JP5427348B2 - Antibodies with improved effector function - Google Patents

Description

  The present invention relates to an antibody with improved effector function.

  Antibodies have high specificity for recognition of antigens and have strong interactions with antigens, so they are widely used for medical treatments, diagnostics, test reagents, etc., and their use will expand in the future. It is expected.

  The structure of an antibody is a polypeptide tetramer composed of two heavy chains and two light chains, and the heavy and light chains are linked by a disulfide bond. The antibody has a variable site corresponding to an antigen-binding site and a constant site, and the C-terminal side of the heavy chain constant site is composed of an Fc region consisting of CH2 and CH3. Although the Fc region is not directly involved in binding to an antigen, various effector functions can be expressed in cells having the same molecule on the surface through binding to an Fc receptor (FcR). Specifically, Fc receptor binding sites on the Fc region bind to FcR present on the effector cell surface, thereby causing phagocytosis and destruction of antibody-coated particles, purification of immune complexes, and activation by activated effector cells. Several important and diverse biological responses including lysis of antibody-coated target cells (referred to as antibody-dependent cellular cytotoxicity (ADCC)), release of inflammatory mediators, placental transfer and control of immunoglobulin production Is caused.

  The expression of the ADCC activity of IgG requires the binding of the Fc region to the FcR present on the surface of the effector cell. For the binding, the second domain (hereinafter referred to as the antibody hinge region and constant region) is used. (Referred to as CH2 domain) has been suggested [Eur. J. Immunol., 23, 1098 (1993) / Non-patent document 1, Immunology, 86, 319 (1995) / Non-patent document 2, Chemical Immunology, 65, 88 (1997) / Non-patent document 3]. In addition, the importance of the sugar chain bound to the CH2 domain has been shown for the binding of the Fc region and FcR [Chemical Immunology, 65, 88 (1997), Abstracts of the 3rd Symposium on Glycoscience Consortium] p30 (2005) / Non-patent document 4], and the production of an antibody having a high effector function by optimizing the sugar chain structure has also been reported [Japanese Patent Laid-Open No. 2005-224240 / Patent Document 1]. These reports show that the CH2 domain of the Fc region containing a sugar chain is extremely important for the effector function of human IgG, and it is possible to produce an antibody having a high effector function by optimizing the amino acid or sugar chain structure of that part. It shows that there is.

  There have been many studies on amino acid substitution of Fc. Presta et al. Comprehensively performed alanine substitution of Fc amino acids, and found that appropriate amino acid substitutions (S298A, E333A, K334A) increased the binding ability to FcγRIIIa and increased ADCC activity [International Publication No. WO00 / 42072]. Pamphlet / patent document 2]. Furthermore, according to Lazar et al., There is data showing that the ADCC activity of different antibodies is increased by simultaneous substitution of S239D / A330L / I332E [US2005 / 0054832A1 / Patent Document 3].

  When producing such an antibody, it takes time and labor to produce cells that produce an antibody that recognizes the target antigen, and a production method using animal cells is performed to ensure the necessary antibody activity. This increases the cost of antibody production and is a major problem in future antibody medicine. In order to efficiently develop antibodies, inexpensive antibody production methods and cost-effective antibody production methods for cost reduction are required.

JP 2005-224240 A International Publication No. 00/42072 Pamphlet US Patent Application Publication No. 2005/0054832 Eur. J. et al. Immunol. , 23, 1098 (1993) Immunology, 86, 319 (1995) Chemical Immunology, 65, 88 (1997) Abstracts of the 3rd Glycoscience Consortium Symposium p30 (2005)

  An object of the present invention is to provide an antibody with enhanced effector function, a method for easily producing the antibody, a method for improving the effector function of the antibody, and a high pharmacological activity of the antibody, which can reduce the dose, and has side effects It is to provide a therapeutic composition that can be used in a wide range of applications.

  The present inventors have conducted extensive studies to achieve the above object, and have found that the effector function can be remarkably improved by substituting a specific amino acid residue for a specific site in the Fc region. It was also found that effector functions can be significantly improved by substituting specific amino acid residues in the Fc region to reduce the fucose of the sugar chain bound to the sugar chain binding site of the antibody.

