fluorescence and HPLC analysis.

(2) Sugar chain structure analysis The structure analysis of sugar chains of glycoprotein compositions and antibody compositions is performed by two-dimensional sugar chain mapping method [Anal. Biochem., 171 , 73 (1988), Biology Chemistry Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center) Takahashi Eiko (1989)]. In the two-dimensional glycan mapping method, for example, the retention time or elution position of reversed-phase chromatography glycans is plotted on the X axis, and the retention time or elution position of glycans by normal phase chromatography is plotted on the Y axis. This is a method for estimating the sugar chain structure by comparing with those of known sugar chains.

Specifically, glycoprotein compositions and compositions are hydrolyzed to liberate sugar chains, and fluorescent labeling of sugar chains with 2-aminopyridine (hereinafter abbreviated as PA) [Journal of Biochemistry (J Biochem.), 95 , 197 (1984)], and then the glycan is separated from excess PA reagent by gel filtration and subjected to reverse phase chromatography. Next, normal phase chromatography is performed on each peak of the separated sugar chain. Based on these results, a plot is made on a two-dimensional glycan map, with glycan standards (TaKaRa), literature [Analytical Biochemistry, 171 , 73 (1988)]. The sugar chain structure can be estimated from spot comparison.
Furthermore, mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain map method.

7). Use of Glycoprotein Composition of the Present Invention Glycoprotein compositions such as the antibody composition of the present invention have a sugar chain structure without fucose modification, for example, improved affinity with receptors, half-blood It exhibits high physiological activity because it can be expected to have effects such as prolongation of phase, improvement of tissue distribution after administration in blood, or improvement of interaction with proteins necessary for expression of pharmacological activity. In particular, the antibody composition produced from the immortalized cell line of the present invention has a high effector function, that is, antibody-dependent cytotoxic activity. These highly bioactive glycoprotein compositions or antibody compositions with high ADCC activity are used in various diseases including cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, and viral or bacterial infections. Useful in the prevention and treatment of disease.

Cancer cells are proliferating in cancer, that is, malignant tumor. Ordinary anticancer agents are characterized by inhibiting the growth of cancer cells. However, an antibody having high antibody-dependent cytotoxic activity is more effective as a therapeutic agent than a normal anticancer agent because it can treat cancer by damaging cancer cells by cell killing effect. Particularly in the case of therapeutic agents for cancer, the anti-tumor effect of antibody drugs alone is often insufficient at present, and combination therapy with chemotherapy is performed [Science, 280 , 1197, 1998]. If a stronger antitumor effect is observed with the antibody composition alone, the dependence on chemotherapy is reduced, and side effects are reduced.

In immune diseases such as inflammatory diseases, autoimmune diseases, and allergies, in vivo reactions in these diseases are triggered by the release of mediator molecules by immune cells, so immune cells using antibodies with high antibody-dependent cytotoxic activity are used. By removing, allergic reaction can be suppressed.
Examples of cardiovascular diseases include arteriosclerosis. Atherosclerosis is currently treated with a balloon catheter, but it prevents and treats cardiovascular disease by suppressing the proliferation of arterial cells in restenosis after treatment with an antibody having high antibody-dependent cytotoxic activity. be able to.

By suppressing the growth of cells infected with viruses or bacteria using an antibody having high antibody-dependent cytotoxic activity, various diseases including viral or bacterial infection can be prevented and treated.
Antibodies that recognize tumor-related antigens, antibodies that recognize antigens related to allergy or inflammation, antibodies that recognize antigens related to cardiovascular disease, antibodies that recognize antigens related to autoimmune diseases, or viruses or bacterial infections Specific examples of antibodies that recognize related antigens are described below.

As antibodies that recognize tumor-associated antigens, anti-GD2 antibodies (Anticancer Res., 13 , 331, 1993), anti-GD3 antibodies (Cancer Immunol. Immunother., 36 , 260, 1993), anti-GM2 antibodies (Cancer Res., 54 , 1511, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci. USA, 89 , 4285, 1992), anti-CD52 antibody (Nature, 332 , 323, 1988), anti-MAGE antibody (British J. Cancer, 83 , 493, 2000), anti-HM1.24 antibody (MolecularImmunol., 36 , 387, 1999), anti-parathyroid hormone related protein (PTHrP) antibody (Cancer, 88 , 2909, 2000), anti-FGF8 antibody (Proc. Natl Acad. Sci. USA, 86 , 9911, 1989) Anti-basic fibroblast growth factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265 , 16455, 1990), anti-basic fibroblast proliferation factor receptor antibody, anti-insulin-like growth factor antibody (J.Neurosci. Res., 40, 647, 1995), anti-insulin-like growth factor receptor antibody (J. Neurosci. Res., 40 , 647, 1995), anti PMSA antibody (J. Urology, 160 , 2396, 1998), increased anti-vascular endothelial cells Growth factor antibody (CancerRes., 57 , 4593, 1997) or anti-vascular endothelial growth factor receptor antibody (Oncogene, 19 , 2138, 2000), anti-CA125 antibody, anti-17-1A antibody, anti-integrin αvβ3 antibody, anti-CD33 Antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD19 antibody, anti-EGF receptor antibody (Immunology Today, 21 , 403, 2000), anti-CD10 antibody (American Journal of Clinical Pathology, 113 , 374, 2000).

Anti-interleukin-6 antibody (Immunol. Rev., 127 , 5, 1992), anti-interleukin-6 receptor antibody (MolecularImmunol., 31 , 371, 1994), as an antibody that recognizes antigens related to allergy or inflammation Anti-interleukin 5 antibody (Immunol. Rev., 127 , 5, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3 , 562, 1991), anti-interleukin 4 receptor antibody (J Immunol. Meth., 217 , 41, 1998), anti-tumor necrosis factor antibody (Hybridoma, 13 , 183, 1994), anti-tumor necrosis factor receptor antibody (Molecular Pharmacol., 58 , 237, 2000), anti-CCR4 antibody (Nature, 400 , 776, 1999), anti-chemokine antibody (J. Immunol. Meth., 174 , 249, 1994), anti-chemokine receptor antibody (J. Exp. Med., 186 , 1373, 1997), anti-IgE Antibody, anti-CD23 antibody, anti-CD11a antibody (Immunology Today, 21 , 403, 2000), anti-CRTH2 antibody (J. Immunol., 162 , 1278, 1999), anti-CCR8 antibody (WO99 / 2) 5734), anti-CCR3 antibody (US6207155) and the like.

Antibodies that recognize antigens associated with cardiovascular diseases include anti-GpIIb / IIIa antibodies (J. Immunol., 152 , 2968, 1994), antiplatelet-derived growth factor antibodies (Science, 253 , 1129, 1991), antiplatelets Examples thereof include a derived growth factor receptor antibody (J. Biol. Chem., 272 , 17400, 1997) or an anti-blood coagulation factor antibody (Circulation, 101 , 1158, 2000).
Antibodies that recognize antigens associated with autoimmune diseases (specific examples include psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis) include anti-self DNA antibodies ( Immunol. Letters, 72 , 61, 2000), anti-CD11a antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin α4β7 antibody, anti-CD40L antibody, anti-IL-2 receptor And antibodies (ImmunologyToday, 21 , 403, 2000).

Antibodies that recognize antigens associated with viral or bacterial infections include anti-gp120 antibodies (Structure, 8 , 385, 2000), anti-CD4 antibodies (J. Rheumatology, 25 , 2065, 1998), anti-CCR5 antibodies, anti-verotoxins And antibodies (J. Clin. Microbiol., 37 , 396, 1999).
The above antibodies can be obtained from public institutions such as ATCC (The American Type Culture Collection), RIKEN Cell Development Bank, Institute of Biotechnology, Dainippon Pharmaceutical Co., Ltd., R & DSYSTEMS, PharMingen, Cosmo Bio. It can be obtained from private reagent sales companies such as Funakoshi Co., Ltd.
Specific examples of the fusion antibody of the binding protein other than the above-described antibody of the present invention and the antibody Fc region are described below.

  Specific examples of proteins and antibody Fc region fusion proteins related to immune diseases such as inflammatory diseases, autoimmune diseases and allergies include etanercept (USP5605690), a fusion protein of sTNFRII and antibody Fc regions, on antigen-presenting cells. Alefacept (USP5914111), a fusion protein of expressed LFA-3 and Fc region, fusion protein of CytotoxicT Lymphocyte-associated antigen-4 (CTLA-4) and antibody Fc region (J. Exp. Med., 181 , 1869, 1995), fusion protein of interleukin-15 and antibody Fc region (J. Immunol., 160, 5742, 1998), fusion protein of factor VII and antibody Fc region (Proc. Natl. Acad. Sci. USA, 98, 12180, 2001), fusion protein of interleukin 10 and antibody Fc region (J. Immunol., 154, 5590, 1995), fusion protein of interleukin 2 and antibody Fc region (J. Immunol., 146, 915, 1991), fusion protein of CD40 and antibody Fc region (Surgery. 132, 149, 2002), Flt-3 (fms-like tyrosine kinase) and antibody Fc Fusion protein with frequency (Acta. Haemato., 95, 218, 1996) and OX40 and fusion protein with an antibody Fc region (J. Leu. Biol., 72, 522, 2002) and the like, such as. In addition to these, various human CD molecules [CD2, CD30 (TNFRSF8), CD95 (Fas), CD106 (VCAM-1), CD137] and adhesion molecules [ALCAM (Activated leukocyte cell adhesion molecule), Cadherins, ICAM (Intercellular adhesionmolecule) ) -1, ICAM-2, ICAM-3], cytokine receptor (hereinafter referred to as R) (IL-4R, IL-5R, IL-6R, IL-9R, IL-10R, IL- 12R, IL-13Rα1, IL-13Rα2, IL-15R, IL-21R), chemokine, cell death induction signal molecule [B7-H1, DR6 (Deathreceptor 6), PD-1 (Programmed death-1), TRAIL R1, ], Costimulatory molecules [B7-1, B7-2, B7-H2, ICOS (Induciblecostimulator)], growth factors (ErbB2, ErbB3, ErbB4, HGFR) or differentiation factor (B7-H3), activator (NKG2D) ), A signal transduction molecule (gp130), a receptor protein of the binding protein, a fusion protein of a ligand and an antibody Fc region, and the like.

The medicament containing the glycoprotein composition or antibody composition of the present invention can be administered alone as a therapeutic agent, but usually together with one or more pharmacologically acceptable carriers. It is desirable to mix and provide as a pharmaceutical formulation produced by any method well known in the pharmaceutical arts.
It is desirable to use the most effective route for treatment, and oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous can be used. In the case of a preparation, intravenous administration can be preferably mentioned.