That is, the present invention provides the following [1] to [20].
[1] An antibody in which the 295th amino acid is replaced with a cysteine residue in the Kabat EU index number, and has the characteristics described in (a) and / or (b) below:
(A) an antibody in which three amino acids other than the 295th amino acid are replaced with other amino acids in the constant region; To which fucose is not bound.
[2] An antibody wherein the amino acids at positions 298, 333, 334, and 295 in Kabat EU index numbers are replaced with alanine, alanine, alanine, and cysteine residues, respectively.
[3] An antibody characterized in that the 239th, 330th, 332th, and 295th amino acids in the Kabat EU index number are replaced with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively.
[4] An antibody in which the 295th amino acid in the Kabat EU index number is replaced with a cysteine residue, and fucose is added to N-acetylglucosamine at the reducing end in the N-glycoside-linked sugar chain bound to the Fc region. Antibody not bound to.
[5] The antibody according to any one of [1] to [4], wherein the constant region is a constant region of human IgG.
[6] The antibody according to any one of [1] to [5], which has a site that recognizes a human cell surface molecule.
[7] Recognizing at least one selected from the group consisting of cytokine receptor, cell adhesion molecule, cancer cell surface molecule, cancer stem cell surface molecule, blood cell surface molecule, virus-infected cell surface molecule, [1] To the antibody of any one of [6].
[8] Antigens CD3, CD11a, CD20, CD22, CD25, CD28, CD33, CD52, Her2 / neu, EGF receptor, EpCAM, MUC1, GD3, CEA, CA125, HLA-DR, TNFalpha receptor, VEGF receptor, The antibody according to any one of [1] to [7], which recognizes at least one selected from the group consisting of CTLA-4, AILIM / ICOS, and an integrin molecule.
[9] An isolated nucleic acid encoding the antibody according to any one of [1] to [8].
[10] A vector comprising the nucleic acid according to [9].
[11] A host cell or host organism having the vector according to [10].
[12] A method for producing an antibody, comprising culturing the host cell or host organism according to [11] so as to express an antibody encoded by a nucleic acid.
[13] A therapeutic composition comprising the antibody according to any one of items [1] to [8].
[14] A cell that expresses an antibody having an effector function in which the 295th amino acid residue in the EU index number of Kabat is replaced with a cysteine residue, and an enzyme that adds fucose to N-acetylglucosamine at the reducing end Cells with reduced or deleted activity.
[15] A method for producing an antibody with improved effector function, comprising the following steps (1) to (3):
(1) a step of replacing amino acids at positions 298, 333, 334, and 295 of Kabat EU index numbers in an antibody having an effector function with alanine, alanine, alanine, and cysteine residues, respectively;
(2) A DNA encoding an H chain in which the 298th, 333, 334, and 295th amino acids in the EU index number of Kabat are substituted with alanine, alanine, alanine, and cysteine residues, respectively, and an L chain Introducing the encoded DNA into a host cell or host organism and expressing the DNA; and (3) recovering the expression product.
[16] A method for producing an antibody with improved effector function, comprising the following steps (1) to (3):
(1) a step of replacing the 239th, 330th, 332th, and 295th amino acids in Kabat EU index numbers in an antibody having an effector function with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively;
(2) DNA encoding the H chain in which the 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers are replaced with aspartic acid, leucine, glutamic acid and cysteine residues, respectively, and the L chain A step of introducing a DNA encoding the DNA into a host cell or host organism and expressing the DNA; and (3) a step of recovering the expression product.
[17] A method for producing an antibody with improved effector function, comprising the following steps (1) to (3):
(1) a step of replacing the 295th amino acid residue in the Kabat EU index number in an antibody having an effector function with a cysteine residue;
(2) DNA encoding the H chain in which the 295th amino acid residue is replaced by a cysteine residue in the EU index number of Kabat, and DNA encoding the L chain are converted to N-acetylglucosamine at the reducing end with fucose. Introducing into the host cell or host organism in which the activity of the enzyme to be added is reduced or deleted, and expressing the DNA; and (3) recovering the expression product.
[18] An antibody effector function comprising the steps of substituting the 298th, 333th, 334th and 295th amino acids in Kabat EU index numbers in an antibody having effector function with alanine, alanine, alanine and cysteine residues, respectively. How to improve.
[19] An antibody effector comprising the steps of substituting aspartic acid, leucine, glutamic acid, and cysteine residues at amino acids 239, 330, 332, and 295 in Kabat EU index numbers in an antibody having an effector function, respectively. How to improve functionality.
[20] A method for improving the effector function of an antibody comprising the following steps (1) to (3);
(1) a step of replacing the 295th amino acid residue in the Kabat EU index number in an antibody having an effector function with a cysteine residue;
(2) DNA encoding the H chain in which the 295th amino acid residue is replaced by a cysteine residue in the EU index number of Kabat, and DNA encoding the L chain are converted to N-acetylglucosamine at the reducing end with fucose. Introducing into the host cell or host organism in which the activity of the enzyme to be added has been reduced or deleted, and expressing the DNA; and (3) recovering the expression product.
[Embodiment of the Invention]

  Herein, “Fc region” is used to define the C-terminal region of an antibody heavy chain. The Fc region is composed of CH2 and CH3 located on the C-terminal side of the heavy chain constant site.

As a part of the present invention, the following antibodies (I) to (IV) are provided.
(I) An antibody in which the 295th amino acid is replaced with a cysteine residue in the EU index number of Kabat, the antibody having the characteristics described in the following (a) and / or (b);
(A) an antibody in which three amino acids other than the 295th amino acid are replaced with other amino acids in the constant region; To which fucose is not bound.
(II) An antibody characterized in that the 298th, 333, 334, and 295th amino acids in the Kabat EU index numbers are replaced with alanine, alanine, alanine, and cysteine residues, respectively (hereinafter referred to as 298Ala / 333Ala / 334Ala / 295Cys type antibody).
(III) An antibody (hereinafter referred to as 239Asp / 330Leu) in which the 239th, 330th, 332th, and 295th amino acids in the Kabat EU index number are replaced with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively. / 332Glu / 295Cys type antibody).
(IV) An antibody in which the 295th amino acid in the EU index number of Kabat is replaced with a cysteine residue, and fucose is added to N-acetylglucosamine at the reducing end in the N-glycoside-linked sugar chain bound to the Fc region. Is not bound (hereinafter also referred to as fucose-reduced 295 Cys type antibody).

298Ala / 333Ala / 334Ala / 295Cys type antibody
(a) The 298th, 333th, 334th, and 295th amino acids in the Kabat EU index numbers correspond to the 303rd, 338th, 339th, and 300th amino acids of SEQ ID NO: 14 (H chain), respectively.
The bases encoding the 298th, 333th, 334th and 295th amino acids in the Kabat EU index numbers are the 907-909, 1012-1014, 1015-1017, and 898-900th positions of SEQ ID NO: 13 (H chain), respectively. Corresponds to the base.
(b) The Kabat EU index numbers 298, 333, 334, and 295th amino acids correspond to amino acids 181, 216, 217, and 178 of SEQ ID NO: 16 (H chain constant region), respectively.
The bases encoding Kabat EU index numbers 298, 333, 334, and 295 amino acids are 541-543, 646-648, 649-651, 532-534 of SEQ ID NO: 15 (H chain constant region), respectively. Corresponds to the second base.
(c) The 298th, 333th, 334th and 295th amino acids in the EU index number of Kabat correspond to the 68th, 103th, 104th and 65th amino acids of SEQ ID NO: 18 (H chain Fc region), respectively.
The bases encoding the amino acids 298, 333, 334, and 295 in the Kabat EU index numbers are 202-204, 307-309, 310-312, and 193-195 of SEQ ID NO: 17 (H chain Fc region), respectively. Corresponds to the second base.
239Asp / 330Leu / 332Glu / 295Cys type antibody
(a) The 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers correspond to the 244th, 335th, 337th and 300th amino acids of SEQ ID NO: 24 (H chain), respectively.
The bases encoding the 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers are the 730-732, 1003-1005, 1009-1011 and 898-900th positions of SEQ ID NO: 23 (H chain), respectively. Corresponds to the base.
(b) The 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers correspond to the 122nd, 213th, 215th and 178th amino acids of SEQ ID NO: 26 (H chain constant region), respectively.
The bases encoding the 239th, 330th, 332th, and 295th amino acids in the Kabat EU index numbers are 364-366, 637-639, 643-645, 532-534 of SEQ ID NO: 25 (H chain constant region), respectively. Corresponds to the second base.
(c) The 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers correspond to the 9th, 100th, 102th and 65th amino acids of SEQ ID NO: 28 (H chain Fc region), respectively.
The bases encoding the 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers are 25-27, 298-300, 304-306, 193-195 of SEQ ID NO: 27 (H chain Fc region), respectively. Corresponds to the second base.
Fucose-reduced 295 Cys type antibody
(a) The 295th amino acid in the EU index number of Kabat corresponds to the 300th amino acid of SEQ ID NO: 30 (H chain).
The base encoding the 295th amino acid in the Kabat EU index number corresponds to the 898th to 900th bases of SEQ ID NO: 29 (H chain).
(b) The 295th amino acid in the EU index number of Kabat corresponds to the 178th amino acid of SEQ ID NO: 32 (H chain constant region).
The base encoding the 295th amino acid in the Kabat EU index number corresponds to the 532-534th bases of SEQ ID NO: 31 (H chain constant region).
(c) The 295th amino acid in the Kabat EU index number corresponds to the 65th amino acid of SEQ ID NO: 34 (H chain Fc region).
The base encoding the 295th amino acid in the Kabat EU index number corresponds to the 193-195th base of SEQ ID NO: 33 (H chain Fc region).