Examples of the dosage form 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 saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.

  Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both. Alternatively, a powder injection can be prepared by freeze-drying a glycoprotein composition or antibody 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 a carrier that does not irritate the glycoprotein composition or antibody composition itself or the recipient's oral cavity and airway mucosa, and facilitates absorption by dispersing the glycoprotein composition or antibody composition as fine particles. Etc. are used.
Specific examples of the carrier include lactose and glycerin. Depending on the nature of the glycoprotein composition or antibody composition and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations 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., but is usually 10 μg / kg to 20 mg / kg per day for an adult.
In addition, as a method for examining the antitumor effect of the antibody composition on various tumor cells, in vitro experiments include CDC activity measurement methods, ADCC activity measurement methods, etc., and in vivo experiments include experimental animals such as mice. Examples include antitumor experiments using tumor systems.
CDC activity, ADCC activity, and anti-tumor experiments are described in the literature [Cancer Immunology Immunotherapy, 36 , 373 (1993); Cancer Research, 54 , 1511 (1994)]. Can be done according to.
The present invention will be described more specifically with reference to the following examples. However, the examples are merely illustrative of the present invention and do not limit the scope of the present invention.

Creation of transgenic mice lacking both α1,6-fucosyltransferase alleles Creation of mice lacking the genomic region including the translation initiation codon for both α1,6-fucosyltransferases (hereinafter also referred to as FUT8) It went as follows.

1. Isolation of the genomic region containing the mouse FUT8 gene translation initiation codon 5 'untranslated region of the full-length porcine FUT8 cDNA [J.Biol. Chem., 271, 27810 (1996)] A fragment (412 bp) containing a translation region of 373 bp was prepared from 39 bp by digestion with restriction enzyme Sac I. Using this as a probe, according to the known method described in Molecular Cloning 2nd edition, a 13.9 Kb genomic clone containing an exon where the mouse FUT8 translation start codon is located from the 129SVJ strain mouse-derived λ-phage genomic library (STRATEGENE) Was isolated (Figure 1).

Next, the obtained genomic clone was digested with various restriction enzymes, and then Southern blotting was performed according to a known method described in Molecular Cloning, Second Edition, using the porcine FUT8 cDNA partial fragment 412 bp as a probe. Among the restriction enzyme fragments that showed positive results, the exon and the downstream intron region where the translation initiation codon is located, and the approximately 3.0 Kb Xba I- Xba I fragment encoding the exon and upstream intron region where the translation initiation codon is located. The encoding Sac I- Sac I fragment of about 6.3 Kb was selected and inserted into pBluescriptIIKS (+) (Strategene) (FIG. 1).

2. Construction of Targeting Vector Plasmid for Disrupting the Mouse FUT8 Gene Translation Start Codon Among the approximately 3.0 Kb Xba I- Xba I region obtained in Example 1 of this Example, the 5 '2.6 Kb Xba I- Sac I 'while the side homologous region of the Sac I- Sac I region of about 6.3Kb obtained in the above item 1 of this example, 3' regions 5 of the side Hin dIII- Xho I region (about 6.1 Kb) 3 by respectively disposed as' side region of homology was constructed lacking targeting vector plasmid Sac I- Hin dIII region (184 bp) encompassing the translation initiation codon. The details are shown below.

First, plasmid pMC1DTpA [Transgenic Research, 8, 215 (1999)] was digested with restriction enzymes Xho I and Not I, and the resulting 1.5 Kb containing the diphtheria toxin A chain (DT-A) gene. Both ends were replaced with Xho I recognition sites by ligating Not I- Xho I adapters to the fragments. On the other hand, the restriction enzyme Xho I was reacted with pBluescriptIIKS (+) containing about 6.3 Kb of Sac I- Sac I region on the 3 ′ side of the FUT8 gene obtained in the first section of this example, and about 6.1 Kb of Hin dIII- An approximately 8.3 Kb fragment containing the Xho I region was obtained. The fragment of about 8.3 Kb and the 1.5 Kb Xho I- Xho I fragment (1.5 Kb) containing the DT-A gene were ligated to construct plasmid I.

Next, pGT1.8IresBgeo [Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 91, 4303 (1994)] was completely digested with restriction enzyme Sal I, and then further digested. By partial digestion with the restriction enzyme Sac I, an internal ribosome translation initiation site (hereinafter referred to as IRES), β-galactosidase gene (hereinafter referred to as LacZ), neomycin resistance gene (hereinafter referred to as Neo ^r ) and poly A A 4.9 Kb fragment containing an expression unit for drug selection (hereinafter referred to as IRES-LacZ-Neor-pA cassette) linked with an additional signal (hereinafter referred to as pA) was obtained.

On the other hand, pBluescriptIIKS (+) containing about 2.9 Kb of Xba I- Xba I region on the 5 ′ side of the mouse FUT8 gene obtained in Example 1 of the present example was digested with restriction enzymes Sac I and Sal I to obtain about 2.6 Kb. An approximately 5.3 Kb fragment containing the Xba I- Sac I region was obtained. By connecting the Sac I- Sal I fragment of about 4.9Kb containing the Sac I- Sal I fragment and IRES-LacZ-Neor-pA cassette from about 5.3Kb containing the 5 'homology region of the mouse FUT8 gene obtained above Plasmid II was constructed.
Finally, the fragment of about 9.4Kb obtained by digesting the plasmid I restriction enzymes Not I and Sal I, ligated a fragment of about 7.6Kb obtained by digesting the plasmid II restriction enzymes Not I and Sal I Thus, the targeting vector plasmid for disrupting the mouse FUT8 gene shown in FIG. 2 was constructed.

3. Homologous recombination of the FUT8 gene genomic region in mouse embryonic stem cells (1) Preparation of feeder cells Mouse embryonic stem cells used for homologous recombination were maintained in culture using mouse primary fibroblasts (hereinafter referred to as EMFI cells) as feeders. . The feeder cells used for culture maintenance of embryonic stem cells were prepared according to the following description.

First, as described in Gene Targeting [GeneTargeting, IRL PRESS at Oxford University Press (1993)], female neomycin-resistant transgenic mice [calmegin knockout mice, Nature, 387 , 607, (1997), 8 weeks of age or older. )], Fetuses from 13.5 days to 15.5 days after fertilization were removed, and primary fibroblasts (EMFI cells) were prepared using the fetuses as materials. Then, the proliferation ability was inactivated by mitomycin C treatment. Subsequently, mitomycin C-treated EMFI cells were treated with FM medium [10% fetal bovine serum (Invitrogen), 55 μM β-mercaptoethanol (Invitrogen), 1 mM MEM sodium pyruvate (Invitrogen), 0.1 mM MEM non-essential amino acid (Invitrogen) ), 3 mM adenosine (SIGMA), 3 mM guanosine (SIGMA), 3 mM cytidine (SIGMA), 3 mM uridine (SIGMA), 1 mM thymidine (SIGMA), 2 mM L-glutamine (Invitrogen), 100 units / ml Suspended in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) containing penicillin and 100 μg / ml streptomycin (Invitrogen)] to 2.5 × 10 ⁵ cells / ml, and then 0.1% gelatin-coated cell culture dish (6cm diameter, 10cm diameter; Asahi Techno Glass Co., Ltd.) or 0.1% gelatin-coated flat bottom plate (96 holes, 24 holes, 6 holes; Asahi Techno Glass Co., Ltd.), 5% CO ₂ at 37 ° C At 24 A feeder plate was prepared by culturing for a period of time. The prepared feeder plate was used within one week after preparation. The feeder cells are not necessarily used as the feeder cells. For example, Neo-resistant primary cultured cells (catalog number YE9284100) sold by Lifetech Oriental Co., Ltd. can be used.

(2) Introduction of targeting vector into embryonic stem cells Mouse embryonic stem cell D3 (provided by Nagoya University School of Medicine, Professor Muramatsu Satoshi and maintained at Osaka University Genetic Information Laboratory) was cultured in ESM medium [20% fetal bovine fetus Serum (Invitrogen), 55 μM β-mercaptoethanol (Invitrogen), 1 mM MEM sodium pyruvate (Invitrogen), 0.1 mM MEM non-essential amino acid (Invitrogen), 3 mM adenosine (SIGMA), 3 mM guanosine (SIGMA), 3 mM cytidine (SIGMA), 3 mM uridine (SIGMA), 1 mM thymidine (SIGMA), 2 mM L-glutamine (Invitrogen), 100 units / ml penicillin and 100 μg / ml streptomycin (Invitrogen), 1000 units / ml ESGRO Dulbecco's modified Eagle medium (DMEM; Invitrogen) containing ^TM (mouse recombinant leukemia inhibitory factor; SIGMA Invitrogen)] was used. First, freeze-thawed D3 cells were seeded on a feeder plate prepared using a 6 cm diameter dish, and cultured under conditions of 5% CO ₂ and 37 ° C. After 24 hours, D3 cells were subcultured to 3 feeder plates prepared using a 10 cm diameter dish.

Gene transfer of the targeting vector plasmid obtained in Example 2 of the present Example into D3 cells was performed according to the following procedure according to the electroporation method [Cytotechnology, 3, 133 (1990)].
First, 20 μg of the targeting vector plasmid was linearized by digestion with restriction enzyme Not I, followed by phenol / chloroform extraction treatment and ethanol precipitation to obtain a 1 μg / μl solution. On the other hand, the culture supernatant was removed from D3 cells that had passed 48 hours after subculture to a 10 cm diameter dish, and replaced with fresh ESM medium. After confirming that D3 cells reached 70% confluence, the cells were suspended in PBS buffer (Invitrogen) to give 1 × 10 ⁷ cells / ml. After 800 μl of cell suspension (8 × 10 ⁶ cells) was mixed with 20 μg of the above linearized plasmid, the entire cell-DNA mixture was transferred to GenePulser Cuvette (electrode distance 4 mm; BIO-RAD), and cell fusion Gene transfer was performed using the apparatus Gene Pulser (BIO-RAD) under the conditions of a pulse voltage of 250 V and an electric capacity of 500 μF. After the introduction, the cell suspension was suspended in 40 ml of ESM medium, and seeded on 3 feeder plates prepared using a 10 cm diameter dish and 2 feeder plates prepared using a 6 cm diameter dish. After culturing for 16 hours or more under conditions of 5% CO ₂ and 37 ° C., the culture supernatant was removed and replaced with an ESM medium supplemented with 150 μg / ml G418 (Invitrogen). While this medium exchange operation was repeated almost every day, the culture was performed for 8 days to obtain a drug resistant strain. As the mouse embryonic stem cells, the above-described mouse embryonic stem cells are not necessarily used. Embryonic stem cells derived from 129 mice, for example, mouse embryonic stem cells D3 (CRL-11632) that can be purchased from ATCC, US Embryonic stem cells derived from 129 strain mice commercially available from Cell & Technologies and DNX Transgenic Sciences can also be used.