  Since the antibody of the present invention has the above configuration, the effector function can be remarkably improved. Effector functions include, for example, phagocytosis and destruction of antibody-coated particles, purification of immune complexes, lysis of antibody-coated target cells by activated effector cells [antibody-dependent cytotoxicity (ADCC)], complement proteins Cell membrane destruction [complement dependent cytotoxicity (CDC)], release of inflammatory mediators, placental transfer and control of immunoglobulin production. Among these, the antibody of the present invention is suitable for improving ADCC and CDC, and particularly suitable for improving ADCC activity.

The antibodies of the present invention can more effectively mediate ADCC in the presence of human effector cells. ADCC means a cell-mediated reaction, in which an effector cell recognizes a target cell and causes lysis of the target cell. Examples of the effector cells include natural killer (NK) cells, neutrophils, macrophages and the like. These effector cells usually express Fc receptors (FcRs) on the cell surface, and depending on the structure of the Fc region, cells expressing a type of FcR that can be bound are recognized and express cytotoxic activity. can do.
The antibody of the present invention is only required to have at least ADCC activity increased, and an antibody having decreased stability, solubility, binding affinity to Fc receptor, or CDC activity is also included in the antibody of the present invention. It is. Examples of the Fc receptor include, but are not limited to, FcγRI, FcγRII, FcγRIII, FcRn, and the like.

  The antibody of the present invention can induce and promote ADCC by recognizing effector cells via the Fc region. In the present invention, an Fc region into which a mutation has been introduced or a mutation has been introduced and the fucose of a sugar chain bound to an antibody has been reduced easily binds to an effector cell surface molecule [FcγR, etc.] for efficient activation. Because of this, high ADCC activity can be rapidly induced with a small amount of antibody.

  CDC means a reaction mediated by complement protein, and is a reaction in which complement protein recognizes a target cell and causes destruction of the cell membrane of the target cell. The complement protein is activated by binding to the Fc region of the antibody. In the present invention, an Fc region in which a mutation is introduced or a mutation is introduced and the fucose of a sugar chain that binds to an antibody is reduced can easily bind to a complement protein [C1q, etc.] and be activated efficiently. Alternatively, high CDC activity can be rapidly induced with a small amount of antibody.

The Fc region in the present invention preferably has a structure capable of effectively mediating effector functions (particularly ADCC, CDC, etc.). From this point of view, an IgG constant region is often used as a constant region (parent constant region) before modification. Among these, human IgG constant regions are preferable, and human IgG1, human IgG2, human IgG3, and human IgG4 constant regions are more preferable. In the present invention, human IgG1 and human IgG3 constant regions originally having ADCC activity are preferably used. Further, CDC activity has a killing effect depending on the subclass, and a constant region such as human IgG1 or human IgG4 can be used.
The sequence of the constant region can be exemplified by the sequence disclosed in [Sequence of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)], but is not limited thereto. Is not to be done.

  The parent constant region is not limited to the constant region of a naturally occurring antibody. When the antibody (II) is produced, the antibody of the 298, 333, 334, and 295th Kabat EU index numbers (III) Kabat EU index numbers 239, 330, 332, and 295, and when manufacturing an antibody (IV), a part of the amino acid sequence other than the 295th amino acid of Kabat EU index number is missing. It may be modified such as loss, addition, substitution, etc., and may be a synthetic polypeptide consisting of the corresponding amino acid sequence. Amino acid substitution in the parent constant region can be performed using a known method using a gene recombination technique.

The Fc region in the present invention contains a sugar chain binding site. In the antibodies described in (II) and (III) above, a sugar chain does not necessarily have to be bound to the sugar chain binding site, but at least one sugar chain is bound to promote ADCC activity. It is preferable. The sugar chain is not particularly limited as long as it is a sugar chain composed of one or more monosaccharides. Also good. For example, a sugar chain represented by the following formula is an example of a sugar chain bound to a sugar chain binding site in the Fc region of an IgG1 antibody heavy chain.

  In general, it is known that the structure of the sugar chain that binds to the Fc region is closely related to the effector function. In the antibodies described in (II) and (III) above, for the purpose of improving the function, It is also preferable to use sugar chains in which natural sugar chains are appropriately modified. As an example of a sugar chain in which a natural sugar chain is modified, for example, in the above formula, fucose bound to N-acetylglucosamine is modified with addition, deletion, substitution or the like. Examples include sugar chains. The sugar chain bound to the sugar chain modification site may be either O-linked or N-linked, but a sugar chain composed of an N-linked sugar chain is preferably used.