(3) Acquisition of targeting vector-introduced strain Arbitrary 343 colonies were collected from the drug resistant strain obtained in (2) in the following procedure.
Remove the culture supernatant from the dish where colonies of drug-resistant clones appeared, inject a phosphate buffer, and then scrape and suck the colonies using a Pipetteman (GILSON) while observing with a stereomicroscope. Collected into 96-well plates. After trypsinization, each clone was inoculated on a feeder plate prepared using a flat-bottom 24-well plate, and cultured until confluent using ESM medium. After culturing, each clone on the plate was treated with trypsin to make a total volume of 350 μl. 300 μl of this was mixed with an equal volume of freezing medium [20% DMSO, 80% FM medium] to prepare a master plate, which was subjected to cryopreservation. The remaining 50 μl of the cell suspension was seeded on a gelatin-coated flat bottom 24-well plate (Asahi Techno Glass Co., Ltd.) to make a replica plate, and cultured using ESM medium until the cells became confluent.

(4) Diagnosis of homologous recombination by genomic Southern blot using FUT8 genomic region 5 'probe Genomic Southern using 5' terminal probe according to the following procedure for 343 clones obtained in this section (3) Diagnosis of homologous recombination by blotting was performed.
First, genomic DNA of each clone was prepared from the replica plate prepared in this section (3) according to a known method [Nucleic Acids Research, 3, 2303, (1976)], and TE buffer ( pH 8.0) [10 mM Tris-HCl, 1 mM EDTA].
On the other hand, a fragment of about 500 bp upstream from the restriction enzyme Xba I recognition site on the 5 ′ end side was prepared from the genomic clone (13.9 Kb) containing the FUT8 translation initiation codon obtained in item 1 of this example (FIG. 2). .
After digesting the genomic DNA with the restriction enzyme Pst I, Southern blotting was performed according to a known method described in Molecular Cloning, 2nd edition, using the above-mentioned FUT8 gene 5′-terminal fragment of about 500 bp as a probe.

Restriction enzyme Pst I treatment generates a DNA fragment of approximately 7.5 Kb from the wild type FUT8 allele. On the other hand, the restriction enzyme treatment produces a DNA fragment of about 11 Kb from the allele that has undergone homologous recombination with the targeting vector (FIG. 2).
By this method, specific fragments of about 7.5 Kb and about 11.0 Kb were found in genomic DNA prepared from 4 clones. Since the quantity ratio of both fragments was 1: 1, this clone was confirmed to be a homologous recombinant in which one FUT8 allele was replaced with a vector sequence.

(5) Diagnosis of homologous recombination by genomic Southern blot using FUT8 genomic region 3 'probe For 343 clones obtained in this section (3), genomic Southern blot using 3' probe in the following procedure A diagnosis of homologous recombination was made.
First, from the approximately 6.3 Kb Sac I- Sac I region obtained in Section 1 of this Example, the restriction enzyme Eco located at about 500 bp from the 3 'terminal restriction enzyme Sac I recognition site to the 5' terminal side. A fragment containing the RV recognition site was prepared.
After digesting the genomic DNA prepared in this section (4) with the restriction enzyme Sac I, Southern blotting is performed according to a known method described in Molecular Cloning 2nd edition using the above-mentioned FUT83 ′ terminal fragment of about 500 bp as a probe. Went.

Restriction enzyme Sac I treatment produces a DNA fragment of approximately 6.6 Kb from the wild type FUT8 allele. On the other hand, the restriction enzyme treatment produces a DNA fragment of about 8.6 Kb from the allele that has undergone homologous recombination with the targeting vector (FIG. 2).
By this method, specific fragments of about 6.6 Kb and about 8.6 Kb were found in the genomic DNA of 4 clones that were positive in this section (4). Since the quantity ratio of both fragments was 1: 1, this clone was confirmed to be a homologous recombinant in which one FUT8 allele was replaced with a vector sequence.

4). Acquisition of mice with disrupted FUT8 gene (1) Acquisition of chimeric mice using embryonic stem cells in which one of both FUT8 alleles was disrupted Embryonicity with one of the FUT8 alleles established in Example 3 For 4 stem cell clones, select 3 clones that maintain normal karyotype according to the known method [Manupirating the Mouse Embryo, Cold Spring Harbor Laboratory Press (1994)] did. Next, according to the injection chimera method described in Guide to Techniques in Mouse Development (Guideto Technuques in Mouse Development, Methods in Enzymology, Volume 225, AcademicPress (1993)), C57BL / 6 strain female mice The three embryonic stem cell clones were each microinjected into the obtained blastocyst space, transplanted into the uterus of a female mouse of MCH strain that was pseudopregnant, and implanted.

  Among male chimera individuals with dark brown hair appearing in brown coat, those with chimera rate exceeding 50% are said that the injected embryonic stem cells contribute to the germline at the same level Judgment was performed, and mating with male mice of C57BL / 6 strain was performed. As a result, it was confirmed that germline chimeras were present in chimeras produced using embryonic stem cells of 2 clones.

(2) Acquisition of heterozygous mice in which one copy of the FUT8 allele was disrupted After breeding the germline chimera obtained in this section (1) up to 8 weeks of age, it was mated with a female individual of the sexually mature C57BL / 6 strain. A litter was obtained. Among the offspring, genomic DNA was prepared from the tail of an individual having brown coat, according to a known method [Nucleic Acids Research, 3, 2303, (1976)]. Southern blotting was performed according to the method described in Section 3 (4).

The heterozygous genomic DNA is subjected to restriction enzyme Pst I treatment to generate a wild-type FUT8 allele-specific fragment of about 7.5 Kb and an allele-specific fragment of about 11 Kb in which homologous recombination has occurred (FIG. 2). . From the result of Southern blotting shown in FIG. 3, the above-mentioned approximately 7.5 Kb fragment and approximately 11 Kb were obtained from the chimeric individuals derived from any of the two embryonic stem cell clones that were found to contribute to the germline in this section (1). It was confirmed that a heterozygote having a 1: 1 quantitative ratio was obtained. The heterozygous mouse is a mouse in which one allele of FUT8 is disrupted. Hereinafter, the mouse is referred to as a FUT8KO heterozygous mouse.

(3) Acquisition of homozygous mice in which both FUT8 alleles were disrupted The heterozygous male and female individuals obtained in this section (2) were raised to 8 weeks of age and then mated to obtain offspring. Among the pups, the genomic DNA of each clone was prepared from the tail of an individual with brown coat according to a known method [Nucleic Acids Research, 3, 2303, (1976)]. Southern blotting was performed according to the method described in Example 3 (4).
The homozygous genomic DNA produces only an allele-specific fragment of about 11 Kb in which homologous recombination has occurred by treatment with the restriction enzyme Pst I (FIG. 2). As shown in FIG. 3, as a result of Southern blotting, it was confirmed that homozygotes satisfying the above criteria were contained in the pups. The homozygous mouse is a double knockout mouse in which both FUT8 alleles are disrupted. Hereinafter, the mouse is referred to as a FUT8KO homozygous mouse.

Establishment of cell lines from FUT8 KO mice Obtaining FUT8 KO homozygous mice The genotypes of the offspring obtained by mating the FUT8 KO heterozygous mice obtained in Example 1, item 4 were determined by the following procedure.
First, genomic DNA was prepared from the tail of a litter according to a known method [Nucleic Acids Research, 3, 2303, (1976)], and each was prepared with TE-RNase buffer (pH 8.0) (10 mM). Tris-HCl, 1 mM EDTA, 200 μg / ml RNase A) was dissolved in 300 μl overnight. In addition, a forward primer (SEQ ID NO: 1) that binds to the internal sequence of the exon where the mouse FUT8 gene translation initiation codon is located, and a reverse that binds to the sequence deleted by homologous recombination described in Example 1, paragraph 3 of the exon. A primer (SEQ ID NO: 2), a forward primer (SEQ ID NO: 3) and a reverse primer (SEQ ID NO: 4) that bind to the IRES sequence in the targeting vector described in Example 1, paragraph 2 were each designed.

Using DNA polymerase ExTaq (Takara Shuzo), 20 μl of the reaction solution containing 1.25 μl of the genomic DNA solution prepared above [ExTaqbuffer (Takara Shuzo), 0.1 mM dNTPs, 0.1 μM Gene-specific primer (SEQ ID NO: 1, SEQ ID NO: 1, 2, 3, 4)] was prepared and subjected to polymerase chain reaction (PCR). PCR was performed under the conditions of 35 cycles, with a reaction consisting of 94 ° C. for 2 minutes, 94 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 30 seconds as one cycle.
From the above PCR, a region-specific 240 bp amplified fragment containing the translation initiation codon was obtained from the FUT8 wild type allele, and an approximately 380 bp fragment specific to the IRES sequence in the targeting vector was obtained from the allele that had undergone homologous recombination. Each is amplified. After PCR, the reaction solution was subjected to agarose gel electrophoresis, and the one that only amplified an about 380 bp fragment specific to the IRES sequence was determined to be a FUT8KO homozygous mouse.

  As a result of genotyping of 137 pups 10 to 14 days after birth, 5 pups were determined to be FUT8KO homozygous mice, 2 of which were 2 to 3 weeks after birth. Died. Of the remaining 3 animals, 1 animal is different for the establishment of the liver-derived cell line described in Section 3 (1) of this Example and the establishment of the bone marrow-derived cell line described in Section 3 (3) of this Example. FUT8KO, which is one more different animal and adult animal, was established for the establishment of the kidney-derived cell line described in the third section (2) of this example and the establishment of the bone marrow-derived cell line described in the third section (3) of the present example. Homozygous mice were used for the establishment of the bone marrow-derived cell line described in item 3 (3) of this Example.

2. Construction of SV40 T antigen expression vector (1) Construction of plasmid pMoAH A protein expression vector pMoAH was constructed by the following procedure.
First, the region containing the restriction enzyme Aat II recognition sequence was amplified by PCR using the plasmid pKANTEX93 [Molecular Immunology (Mol. Immunol.), 37, 1035 (2000)] as a template, and the amplified DNA fragment was amplified with the restriction enzyme Mlu of pKANTEX93. Inserted into I site. Subsequently, the ampicillin resistant gene coding region was amplified by PCR using pKANTEX93 as a template, and the amplified DNA fragment was combined with the Aat II fragment (11.7 kb) of the plasmid prepared above. Further, a region containing restriction enzyme Sal I and Not I recognition sequences at both ends was amplified by PCR using pKANTEX93 as a template, and the amplified DNA fragment was subjected to 11.9 kb Sal I- Not I of the plasmid prepared above. Combined with fragments. This plasmid is hereinafter referred to as pKANTEX93 '.

Subsequently, the Eco RI- Bam HI fragment containing the antibody expression unit was removed from the plasmid pKANTEX93 ', and the resulting Eco RI- Bam HI fragment (8.8 kb) was replaced with the recognition sequences for the restriction enzymes Eco RI and Bam HI. A multicloning site was introduced by binding to a synthetic DNA fragment at the end. This plasmid is hereinafter referred to as pPAH.
Further, from plasmid PPAH, removing the Mlu I- Hin dIII fragment containing a dihydrofolate reductase (dhfr) gene and the G418 resistance gene coding region, resulting 5.3kb Both ends of the Mlu I- Hin dIII fragment was self-ligated and smoothed. This plasmid is hereinafter referred to as pMoAH.
The nucleotide sequence of each plasmid was determined using a DNA sequencer 377 (ParkinElmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Parkin Elmer), and the method was in accordance with the attached manual. By this method, it was confirmed that the sequence of the inserted DNA and the binding site was consistent with the target sequence.

(2) Acquisition of SV40 large T antigen and small t antigen coding region Plasmid pBlu-SV40Tag having the coding region of SV40 large T antigen and small t antigen was constructed by the following procedure (FIG. 4).

Plasmid SVori-8-16 (Human Science Promotion Foundation Research Resource Bank; Resource No. VG025) was digested overnight with restriction enzymes Bam HI (New England Biolabs) and Stu I (Takara Shuzo), and the solution was agarose gel It was subjected to electrophoresis, and a fragment of about 2.6 Kb containing coding regions of SV40 large T antigen and small t antigen was purified.
On the other hand, plasmid pBluescriptIIKS (+) (Strategene) was added with restriction enzymes Bam HI and Eco RV (New England Biolabs) and digested overnight at 37 ° C. After the digestion reaction, the solution was subjected to 1.0% (w / v) agarose gel electrophoresis, and a DNA fragment of about 2.9 Kb was purified.
About 2.6 Kb Stu I- Bam HI fragment derived from plasmid SVori-8-16 obtained above and about 2.9 Kb Eco RV- Bam HI fragment derived from plasmid pBluescriptIIKS (+) were ligated to construct pBlu-SV40Tag. .

(3) Construction of SV40 T antigen expression vector pMo-SV40Tag Using pMoAH obtained in this section (1) and plasmid pBlu-SV40Tag obtained in this section (2), the SV40T antigen expression vector pMo-SV40Tag is obtained as follows. Was constructed (FIG. 5).
After digesting plasmid pBlu-SV40Tag with restriction enzymes Bam HI and restriction enzyme Sal I, the solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 2.6 Kb containing the coding regions of SV40 large T antigen and small t antigen. Was purified.

On the other hand, plasmid pMoAH was digested with restriction enzyme Bam HI and restriction enzyme Sal I, and then the solution was subjected to agarose gel electrophoresis to purify a DNA fragment of about 5.3 Kb.
Plasmid pBluescriptIIKS obtained above (+) - Construction Sal I- Bam HI fragment of about 2.6Kb from SV40Tag, about connecting the Sal I- Bam HI fragment of 5.3 kb SV40T antigen expression vector PMO-SV40Tag from plasmid pMoAH did.
The base sequence of the constructed SV40 T antigen expression vector pMo-SV40Tag was determined using DNA Sequencer 377 (Parkin Elmer) and BigDye Terminator Cycle Sequencing FSReady Reaction Kit (Parkin Elmer), and the method was in accordance with the attached manual. . By this method, it was confirmed that the inserted DNA matched the sequence containing the large T antigen and the small t antigen coding region of the SV40 genomic DNA sequence [GenBankJ02400].

3. Establishment of cell lines from FUT8 KO homozygous mice From the FUT8 KO homozygous mice obtained in item 2 above, liver, kidney, lung, heart, abdominal muscle, brain, skull muscle, skin, bone marrow, spleen, thymus Each organ was removed and subjected to primary culture for each organ. Among the cells derived from the organs subjected to the primary culture, cells derived from the bone marrow, spleen, and thymus organs did not grow at the time of the primary culture, and therefore cell lines could not be established. In addition, among the cells derived from organs subjected to primary culture, cell lines were established as follows from the liver and kidney, which were better proliferative compared to other cell lines by microscopic observation. It was. In addition, cell lines were established for bone marrow-derived cells that are frequently used in substance production by mammalian cells as follows.

(1) Establishment of liver-derived cell line A liver-derived immortalized cell line was established from the FUT8KO homozygous mouse obtained in Example 1 of the present example by the following procedure.
First, primary cultured cells were prepared from the livers of FUT8 KO homozygous mice by the following procedure.
From 2-week-old FUT8 KO homozygous mice, the liver was removed, washed with Dulbecco's PBS (Invitrogen), and then minced in a 2cm dish (Falcon) containing 2mL of trypsin-EDTA (Invitrogen) solution. Then, it was incubated for 15 minutes under the conditions of 5% CO ₂ and 37 ° C. Subculture medium [DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 50 μg / mL gentamicin sulfate solution (Nacalai Tesque)] mixed with 4 mL, then added to a 15 mL tube (Falcon) Collected and pipetted. The tissue mass was precipitated by allowing to stand for about 5 minutes, and about 5 mL of the supernatant was placed in another 15 mL tube and centrifuged at 1000 rpm for 5 minutes to collect cells. When the number of cells was counted, the number of viable cells was about 6 × 10 ⁶ . Therefore, after suspending in a culture medium for subculture so that the cell density is about 3 × 10 ⁵ cells / mL, and dispensing 10 mL of cell suspension into two T75 flasks (Greiner), 5% CO ₂ , The culture was started at 37 ° C. This primary cultured cell is hereinafter referred to as FUT8KO-LIV cell.

When FUT8KO-LIV cells were cultured for 24 hours and examined under a microscope, many dead cells were observed, and the number of living cells in which adhesion to the flask was observed was 10% or less of the total cells. Therefore, the culture supernatant was removed, and dead cells were removed by washing with Dulbecco PBS (Invitrogen), and then 10 mL of a new subculture medium was dispensed. This medium exchange operation was repeated every two days. Seven days after the start of culture, the number of cells was counted when the cell density of adherent cells reached 80% confluence. The total number of living cells was about 7 × 10 ⁵ , and the cell density was diluted about 2-fold. Cells were passaged as follows. After that, since the cell growth was poor, the medium was changed every 3 to 4 days.When the cell density became 70-80% confluent by microscopic examination, the cell density was about 2 times diluted. Repeated the work of subculture. At the time of cell passage, the number of cells was counted, and the generation number from the start of the culture of FUT8KO-LIV cells was calculated. Since the appearance of gradually proliferating cells was observed from about two months after the start of culture, the dilution of the cell density at the time of passage was increased to 5 to 10 times, and for another 4 months, The medium exchange work or subculture work every 3 to 4 days was continuously repeated. Six months after the start of culture, the number of generations reached about 120 generations, and an immortalized cell line showing stable cell growth with a doubling time of about 25 hours was obtained. This immortalized cell line is hereinafter referred to as LIV86 strain.

On the other hand, 17 days after the start of culture of FUT8KO-LIV cells, the SV40T antigen expression vector pMo-SV40Tag constructed in Example 2 (2) of this example was introduced into some cells according to the following procedure. It was. Gene transfer of the SV40 T antigen expression vector pMo-SV40Tag into FUT8KO-LIV cells was carried out by the lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the following procedure.

First, FUT8KO-LIV cells were seeded at 10 ml each in two T75 flasks so that the cell density was about 2 × 10 ⁴ cells / mL 18 hours before gene transfer, under conditions of 5% CO ₂ and 37 ° C. After culturing below, the medium was replaced with a serum-free DMEM medium (Invitrogen) 6.4 mL / T75 flask immediately before gene introduction. On the other hand, the SV40 T antigen expression vector pMo-SV40Tag was linearized with the restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 0.5 μg / μL aqueous solution. 16 μL (8 μg) of the linearized vector solution was mixed with 0.8 mL of serum-free DMEM medium, and then mixed with 50 μL of lipofectamine ^™ reagent and 0.8 mL of serum-free DMEM medium. After standing at room temperature for 15 minutes, 1.6 mL of the solution was added to the above T75 flask, and FUT8KO-LIV cells were cultured for 6 hours under conditions of 5% CO ₂ , 37 ° C. and DNA-liposomecomplex coexistence. After culturing, 8 mL of DMEM medium (Invitrogen) supplemented with 20% fetal calf serum (Invitrogen) was added and cultured under conditions of 5% CO ₂ and 37 ° C. After culturing for 24 hours, the supernatant was removed, and 15 mL of a new subculture medium was dispensed, and further cultured for 5 days, and then subcultured so that the cell density was about 2-fold diluted. When further cultured for 4 days, the cell density was about 80% confluent, the total number of living cells reached about 2 × 10 ⁶ cells, compared with cells not transfected with the SV40T antigen expression vector pMo-SV40Tag. Increased growth rate was observed. Then, repeat the medium exchange every 2 to 4 days, and when the cell density becomes 70-80% confluent by microscopic examination, pass the cells so that the cell density is about 10 to 100 times diluted. It was. This medium exchange and cell passage work were continued for about 3 months. The number of generations reached about 120 generations, and an immortalized cell line showing stable cell growth with a doubling time of about 12 hours was obtained. This immortalized cell line is hereinafter referred to as LIV86-SV strain.

(2) Establishment of kidney-derived cell line A kidney-derived immortalized cell line was established from the FUT8KO homozygous mouse obtained in Section 1 of this Example by the following procedure.
First, primary cultured cells were prepared from kidneys of FUT8 KO homozygous mice by the following procedure.
From 2 weeks old FUT8 KO homozygous mice, the kidneys were removed, washed with Dulbecco's PBS (Invitrogen), then minced in a 2cm dish (Falcon) containing 1mL of trypsin-EDTA (Invitrogen) solution. After that, it was incubated for 10 minutes under the conditions of 5% CO ₂ and 37 ° C. Add 2 mL of trypsin-EDTA solution, incubate for 10 minutes under 5% CO ₂ at 37 ° C, mix with 10 mL of subculture medium, collect in 50 mL tube (Falcon), and pipette. . The tissue mass was precipitated by allowing to stand for about 5 minutes, and about 12 mL of the supernatant was placed in a 15 mL tube (Falcon) and centrifuged at 800 rpm for 5 minutes to collect cells. When the number of cells was counted, the number of viable cells was about 3 × 10 ⁶ . Therefore, after suspending in a subculture medium so that the cell density is about 2 × 10 ⁵ cells / mL, and dispensing 15 mL cell suspension into one T75 flask (Greiner), 5% CO ₂ , The culture was started at 37 ° C. This primary cultured cell is hereinafter referred to as FUT8KO-KID cell.