In the antibody described in (IV) above, at least one sugar chain is bound to the sugar chain binding site of the Fc region. The sugar chain to be bound is an N-glycoside-linked sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end. In general, an N-glycoside-linked sugar chain includes the following core structure. In the following structure, the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
The N-glycoside-linked sugar chain has a high mannose type in which only mannose binds to the non-reducing end of the core structure, and a galactose-N-acetylglucosamine (hereinafter referred to as Gal-GlnNAc) branch on the non-reducing end of the core structure. And a complex type having a structure such as sialic acid and bisecting N-acetylglucosamine on the non-reducing end side of Gal-GlnNAc, and a high-mannose type on the non-reducing end of the core structure. Hybrid type with both branches of complex type.
The N-glycoside-linked sugar chain contained in the antibody of (IV) has various structures. Preferably, in the structure of [Chemical Formula 2], fucose is bound to N-acetylglucosamine at the reducing end. Is a sugar chain containing no sugar chain.

IgG is composed of two heavy chains. The antibody (IV) is an antibody in which an N-glycoside-linked sugar chain in which fucose is not bound to N-acetylglucosamine is added to at least one sugar chain modification site in the H chain Fc region. More preferred is an antibody in which an N-glycoside-linked sugar chain to which fucose is not bound to N-acetylglucosamine is added at all sugar chain modification sites in the H chain Fc region.
The composition containing the antibody of (IV) above is 20% or more, preferably 50% or more, more preferably 70% or more, more preferably 90%, of the N-glycoside-linked sugar chain of the antibody in the composition. It refers to a composition in which fucose is deficient in the above N-glycoside-linked sugar chain.

  In general, the sugar chain in the Fc region of a human IgG antibody is extremely important for the ADCC activity of the IgG antibody, and it is known that the sugar chain itself and the sugar chain structure are closely related to the ADCC activity. Yes. In addition, an amino acid sequence of Asn-X-Ser / Thr (X is an arbitrary amino acid residue) is required for binding of a sugar chain to a sugar chain modification site (particularly, expression of an N-type sugar chain). In the antibody of the present invention, the amino acid sequence (for example, amino acids Asn and Thr at the 297th and 299th amino acids) existing on the C-terminal side including the sugar chain modification site in the Fc region of IgG is conserved, and N at the sugar chain binding site. Expression of sugar chains such as type sugar chains is not impaired, and a more excellent ADCC activity improving effect can be obtained. For example, even when the substitution site of a cysteine residue is at the 296th position in the Kabat EU index in IgG, the sugar chain is difficult to bind to the sugar chain modification site or even when bound, the three-dimensional structure of the sugar chain is not retained. Therefore, the effect of improving ADCC activity cannot be obtained sufficiently.

  The antibody of the present invention preferably recognizes a cell surface molecule. More preferably, the cell surface molecule recognized by the antibody of the present invention is a human cell surface molecule.

Examples of the human cell surface molecule include cytokine receptors, cell adhesion molecules, cancer cell surface molecules, cancer stem cell surface molecules, blood cell surface molecules, and virus-infected cell surface molecules. Specific examples of such human cell surface molecule include CD3, CD11a, CD20, CD22, CD25, CD28, CD33, CD52, Her2 / neu, EGF receptor, EpCAM, MUC1, GD3, CEA, CA125, HLA. -DR, TNFalpha receptor, VEGF receptor, CTLA-4, AILIM / ICOS, and integrin molecules.
The antibody of the present invention has an improved effector function compared to the wild type.

  The antibody in the present invention is not particularly limited as long as it has an antigen-binding site and can induce an effector function (particularly ADCC). For example, natural antibodies such as human antibodies, non-human antibodies such as mouse antibodies, and the like These derivatives include recombinant antibodies capable of inducing an antigen-antibody reaction such as chimeric antibodies, humanized antibodies, and single chain antibodies. These may be either monoclonal antibodies or polyclonal antibodies, and may be used alone or in combination of two or more. The derivative of the natural antibody means an antibody in which mutations such as deletion, addition, substitution, etc. are introduced into a part of the amino acid sequence of the natural antibody, and these mutations may occur naturally. Well, it can be artificial. The antigen-binding site includes variable sites VH and VL (especially hypervariable sites CDR1 to 3) in the Fab region of a natural antibody and a region composed of a sequence homologous thereto.

  Among these, recombinant antibodies, particularly chimeric antibodies, humanized antibodies, and the like are preferably used because they can exhibit good ADCC activity and are easily applied to antibody drugs. The chimeric antibody means an antibody composed of two or more heterogeneous or homologous fusion proteins. For example, an antibody composed of an Fc region of human IgG and a Fab region of non-human IgG (such as mouse IgG) Can be mentioned. Examples of the humanized antibody include an antibody obtained by fusing a hypervariable region of non-human IgG (such as mouse IgG) with the remaining region including an Fc region derived from human IgG. For example, a chimeric antibody characterized in that the Fab region is a Fab region derived from a non-human antibody and the Fc region is a Fc region derived from a human antibody, the variable region is a variable region derived from a non-human antibody, and is constant A chimeric antibody characterized in that the region is a constant region derived from a human antibody, a CDR is a CDR derived from a non-human antibody, an FR is an FR derived from a human antibody, and the constant region is a constant region derived from a human antibody There are humanized antibodies that are characterized.