When FUT8KO-KID cells were cultured for 24 hours and examined under a microscope, many dead cells were observed, but many viable adherent cells with good proliferation were observed. Therefore, the culture supernatant was removed, and dead cells were removed by washing with Dulbecco PBS (Invitrogen), and then 10 mL of a new subculture medium was dispensed. Three days after the start of culture, the cell density of the living cells reached 80% confluence. When the number of cells was counted at this point, the total number of living cells was about 9 × 10 ⁶ , and the cells were passaged so that the cell density was about 6-fold dilution. After that, the medium is changed every 3 to 4 days, and the cells are subcultured so that the cell density is about 2 to 5 times diluted when the cell density becomes 70-80% confluent by microscopic examination. Was repeated. At the time of cell passage, the number of cells was counted, and the number of generations from the start of culture of FUT8KO-KID cells was calculated. From about 3 weeks after the start of the culture, the proliferation of the cells deteriorated, and only about 3 to 4 days thereafter, the medium was changed for about 1 month. Since the appearance of gradually proliferating cells was observed from about 2 months after the start of culture, the cell density was about 2 to 5 times when the cell density became 70-80% confluent by microscopic examination The operation of subculturing cells so as to achieve dilution was repeated. Furthermore, for 4 months, medium replacement work or subculture work every 3 to 4 days was repeatedly performed. Six months after the start of culture, the number of generations reached about 100 generations, and an immortalized cell line showing stable cell growth with a doubling time of about 18 hours was obtained. This immortalized cell line is hereinafter referred to as KID86 strain.

On the other hand, 5 days after the start of the culture of FUT8KO-KID cells, the SV40T antigen expression vector pMo-SV40Tag constructed in Example 2 (2) of this example was introduced into some cells according to the following procedure. .
Gene transfer of the SV40 T antigen expression vector pMo-SV40Tag into FUT8KO-KID cells was performed by the lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the following procedure.

First, FUT8KO-KID cells were seeded 5 mL in one T25 flask (Greiner) so that the cell density was about 1 × 10 ⁵ cells / mL at least 18 hours before gene transfer, 5% CO ₂ , The cells were cultured at 37 ° C. On the other hand, the SV40 T antigen expression vector pMo-SV40Tag was linearized with the restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 0.5 μg / μL aqueous solution. 10 μL (5 μg) of the linearized vector solution was mixed with 0.3 mL of serum-free DMEM medium, and then mixed with 30 μL of lipofectamine ^™ reagent and 0.3 mL of serum-free DMEM medium. After standing at room temperature for 15 minutes, the solution was mixed with 2.4 mL of serum-free DMEM medium. The solution was added to the above T25 flask after removing the culture supernatant, cultured for 6 hours in the presence of 5% CO ₂ , 37 ° C. and DNA-liposome complex, and then replaced with a subculture medium. The cells were cultured under conditions of 5% CO ₂ and 37 ° C. After culturing for 24 hours, the supernatant was removed, and 4 mL of a new subculture medium was dispensed. After further culturing for 4 days, the cells were subcultured so that the cell density was about 6-fold dilution. After that, the medium is changed every 3 to 4 days, and the cells are subcultured so that the cell density is about 2 to 5 times diluted when the cell density becomes 70-80% confluent by microscopic examination. Was repeated. Since about 2 weeks after the gene transfer, the deterioration of cell proliferation was observed, and for about 2 weeks thereafter, only the medium was changed every 3 to 4 days. The passage work was resumed since the appearance of proliferating colonies was observed about 1 month after the gene transfer. Thereafter, for about 3 months, medium replacement work or subculture work every 3 to 4 days was repeatedly performed. Four months after the start of culture, the number of generations reached about 110 generations, and an immortalized cell line showing stable cell growth with a doubling time of about 16 hours was obtained. This immortalized cell line is hereinafter referred to as KID86-SV strain.

(3) Establishment of Bone Marrow-Derived Cell Line A bone marrow-derived immortalized cell line was established from the FUT8KO homozygous mouse obtained in Section 1 of this Example by the following procedure.
First, primary cultured cells were prepared from bone marrow cells of FUT8 KO homozygous mice by the following procedure.

Femurs were removed from FUT8 KO homozygous mice. Cut both ends of the femur, insert an injection needle (Terumo) into the bone from one end, flow 10 mL of RPMI1640 medium (Invitrogen), drain bone marrow cells, and collect in 15 mL tube (Falcon) . After collecting cells by centrifugation at 800 rpm for 5 minutes, the number of cells was counted, and the culture medium for subculture [10% fetal bovine serum (Invitrogen) so that the cell density was 1 to 10 × 10 ⁵ cells / mL And RPMI1640 medium (Invitrogen) supplemented with 50 μg / mL gentamicin sulfate solution (Nacalai Tesque). The cell suspension was dispensed into T75 flasks (Asahi Techno Glass) at a rate of 10 to 15 mL, and culture was started under conditions of 5% CO ₂ and 37 ° C. About one of the FUT8KO homozygous mice obtained in the first section of this example, the number of bone marrow cells obtained was as low as about 1 × 10 ⁶ , and the medium was cultured every 3 to 4 days for 1 month from the start of the culture. The exchange was repeated, but all cells were killed.

In addition, bone marrow cells prepared from another FUT8 KO homozygous mouse had about 7 × 10 ⁶ cells in primary culture. Bone marrow cells after branching into 3 lines, subculture medium, IL-3 supplemented medium [subculture medium supplemented with 200pMrecombinant murine IL-3 (Peprotech)], or IL-3 supplemented X-VIVO15 medium [ Culture using X-VIVO15 medium (BioWhittaker) supplemented with 200pMrecombinant murine IL-3 (Peprotech), 10% fetal bovine serum (Invitrogen) and 50μg / mL gentamicin sulfate solution (Nacalai Tesque)] However, the medium exchange operation was repeated every 3 to 4 days, but after about 1 month from the start of the culture, all cells were killed.

Furthermore, a bone marrow-derived cell line was established from one of the FUT8KO homozygous mice obtained in item 1 of this Example that reached adulthood. The number of bone marrow cells prepared from the mice was about 2 × 10 ⁷ at the time of primary culture. This primary cultured cell is hereinafter referred to as FUT8KO-Bom cell. After culturing FUT8KO-Bom cells for 24 hours, the cell culture solution was diluted 2-fold with IL-3 supplemented medium. Thereafter, every 3 to 7 days, the cell suspension is collected in a 50 mL tube (Falcon), centrifuged at 800 rpm for 5 minutes to collect cells, and then 1/3 to half of the culture supernatant is renewed. The medium was replaced with IL-3 supplemented medium. During about 3 months from the start of culture, the cell growth was poor and the proportion of dead cells was high, but thereafter the proportion of dead cells gradually decreased and the proportion of cells with good proliferation increased. Therefore, if the medium change operation is continued every 3-7 days, the cell growth is good, and the cell density exceeds 1 × 10 ⁶ cells / mL, the cell density is diluted about 2 to 10 times. In this case, the culture supernatant was added in an amount of one third to half of the amount of the culture solution. This medium exchange and subculture were repeated every 4 to 7 days, and long-term subculture was performed for about 6 months. Immortalized cells showing stable cell growth were obtained. This immortalized cell line is hereinafter referred to as Bom86 strain.

On the other hand, 6 days after the start of culture of FUT8KO-Bom cells, the SV40T antigen expression vector pMo-SV40Tag constructed in Example 2 (2) of this example was introduced into some cells by the following procedure. .
Gene transfer of the SV40 T antigen expression vector pMo-SV40Tag into FUT8KO-Bom cells was performed according to the following procedure in accordance with the electroporation method [Cytotechnology, 3, 133 (1990)].

First, the SV40 T antigen expression vector pMo-SV40Tag was linearized with the restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 0.5 μg / μl aqueous solution. On the other hand, FUT8KO-Bom cells were suspended in RPMI1640 medium to give 7 × 10 ⁶ cells / ml. After 800 μl of cell suspension (5.6 × 10 ⁶ cells) is mixed with 20 μl (10 μg) of the above linearized vector, the total amount of the cell-DNA mixture is Gene Pulser Cuvette (electrode distance 4 mm) (BIO-RAD) Then, gene transfer was performed using a cell fusion device GenePulser (BIO-RAD) under the conditions of a pulse voltage of 300 V and an electric capacity of 500 μF. After gene transfer, the cell suspension was suspended in 10 mL of IL-3 supplemented medium and seeded in a T75 flask (Asahi Techno Glass). After culturing under conditions of 5% CO ₂ and 37 ° C for 24 hours, collect the cell suspension in a 50 mL tube (Falcon), centrifuge at 800 rpm for 5 minutes to collect the cells, and then collect half of the culture supernatant. The medium was replaced with fresh IL-3 supplemented medium. Thereafter, every 3 to 7 days, the cell suspension is collected in a 50 mL tube (Falcon), centrifuged at 800 rpm for 5 minutes to collect cells, and then 1/3 to half of the culture supernatant is renewed. The medium was replaced with IL-3 supplemented medium. During about 3 months from the start of culture, cell proliferation was poor and the proportion of dead cells was high, but thereafter the proportion of dead cells gradually decreased and the proportion of cells with good proliferation increased. Therefore, if the medium change operation is continued every 3-7 days, the cell growth is good, and the cell density exceeds 1 × 10 ⁶ cells / mL, the cell density is diluted about 2 to 10 times. In this case, the culture supernatant was added in an amount of one third to half of the amount of the culture solution. This medium exchange and subculture were repeated every 4 to 7 days, and long-term subculture was performed for about 6 months. Immortalized cells showing stable cell growth were obtained. This immortalized cell line is hereinafter referred to as Bom86-SV strain.

Expression of antibody composition using established cell line derived from FUT8 KO zygote mouse Expression of anti-CD20 chimeric antibody composition (1) Expression of anti-CD20 chimeric antibody composition using LIV86-SV strain For the LIV86-SV strain established in Example 2, item 3 (1), the expression of WO03 / 55993 An anti-CD20 chimeric antibody expression vector pKANTEX2B8P and a hygromycin resistant gene expression vector pAGE249 were introduced to produce a stable expression strain of the anti-CD20 chimeric antibody composition. pAGE249 is a derivative of pAGE248 [Journal of Biological Chemistry (J. Biol. Chem., 269, 14730 (1994))], and it contains 2.7 Kb containing a dihydrofolate reductase gene (dhfr) expression unit from pAGE248. This is a vector from which the Sph I- Sph I fragment has been removed.

Gene transfer of the vector pKANTEX2B8P and the vector pAGE249 into the LIV86-SV strain was performed according to the following procedure in accordance with the electroporation method [Cytotechnology, 3, 133 (1990)]. First, vector pKANTEX2B8P was linearized with restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 1 μg / μl aqueous solution. The vector pAGE249 was linearized with the restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μl aqueous solution.

On the other hand, the LIV86-SV strain was suspended in K-PBS buffer [137 mM KCl, 2.7 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , ₄ mM MgCl ₂ ] to make 8 × 10 ⁶ cells / ml. After mixing 200 μL of cell suspension (1.6 × 10 ⁶ cells) with 4 μL (4.4 μg) of each of the above linearized vectors, the total amount of the cell-DNA mixture is GenePulser Cuvette (distance between electrodes 2 mm) (BIO-RAD) ), And gene transfer was performed using a cell fusion device Gene Pulser (BIO-RAD) under conditions of a pulse voltage of 350 V and an electric capacity of 250 μF. After gene transfer, the cell suspension was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and seeded in a 96-well plate (Greiner) for adherent cell culture. After incubation for 24 hours under conditions of 5% CO ₂ and 37 ° C, remove the culture supernatant and add DMEM medium containing 10% fetal calf serum (Invitrogen) and 300μg / ml hygromycin B (Wako Pure Chemical Industries) The medium was changed to (Invitrogen). While this medium exchange operation was repeated every 3 to 4 days, culture was performed for 8 days to obtain a drug resistant strain. Thereafter, the drug-resistant strain was expanded to a 24-well plate (Greiner) and cultured for 2 weeks in a DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 50 nM MTX (SIGMA). Furthermore, the anti-CD20 chimeric antibody stable expression strain LIV86-SV-CD20 was obtained by culturing in DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 250 nM MTX (SIGMA). The acquired LIV86-SV-CD20 strain was transferred to FERM BP- on March 4, 2003 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki Pref. Deposited as 8314.

(2) Expression of anti-CD20 chimeric antibody composition using KID86-SV strain For the KID86-SV strain established in Example 2, item 3 (2), anti-CD20 chimeric antibody expression vector pKANTEX2B8P described in WO03 / 55993 and The hygromycin resistant gene expression vector pAGE249 was introduced to prepare a stable expression strain of the anti-CD20 chimeric antibody composition.
The gene pKANTEX2B8P and the vector pAGE249 were introduced into the KID86-SV strain by the lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the following procedure. First, KID86-SV strain was seeded in a T25 flask (Greiner) at a cell density of about 1 × 10 ⁵ cells / mL at least 18 hours before gene transfer, 5% CO ₂ , 37 ° C. Cultured under conditions. On the other hand, vector pKANTEX2B8P was linearized with restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 1 μg / μL aqueous solution. Further, vector pAGE249 was linearized with restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μL aqueous solution. 5 μL (5.5 μg) of each of the linearized vector solutions was mixed with 0.3 mL of serum-free DMEM medium, and then mixed with 30 μL of lipofectamine ^™ reagent and 0.3 mL of serum-free DMEM medium.

After standing at room temperature for 15 minutes, the solution was mixed with 2.4 mL of serum-free DMEM medium. The solution was added to the above T25 flask after removing the culture supernatant, and cultured for 6 hours in the presence of 5% CO ₂ , 37 ° C. and DNA-liposome complex, and then 10% fetal calf serum (Invitrogen) Was replaced with DMEM medium (Invitrogen) supplemented with. After culturing for 24 hours under conditions of 5% CO ₂ and 37 ° C, the culture supernatant was removed, and DMEM medium supplemented with 10% fetal calf serum (Invitrogen) and 400 µg / ml hygromycin B (Wako Pure Chemical Industries, Ltd.) The medium was changed to (Invitrogen). By repeating this medium exchange operation every 3 to 4 days, culturing was performed for 14 days to obtain an anti-CD20 chimeric antibody stable expression strain KID86-SV-CD20. The acquired KID86-SV-CD20 strain was transferred to the FERM BP- as of March 4, 2003 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1st, 1st East, Tsukuba City, Ibaraki Prefecture, 6th Central). Deposited as 8315.

3. Expression of anti-ganglioside GD3 chimeric antibody composition (1) Expression of anti-ganglioside GD3 chimeric antibody composition using LIV86-SV strain For the LIV86-SV strain established in Example 3, item 3 (1), WO00 / 61739 The described anti-GD3 chimeric antibody expression vector pChi641LHGM4 and hygromycin resistance gene expression vector pAGE249 were introduced to prepare a stable expression strain of the anti-ganglioside GD3 chimeric antibody composition.
Gene transfer of the vector pChi641LHGM4 and the vector pAGE249 into the LIV86-SV strain was performed according to the following procedure in accordance with the electroporation method [Cytotechnology, 3, 133 (1990)]. First, the vector pChi641LHGM4 was linearized with the restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 1 μg / μL aqueous solution. Further, vector pAGE249 was linearized with restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μL aqueous solution.

On the other hand, the LIV86-SV strain was suspended in K-PBS buffer [137 mM KCl, 2.7 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , ₄ mM MgCl ₂ ] to make 8 × 10 ⁶ cells / mL. After mixing 200 μL of cell suspension (1.6 × 10 ⁶ cells) with 4 μL (4.4 μg) of each of the above linearized vectors, the total amount of the cell-DNA mixture is GenePulser Cuvette (distance between electrodes 2 mm) (BIO-RAD) ), And gene transfer was performed using a cell fusion device Gene Pulser (BIO-RAD) under conditions of a pulse voltage of 350 V and an electric capacity of 250 μF. After gene transfer, the cell suspension was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and seeded in a 96-well plate (Greiner) for adherent cell culture. After incubation for 24 hours under conditions of 5% CO ₂ and 37 ° C, remove the culture supernatant and add DMEM medium containing 10% fetal calf serum (Invitrogen) and 300 μg / mL hygromycin B (Wako Pure Chemical Industries) The medium was changed to (Invitrogen). While this medium exchange operation was repeated every 3 to 4 days, culture was performed for 8 days to obtain a drug resistant strain. Thereafter, the drug-resistant strain was expanded to a 24-well plate (Greiner) and cultured for 2 weeks in a DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 50 nM MTX (SIGMA). Furthermore, the anti-ganglioside GD3 chimeric antibody stable expression strain LIV86-SV-2871 was obtained by culturing in DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 250 nM MTX (SIGMA). .

(2) Expression of anti-ganglioside GD3 chimeric antibody composition using KID86-SV strain The anti-GD3 chimeric antibody expression vector pChi641LHGM4 described in WO00 / 61739 is used against the KID86-SV strain established in Example 2, item 3 (2). Then, the hygromycin resistance gene expression vector pAGE249 was introduced to prepare a stable expression strain of the anti-ganglioside GD3 chimeric antibody composition.
Gene transfer of the vector pChi641LHGM4 and the vector pAGE249 into the KID86-SV strain was carried out by the lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the following procedure. First, KID86-SV strain was seeded in a T25 flask (Greiner) at a cell density of about 1 × 10 ⁵ cells / mL at least 18 hours before gene transfer, 5% CO ₂ , 37 ° C. Cultured under conditions. On the other hand, the vector pChi641LHGM4 was linearized with the restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 1 μg / μL aqueous solution. Further, vector pAGE249 was linearized with restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μL aqueous solution. 5 μL (5.5 μg) of each of the linearized vector solutions was mixed with 0.3 mL of serum-free DMEM medium, and then mixed with 30 μL of lipofectamine ^™ reagent and 0.3 mL of serum-free DMEM medium. After standing at room temperature for 15 minutes, the solution was mixed with 2.4 mL of serum-free DMEM medium. The solution was added to the above T25 flask after removing the culture supernatant, and cultured for 6 hours in the presence of 5% CO ₂ , 37 ° C. and DNA-liposome complex, and then 10% fetal calf serum (Invitrogen) Was replaced with DMEM medium (Invitrogen) supplemented with. After culturing for 24 hours under conditions of 5% CO ₂ and 37 ° C, remove the culture supernatant and add DMEM medium containing 10% fetal calf serum (Invitrogen) and 400 μg / mL hygromycin B (Wako Pure Chemical Industries) The medium was changed to (Invitrogen). By repeating this medium exchange operation every 3 to 4 days, culturing was performed for 14 days to obtain an anti-ganglioside GD3 chimeric antibody stable expression strain KID86-SV-2871.

4). Expression of anti-CCR4 chimeric antibody composition (1) Expression of anti-CCR4 chimeric antibody composition using LIV86-SV strain For the LIV86-SV strain established in Example 2, item 3 (1), the method described in WO01 / 64754 An anti-CCR4 chimeric antibody expression vector pKANTEX2160 and a hygromycin resistant gene expression vector pAGE249 were introduced to prepare a stable expression strain of the anti-CCR4 chimeric antibody.
Gene transfer of the vector pKANTEX2160 and the vector pAGE249 into the LIV86-SV strain was performed according to the following procedure in accordance with the electroporation method [Cytotechnology, 3, 133 (1990)]. First, vector pKANTEX2160 was linearized with restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 1 μg / μL aqueous solution. Further, vector pAGE249 was linearized with restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μL aqueous solution.

On the other hand, the LIV86-SV strain was suspended in K-PBS buffer [137 mM KCl, 2.7 mM NaCl, 8.1 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , ₄ mM MgCl ₂ ] to make 8 × 10 ⁶ cells / ml. After mixing 200 μl of cell suspension (1.6 × 10 ⁶ cells) with 4 μL (4.4 μg) of each of the above linearized vectors, the total amount of the cell-DNA mixture was GenePulser Cuvette (distance between electrodes 2 mm) (BIO-RAD) ), And gene transfer was performed using a cell fusion device Gene Pulser (BIO-RAD) under conditions of a pulse voltage of 350 V and an electric capacity of 250 μF. After gene transfer, the cell suspension was suspended in DMEM medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and seeded in a 96-well plate for adherent cell culture (Greiner). After incubation for 24 hours under conditions of 5% CO ₂ and 37 ° C, remove the culture supernatant and add DMEM medium containing 10% fetal calf serum (Invitrogen) and 300 μg / mL hygromycin B (Wako Pure Chemical Industries) The medium was changed to (Invitrogen). While this medium exchange operation was repeated every 3 to 4 days, culture was performed for 8 days to obtain a drug resistant strain. Thereafter, the drug-resistant strain was expanded to a 24-well plate (Greiner) and cultured for 2 weeks in a DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 50 nM MTX (SIGMA). Furthermore, the cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine dialysis serum (Invitrogen) and 250 nM MTX (SIGMA) for 2 weeks to obtain anti-CCR4 chimeric antibody stable expression strain LIV86-SV-2760.