  As the antibody, antibodies against various antigens can be used. Antigens recognized by these antibodies include polypeptides such as receptors such as transmembrane molecules and ligands such as growth factors; non-polypeptide antigens described in US Pat. No. 5,091,178 such as tumor-associated glycolipid antigens, and the like. . Specific antigens include, for example, renin; growth hormone including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α-1-antitrypsin; insulin A-chain Insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anticoagulation factors such as protein C; Pulmonary surfactant; urokinase or plasminogen activator such as human urine or tissue type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor necrosis factor-α and -β; Enkephalinase; RANTES (regulated on activation normally T-cell expresse d and secreted); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Mueller inhibitor; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin related peptide; Microbial protein; DNase; IgE; cytotoxic T lymphocyte associated antigen (CTLA) such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); hormone or growth factor receptor; protein A or D; Rheumatoid factor; brain-induced neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5, or NT-6), or NGF-β Nerve growth factors such as nerve growth factors such as: platelet-derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF Epidermal growth factor (EGF); transforming growth factors (TGF) such as TGF-α and TGF-β (such as TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5); insulin-like Growth factors-I and -II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD3, CD4, CD8, CD19 and CD20 CD protein such as erythropoietin; osteoinductor; immunotoxin; bone morphogenetic protein (BMP); interferon such as interferon-α, -β, and -γ; colonies such as M-CSF, GM-SCF, and G-CSF Stimulating factors (CSFs); interleukins (IL) such as IL-1 to IL-10; superoxide dismutase; T cell receptor; surface membrane protein; Viral antigens such as part of AIDS envelope; transport protein; homing receptor; addressin; regulatory protein; integrin such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; HER2 Tumor-associated antigens such as HER3 or HER4 receptors; and fragments of these polypeptides.

  Suitable molecular targets for the antibodies of the invention include, for example, CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; ErbB receptor families such as HER2, HER3 or HER4 receptors; LFA-1, Mac1, p150.95 Cell adhesion molecules such as αv / β3 integrin (for example, anti-CD11a, anti-CD18 or anti-CD11b antibody) containing any one of VLA-4, ICAM-1, VCAM and α or β subunit thereof; growth factors such as VEGF; IgE; blood group antigen; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C and the like.

  Among the antigens exemplified above, soluble antigens and fragments thereof can be used as an immunogen for producing antibodies. For example, for a transmembrane molecule such as a receptor, for example, a fragment such as the extracellular domain of the receptor can be used as an immunogen. Alternatively, cells that express transmembrane molecules can be used as immunogens. Such cells can be removed from natural sources (eg, cancer cell lines), or are cells that have been transformed to express an antigen recognition region, such as a transmembrane molecule, using a recombinant vector. Also good.

  These antibodies may have a site capable of binding a cell surface molecule in addition to the antigen binding site. Since the antibody having such a configuration can improve the recognition efficiency of the target cell, it is possible to exert a high effector function with a small amount of antibody.

  Further, the antibody of the present invention may have other peptides or polypeptides in a part thereof. Other peptides or polypeptides include, for example, FLAG (Hopp, TP et al., BioTechnology (1988) 6, 1204-1210), 6 × His consisting of 6 His (histidine) residues, 10 × His, Influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, Known peptides such as B-tag and protein C fragments can be used. Further, examples of other polypeptides to be subjected to fusion with the antibody of the present invention include GST (glutathione-S-transferase), HA (influenza agglutinin), β-galactosidase, MBP (maltose binding protein) and the like. Can be mentioned.

In the present invention, for example, the 298Ala / 333Ala / 334Ala / 295Cys type anti-CD20 antibody described in any of (1) to (4) below is provided.
(1) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 16 as CH An antibody comprising an H chain having a sequence;
(2) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 14 (amino acid sequence of the entire H chain),
(3) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid sequence described in SEQ ID NO: 18 as Fc region An antibody comprising an H chain having an amino acid sequence,
(4) An antibody having a pair of the H chain according to any of (1), (2) or (3) above and the L chain according to (i) or (ii) below,
(I) the amino acid sequence described in SEQ ID NO: 8 as CDR1, the amino acid sequence described in SEQ ID NO: 10 as CDR2, the amino acid sequence described in SEQ ID NO: 12 as CDR3, and the amino acid described in SEQ ID NO: 22 as CL A light chain having a sequence;
(Ii) An L chain having the amino acid sequence of SEQ ID NO: 20 (an amino acid sequence of the entire L chain).