(2) Expression of anti-CCR4 chimeric antibody composition using KID86-SV strain For KID86-SV strain established in Example 2, item 3 (2), anti-CCR4 chimeric antibody expression vector pKANTEX2160 described in WO01 / 64754 and The hygromycin resistant gene expression vector pAGE249 was introduced to prepare a stable expression strain of the anti-CCR4 chimeric antibody composition.
Gene transfer of the vector pKANTEX2160 and the vector pAGE249 into the KID86-SV strain was performed by the lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the following procedure. First, KID86-SV strain was seeded in a T25 flask (Greiner) at a cell density of about 1 × 10 ⁵ cells / mL at least 18 hours before gene transfer, 5% CO ₂ , 37 ° C. Cultured under conditions. On the other hand, the vector pKANTEX2160 was linearized with the restriction enzyme Acl I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was used as a 1 μg / μL aqueous solution. Further, vector pAGE249 was linearized with restriction enzyme Xho I, followed by phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector was made into a 0.1 μg / μL aqueous solution. 5 μL (5.5 μg) of each of the linearized vector solutions was mixed with 0.3 mL of serum-free DMEM medium, and then mixed with 30 μL of lipofectamine ^™ reagent and 0.3 mL of serum-free DMEM medium. After standing at room temperature for 15 minutes, the solution was mixed with 2.4 mL of serum-free DMEM medium. The solution was added to the above T25 flask after removing the culture supernatant, and cultured for 6 hours in the presence of 5% CO ₂ , 37 ° C. and DNA-liposome complex, and then 10% fetal calf serum (Invitrogen) Was replaced with DMEM medium (Invitrogen) supplemented with. After culturing for 24 hours under conditions of 5% CO ₂ and 37 ° C, remove the culture supernatant and add DMEM medium containing 10% fetal calf serum (Invitrogen) and 400 μg / mL hygromycin B (Wako Pure Chemical Industries) The medium was changed to (Invitrogen). The culture was performed for 14 days while repeating this medium exchange operation every 3 to 4 days to obtain an anti-CCR4 chimeric antibody stable expression strain KID86-SV-2760.

4). Purification of the antibody composition The anti-CD20 chimeric antibody expression strain LIV86-SV-CD20 obtained in Example 1 (1) of this Example was treated with 10% Ultra-low IgG fetal calf serum (Invitrogen) and 250 nM MTX (SIGMA). Was suspended in DMEM medium (Invitrogen) with a density of 3 × 10 ⁴ cells / ml, and 1 L was seeded on 40 T182 flasks (Greiner) for culture of adherent cells. After culturing in a 5% CO ₂ incubator at 37 ° C. for 7 days, the cell suspension was recovered. In addition, the anti-CD20 chimeric antibody expression strain KID86-SV-CD20 obtained in the first section (2) of this Example was obtained by using 10% Ultra-low IgG fetal calf serum (Invitrogen) and 400 μg / mL hygromycin B (Wako Jun) After suspension at a density of 3 × 10 ⁴ cells / ml in DMEM medium (Invitrogen) supplemented with (medicine), 1 L was seeded onto 40 T182 flasks (Greiner) for adherent cell culture. After culturing in a 5% CO ₂ incubator at 37 ° C. for 8 days, the cell suspension was recovered.

Each collected cell suspension was centrifuged at 3000 rpm and 4 ° C. for 10 minutes to collect the supernatant, and then filtered using a 0.22 μm pore size 500 mL PES Membrane (Asahi Techno Glass).
A 0.8 cm diameter column was filled with 0.5 mL of PROSEP-AHigh Capacity (MILLIPORE), and 3 ml of 1 M glycine-0.15 M NaCl buffer (pH 8.6) was passed through it. Furthermore, the carrier was equilibrated by washing sequentially with 2 ml of 0.1 M citrate buffer (pH 3.5) and 2 ml of 1 M glycine-0.15 M NaCl buffer (pH 8.6). Next, 500 ml of the culture supernatant was passed through the column, and then washed with 20 ml of 1M glycine-0.15M NaCl buffer (pH 8.6). After washing, the antibody adsorbed on the carrier was eluted using 1.25 ml of 0.1 M citrate buffer (pH 3.5). First, 250 μl of the eluted fraction was discarded, and then 1 mL of the resulting eluted fraction was collected and neutralized by mixing with 200 μL of 2M Tris-HCl (pH 8.5). The obtained eluate was dialyzed overnight at 4 ° C. using 10 mM citric acid-0.15 M NaCl buffer (pH 6.0). After dialysis, the antibody solution was collected and sterilized by filtration using a 0.22 μm pore size Millex GV (MILLIPORE).

In vitro cytotoxic activity of the antibody composition FUT8 knockout mice derived cells produced (ADCC activity)
(1) To assess the invitro cytotoxicity of Example 3 Anti-CD20 chimeric antibody composition was purified by Section 4 of the target cell solution was measured ADCC activity according to the following description.

Human B lymphocyte culture cell line Raji cells (JCRB9012) cultured in RPMI1640-FCS (10) medium (RPMI1640 medium (GIBCO BRL) containing 10% FCS) was centrifuged and suspended to RPMI1640-FCS (5 ) After washing with a medium (RPMI1640 medium containing 5% FCS (GIBCOBRL)), it was adjusted to 2 × 10 ⁵ cells / mL with RPMI1640-FCS (5) medium to obtain a target cell solution.

(2) Preparation of effector cell solution 50 mL of healthy human venous blood was collected and 0.5 mL of heparin sodium (manufactured by Shimizu Pharmaceutical Co., Ltd.) was added and gently mixed. This was subjected to centrifugation (800 g, 20 minutes) using Lymphoprep ^™ (manufactured by AXIS SHIELD) according to the instruction manual to separate the mononuclear cell layer. After washing by centrifugation three times with RPMI1640-FCS (5) medium, the suspension was resuspended at a concentration of 4 × 10 ⁶ cells / mL using the same medium to obtain an effector cell solution.

(3) Measurement of ADCC activity
50 μL (1 × 10 ⁴ cells / well) of the target cell solution prepared in (1) above was dispensed into each well of a 96-well U-shaped bottom plate (Falcon). Next, 50 μL of the effector cell solution prepared in (2) was added (2 × 10 ⁵ cells / well, the ratio of effector cells to target cells was 20: 1). Furthermore, various anti-CD20 chimeric antibodies were added so that each final concentration would be 0.3 to 3000 ng / mL to make a total volume of 150 μL, and reacted at 37 ° C. for 4 hours. After the reaction, the plate is centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant is measured by using CytoTox96Non-Radioactive Cytotoxicity Assay ^TM (Promega) and acquiring absorbance data according to the attached instructions. did. Absorbance data of spontaneous release of target cells uses only medium instead of effector cell solution and antibody solution. Absorbance data of spontaneous release of effector cells uses only medium instead of target cell solution and antibody solution. Obtained by performing the same operation as above. Absorbance data for target cell total release was obtained by using medium instead of antibody solution and effector cell solution, adding 15 μL of 9% Triton X-100 solution 45 minutes before the end of the reaction, and performing the same operation as above. It was determined by measuring LDH activity of Qing. Using these values, ADCC activity was determined by the following equation.

FIG. 6 shows the ADCC activity of each anti-CD20 chimeric antibody. Antibodies obtained from FUT8 knockout mouse-derived cells LIV86-SV and KID86-SV showed ADCC activity higher than that of Rituxan ^{™ at} any antibody concentration, and the highest cytotoxic activity value was also high.
From the above results, it was confirmed that an antibody composition exhibiting high ADCC activity can be produced by using the immortalized cell line of the present invention as a host cell.

Monosaccharide composition analysis of antibody composition produced by FUT8 knockout mouse-derived cells For the anti-CD20 antibody composition, anti-GD3 antibody composition and anti-CCR4 antibody composition obtained in Example 3, a known method [Journal of Monosaccharide composition analysis was performed according to Journal of Liquid Chromatography, 6, 1577, (1983)]. As a result, all the antibody compositions obtained from the FUT8 knockout mouse-derived cell lines LIV86-SV and KID86-SV had a fucose content below the detection limit.
From the above results, in the immortalized cell line of the present invention, it was found that the function of α-bonding the 1-position of fucose to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing end was deleted. Indicated.

Establishment of cell lines from fetuses of FUT8 KO mice Construction of SV40T antigen expression vector pcDNA-SV40largeT that can be selected by zeocin An SV40 T antigen expression vector pcDNA-SV40largeT that can be selected by zeocin was constructed by the following procedure (FIG. 7).
After digesting plasmid SVori-8-16 (Human Science Promotion Foundation Research Resource Bank; Resource No. VG025) with restriction enzymes Bam HI (New England Biolabs) and Stu I (New England Biolabs), the solution was subjected to agarose gel electrophoresis. The fragments of about 2.6 Kb containing the coding regions of SV40 large T antigen and small t antigen were recovered.

On the other hand, plasmid pcDNA3.1 / Zeo (Invitrogen) was digested with restriction enzymes Bam HI and Eco RV (New England Biolabs), and then the solution was subjected to agarose gel electrophoresis to recover a fragment of about 5 Kb.
Connecting the Bam HI- Eco RV fragment of about 5kbp from about 2.6Kb Bam HI- Stu I fragment and the plasmid pcDNA3.1 / Zeo from plasmid SVori-8-16 obtained above, SV40 large T antigen expression vector pcDNA -Constructed SV40largeT.

  The nucleotide sequence of the constructed SV40 large T antigen expression vector pcDNA-SV40largeT was determined using a DNA sequencer 377 (manufactured by Parkin Elmer) and BigDye Terminator v3.0 Cycle Sequencing Kit (manufactured by Applied Biosytems) according to the attached manual. By this method, it was confirmed that the inserted DNA matched the sequence containing the large T antigen coding region of the SV40 genomic DNA sequence [GenBankJ02400].

2. Collection of FUT8 KO homozygous mouse fetal fibroblasts The FUT8KO heterozygous mouse obtained in Example 4, item 4 was mated. Fetal fibroblasts were collected from fetuses at 18.5 days of age by the following procedure.

The uterus was aseptically removed from a pregnant FUT8KO heterozygous female mouse at 18.5 days of gestation. The uterus was opened and the fetus was taken out and washed with Dulbecco PBS (Invitrogen), and then the head and internal organs were removed under a stereomicroscope. Each fetus was transferred to a dish containing Dulbecco's PBS (Invitrogen) supplemented with 0.05% trypsin (Invitrogen), 0.53 mM EDTA (Wako Pure Chemicals) and 40 μg / mL DNase (Invitrogen). After cutting, the cells were incubated for 30 minutes under conditions of 5% CO ₂ and 37 ° C. After the incubation, a subculture medium [DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 50 μg / mL gentamicin sulfate solution (Nacalai Tesque)]] was added to each dish and mixed. The contents of each dish are collected in a 15 mL tube (Falcon), mixed by pipetting, allowed to stand for 5 minutes to precipitate a tissue mass, the supernatant is placed in another 15 mL tube, and centrifuged at 1000 rpm for 5 minutes. The cells were separated and collected. Each of the collected cells was suspended in a subculture medium, seeded in a 10 cm dish (Falcon), and cultured under conditions of 5% CO ₂ and 37 ° C. The cells were used as the primary cultured mouse fetal fibroblasts for the following studies.

3. Genotype check of primary cultured mouse fetal fibroblasts The primary cultured mouse fetal fibroblasts collected in Section 2 of this Example were subjected to genotype check by the method described in Section 1 of Example 2.
PCR amplified a region-specific 240 bp amplified fragment containing the translation initiation codon from the FUT8 wild-type allele, and an IRES sequence-specific fragment of about 380 bp in the targeting vector from the allele that had undergone homologous recombination. Is done. After PCR, the reaction solution was subjected to agarose gel electrophoresis, and the primary cultured mouse embryo fibroblasts in which only IRES sequence-specific amplification of about 380 bp was observed was determined to be fetal fibroblasts of FUT8 KO homozygous mice. . The primary cultured mouse fetal fibroblasts are hereinafter referred to as FUT8KO-MEF cells. On the other hand, from the FUT8 wild type allele, those in which only a region-specific 240 bp amplification including the translation initiation codon was observed were determined as wild type mouse embryo-derived fibroblasts.

4). Establishment of immortalized cell line from FUT8 KO homozygous mouse fetal fibroblasts From FUT8KO-MEF cells determined as fetal fibroblasts of FUT8 KO homozygous mice by genotype check in section 3 of this example The immortalized cell line was established by the following procedure.

The SV40 large T antigen expression vector pcDNA-SV40largeT constructed in section 1 of this example was introduced into FUT8 KO-MEF cells. The vector pcDNA-SV40largeT was linearized with the restriction enzyme Sca I, then subjected to phenol / chloroform extraction treatment and ethanol precipitation, and the recovered linearized vector dissolved in sterilized water was used for gene introduction. The gene was introduced by a lipofection method using lipofectamine ^™ reagent (Invitrogen) according to the attached manual.

One to two days after gene transfer, the medium was replaced with a subculture medium containing zeocin (Invitrogen) at a concentration of 400 μg / mL, and cultured under conditions of 5% CO ₂ and 37 ° C. After that, the medium is changed every 3 to 4 days, and the cells are subcultured so that the cell density is about 2 to 5 times diluted when the cell density becomes 70-80% confluent by microscopic examination. Was repeated. Cell growth began to be observed stably after about one month from the start of culture in a subculture medium containing zeocin, and as a result of continuing the culture for about one month, stable cell growth was exhibited. An immortal cell line (hereinafter referred to as Fut8 − / − cells) was obtained.
Similarly, as a control cell line for Fut8 − / − cells, an immortalized cell line (from a primary cultured fibroblast determined as a wild-type mouse embryo-derived fibroblast by genotype check in Example 3) was used. (Hereinafter referred to as Fut8 + / + cells).

5). AAL staining analysis of immortalized cell lines derived from FUT8 knockout homozygous mouse fetuses A lectin that specifically recognizes α1,6-fucose for Fut8 − / − cells and Fut8 + / + cells established in Section 4 of this Example The staining property with AAL was analyzed by the following procedure.

Fut8-/-and Fut8 + / + cells in subconfluent state were detached from the dish using 0.2% EDTA, then the number of cells was counted, and 5 × 10 ⁶ cells were centrifuged at 1000 rpm for 5 minutes. It was collected. The collected cells were suspended using Dulbecco's PBS (Invitrogen), and then the cells were collected by centrifugation at 1000 rpm for 5 minutes. The collected cells were suspended in Dulbecco's PBS (Invitrogen) containing AAL-FITC (Seikagaku Corporation) at a concentration of 2 μg / mL and incubated on ice for 30 minutes. After the incubation, the cells were washed twice using Dulbecco's PBS (Invitrogen) cooled with ice, and the cells were collected. The collected cells were suspended in 500 μL of Dulbecco's PBS (Invitrogen) and subjected to FACS analysis. Similarly, as a negative control for AAL staining, unstained cells that were not stained with AAL-FITC were also used for FACS analysis. FACS analysis was performed using FACScan ^™ and CELLQuest ^™ (Becton Dickinson), and the results are shown in FIG.
The staining with AAL of Fut8 − / − cells was significantly lower than that of Fut8 + / + cells, which was comparable to the fluorescence intensity of unstained cells. Therefore, it was shown that α1,6 fucose cannot be added to the N-glycoside-linked sugar chain of glycoprotein in Fut8 − / − cells.

It is the figure which showed the genomic region containing the exon in which the translation start codon of a mouse | mouth FUT8 gene is located. It is the figure which showed the determination method of the structure of the targeting vector for mouse | mouth FUT8 gene destruction, and a Southern blot.

FIG. 5 shows a genomic Southern blot of mice in which the FUT8 allele was disrupted. FIG. 3 is a diagram showing an outline of the production of the SV40T antigen expression vector pBlu-SV40Tag having the coding regions of the produced SV40 large T antigen and small t antigen. FIG. 3 is a diagram showing an outline of the production of the produced SV40 T antigen expression vector pMo-SV40Tag.

It is the figure which showed ADCC activity measured by changing the antibody concentration of two types of purified anti-CD20 chimeric antibodies. The horizontal axis represents the antibody concentration, and the vertical axis represents the cytotoxic rate at each antibody concentration. □ indicates LIV86-SV cell-producing antibody, ■ indicates KID86-SV cell-producing antibody, and ○ indicates cytotoxic activity when the commercially available antibody Rituxan ^™ , which is an antibody produced in CHO cells, is used.

It is the figure which showed the outline of preparation of SV40T antigen expression vector pcDNA-SV40largeT which can be selected by the produced zeocin. It is the figure which showed the dyeing | staining property by AAL which is a lectin specific to (alpha) 1,6 fucose of Fut8 + / + cell and Fut8-/-cell. The horizontal axis indicates the fluorescence intensity, and the vertical axis indicates the number of cells. The solid line shows the result of staining with AAL-FITC, and the dotted line shows the result of unstained cells.

Claims (25)

Immortalization established from a non-human animal in which the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is alpha-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex-chain reducing end is knocked out Cell line. The immortalized cell line according to claim 1, wherein the non-human animal is a mouse. All of the alleles on the genome of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-binding complex sugar chain reducing terminal is knocked out. Or the immortalized cell line of 2. N-glycosidic complex type sugar chain reducing terminal N-acetylglucosamine 6-position of fucose 1-position α-bonded sugar chain modification The enzyme is involved in the exon region including the start codon of the genomic gene The immortalized cell line according to any one of claims 1 to 3. The enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is α1,6-fucosyltransferase. The immortalized cell line according to any one of the above. The immortalized cell line according to claim 5, wherein the α1,6-fucosyltransferase is a protein encoded by a DNA selected from the following (a) or (b):
(a) DNA consisting of the base sequence represented by SEQ ID NO: 5;
(b) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity. The immortalized cell line according to claim 5, wherein the α1,6-fucosyltransferase is a protein selected from the group consisting of the following (a) to (c).
(a) a protein comprising the amino acid sequence represented by SEQ ID NO: 6;
(b) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity;
(c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having α1,6-fucosyltransferase activity. A transformant in which a gene encoding a glycoprotein is introduced into the immortalized cell line according to any one of claims 1 to 7. The transformant according to claim 8, wherein the glycoprotein is an antibody. The transformant according to claim 9, wherein the antibody is selected from the group consisting of the following (a) to (d).
(a) a human antibody;
(b) a humanized antibody;
(c) an antibody fragment comprising the Fc region of (a) or (b);
(d) A fusion protein having the Fc region of (a) or (b). The transformant according to claim 9 or 10, wherein the antibody class is IgG. A method for producing a glycoprotein, comprising the steps of culturing the transformant according to claim 8 in a medium, producing and accumulating glycoprotein in the culture, collecting the glycoprotein from the culture, and purifying it. The method includes culturing the transformant according to any one of claims 9 to 11 in a medium, producing and accumulating the antibody composition in the culture, collecting the antibody composition from the culture, and purifying the antibody composition. The manufacturing method of an antibody composition. A glycoprotein or antibody composition in which the N-glycoside-linked complex type sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain is produced, or 14. The method according to 13. The method according to claim 14, wherein the N-glycoside-linked complex type sugar chain is a sugar chain in which the 1-position of fucose is not α-bonded to the 6-position of N-acetylglucosamine at the reducing end of the sugar chain. A glycoprotein produced using the method according to any one of claims 12, 14 and 15. The antibody composition manufactured using the method of any one of Claims 13-15. A medicament comprising the glycoprotein according to claim 16 as an active ingredient. A medicament comprising the antibody composition according to claim 17 as an active ingredient. The medicine is a diagnostic, prophylactic or therapeutic agent for diseases involving tumors, diseases involving allergies, diseases involving inflammation, cardiovascular diseases, autoimmune diseases, diseases involving viral infections or diseases involving bacterial infections Item 20. The pharmaceutical according to Item 18 or 19. Use of the glycoprotein according to claim 16 for the manufacture of a medicament according to claim 18 or 20. Use of the antibody composition according to claim 17 for the manufacture of a medicament according to claim 19 or 20. The method to establish the immortalized cell line of any one of Claims 1-7 including the process of the following (a)-(c).
(a) An organ from a non-human animal in which the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is knocked out Extraction step;
(b) making the extracted organ a non-aggregated cell population;
(c) A step of culturing a non-aggregated cell population. The method to establish the immortalized cell line of any one of Claims 1-7 including the process of the following (a)-(d).
(a) An organ from a non-human animal in which the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is knocked out Extraction step;
(b) making the extracted organ a non-aggregated cell population;
(c) introducing an oncogene into a non-aggregated cell population;
(d) A step of culturing a cell population into which an oncogene has been introduced. The method for establishing an immortalized cell line according to claim 23 or 24, wherein the organ is an organ selected from the group consisting of the following (a) to (d).
(a) liver;
(b) kidney;
(c) bone marrow;
(d) Fetus.

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