The present invention provides, for example, the 298Ala / 333Ala / 334Ala / 295Cys type anti-CD20 antibody described in any of (5) to (8) below.
(5) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 15;
(6) an antibody comprising an H chain encoded by the base sequence set forth in SEQ ID NO: 13;
(7) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 17;
(8) An antibody having a pair of the H chain according to any of (5), (6) or (7) above and the L chain according to (i) or (ii) below,
(I) CDR1 encoded from the nucleotide sequence set forth in SEQ ID NO: 7, CDR2 encoded from the nucleotide sequence set forth in SEQ ID NO: 9; CDR3 encoded from the nucleotide sequence set forth in SEQ ID NO: 11; and sequence L chain having CL encoded by the nucleotide sequence of number: 21;
(Ii) An L chain encoded from the nucleotide sequence set forth in SEQ ID NO: 19.

In the present invention, for example, the 239Asp / 330Leu / 332Glu / 295Cys type anti-CD20 antibody described in any of (9) to (12) below is provided.
(9) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 26 as CH An antibody comprising an H chain having a sequence;
(10) an antibody comprising an H chain having the amino acid sequence of SEQ ID NO: 24 (amino acid sequence of the entire H chain),
(11) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid sequence described in SEQ ID NO: 28 as Fc region An antibody comprising an H chain having an amino acid sequence,
(12) An antibody having a pair of the H chain according to any of (9), (10) or (11) above and the L chain according to (i) or (ii) of (4) above.

The present invention provides, for example, the 239Asp / 330Leu / 332Glu / 295Cys type anti-CD20 antibody described in any of (13) to (16) below.
(13) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 25;
(14) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 23,
(15) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 27;
(16) An antibody having the pair of the H chain according to any of (13), (14) or (15) and the L chain according to (i) or (ii) of (8) above.

In the present invention, for example, the fucose-reduced 295 Cys-type anti-CD20 antibody described in any of (17) to (20) below is provided.
(17) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid described in SEQ ID NO: 32 as CH3 An antibody comprising an H chain having a sequence;
(18) an antibody comprising an H chain having the amino acid sequence set forth in SEQ ID NO: 30 (amino acid sequence of the entire H chain),
(19) The amino acid sequence described in SEQ ID NO: 2 as CDR1, the amino acid sequence described in SEQ ID NO: 4 as CDR2, the amino acid sequence described in SEQ ID NO: 6 as CDR3, and the amino acid sequence described in SEQ ID NO: 34 as Fc region An antibody comprising an H chain having an amino acid sequence,
(20) An antibody having a pair of the H chain according to any of (17), (18) or (19) and the L chain according to (4) (i) or (ii) above.

The present invention provides, for example, the fucose-reduced 295 Cys-type anti-CD20 antibody described in any of (21) to (24) below.
(21) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having CH encoded by the nucleotide sequence of No. 31;
(22) an antibody comprising an H chain encoded from the base sequence set forth in SEQ ID NO: 29,
(23) CDR1 encoded from the base sequence set forth in SEQ ID NO: 1, CDR2 encoded from the base sequence set forth in SEQ ID NO: 3, CDR3 encoded from the base sequence set forth in SEQ ID NO: 5, and sequence An antibody comprising an H chain having an Fc region encoded by the nucleotide sequence of No. 33;
(24) An antibody having a pair of the H chain according to any of (21), (22) or (23) and the L chain according to (i) or (ii) of (8) above.

  The antibody of the present invention can be produced by a known method using a gene recombination technique or the like. The antibody of the present invention uses an isolated nucleic acid encoding the antibody of the present invention, creates a vector containing the nucleic acid, obtains a host cell or host organism having the vector, and selects these host cells or host organisms. It can be produced using a method of culturing to express a polypeptide encoded by a nucleic acid.

  The antibody of the present invention can be produced using a general antibody production method. The antibody production method is described in Molecular Cloning 2nd edition, Current Protocols in Molecular Biology, Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, 1988, Monoclonal Antibodies: principals and practice, Third Edition, Acad. The method described in Press, 1993, Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996, etc. can be used.

  For example, in the production of an antibody, a step of isolating a nucleic acid comprising a sequence in which an amino acid in an Fc region in IgG is substituted to encode a target amino acid in a nucleic acid encoding a polypeptide containing a parent Fc region; Incorporating the encoded isolated nucleic acid into an expression vector containing an appropriate promoter to produce a replicable recombinant vector; transforming a host cell or host organism with the resulting recombinant vector; And culturing the transformed host cell or host organism to produce antibodies.

  Polypeptides containing a parent Fc region may be modified such as deletion, addition, substitution, etc. at amino acids other than the 239, 295, 298, 330, and 332-334 amino acids in Kabat's EU index number in the Fc region of IgG. It may have been made.

The nucleic acid encoding the antibody of the present invention can be obtained by a conventional method for introducing a mutation into the base sequence. For example, the nucleic acid sequence encoding the substituted amino acid residue in the parent Fc region is represented by “TGC” or “TCT”, “GAT” or “GAC”, “CTT” or “CTC” or “CTA” or “CTG”, “GAA” or “GAG”, “GCT” or “GCC” or “GCA” or “GCG”, “TCT” ”Or“ TCC ”or“ TCA ”or a synthetic oligonucleotide containing a nucleic acid sequence replaced with“ TCG ”and can be prepared by PCR using cDNA containing the parent Fc region as a template.
In the antibodies described in (II) and (III) above, during or during the process of introducing the mutation described in (II) or (III) into the amino acid sequence of the parent Fc region within a range not inhibiting the effector function Later, other modifications may be applied. For example, modifications such as deletion, addition, and substitution may be applied to a part of the sugar chain that binds to the amino acid sequence or the sugar chain binding site. In particular, in the present invention, it is preferable to add a known modification to the sugar chain that binds to the sugar chain binding site of the Fc region, whereby the effect of improving the effector function can be obtained.

  A replicable recombinant vector can be prepared by inserting a nucleic acid (DNA fragment or full-length cDNA) encoding the antibody of the present invention downstream of the promoter of an appropriate expression vector. The expression vector can be appropriately selected according to the type of host cell or host organism to be introduced, and can be replicated autonomously in the host cell or can be integrated into the chromosome, and a nucleic acid encoding the desired antibody can be obtained. Those containing a promoter capable of transcription are used. These recombinant vectors may be used alone or in combination of two or more.

  In the present invention, two or more recombinant vectors may be used in combination. Examples of the recombinant vector to be used in combination include a suitable antibody light chain or heavy chain, or a nucleic acid encoding a binding domain of a desired receptor or ligand, a nucleic acid encoding a desired sugar chain modifying enzyme, etc. An expression vector can be used. By using these recombinant vectors in combination, an antibody capable of expressing a higher effector function can be obtained. When two or more recombinant vectors are used, the host cell or host organism may be the same or different.

  As the host cell, any yeast cell, animal cell, insect cell, plant cell and the like that can express the target gene can be used. In the present invention, the host cell is selected from cells in which the activity of the modifying enzyme for the sugar chain that binds to the sugar chain binding site that affects the effector function is reduced or deleted, or the same activity is obtained by an artificial method. Reduced or deleted cells can be used. Such enzymes include enzymes involved in the modification of N-glycoside-linked sugar chains. For example, enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose, N-glycoside-linked complex type sugar chain reducing terminal Examples include enzymes involved in sugar chain modification in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine. Examples of host organisms include animal individuals and plant individuals.

  When yeast is used as a host cell, YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), etc. can be used as an expression vector. Any promoter can be used as long as it can be expressed in yeast strains. For example, a glycolytic gene promoter such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 Examples include promoters, gal10 promoters, heat shock protein promoters, MFα1 promoters, CUP1 promoters and the like. Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Cluyveromyces, Trichosporon, Schwaniomyces, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromes Can do.

  As a method for introducing a recombinant vector into yeast, any method can be used as long as it is a method for introducing DNA into yeast. For example, electroporation method [Methods in Enzymology], 194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)] Lithium Acetate Method [J. Bacteriology, 153, 163 (1983)], Proceedings of the National Academy of Science (Proc. Natl. Acad. Sci. U.). S.A), 75, 1929 (1 And a method such as described in 78).

  When animal cells are used as hosts, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnology, 3, 133, (1990)], pAS3. -3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [Journal of Biochemistry (J) Biochemistry), 101, 1307 (1987)], pAGE210, and the like. Any promoter can be used so 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 enhancer of human CMV IE gene may be used together with a promoter. Examples of host cells include human cells such as Namalwa cells, COS cells that are monkey cells, CHO cells that are Chinese hamster cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), rat myeloma cells, and mouse myeloma. Examples include cells, Syrian hamster kidney-derived cells, embryonic stem cells, fertilized egg cells, and the like.

  As a method for introducing a recombinant vector into animal cells, 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 [Japanese Patent Laid-Open No. 2-227075], lipofection method [Proc. Natl. Acad. Sci. USA,] 84, 7413 (Proc. Natl. Acad. Sci. USA) 1987)], injection method [manipulating the mouse embryo a laboratory manual], method using particle gun (gene gun) [patent 2606856, patent 2517813], DEAE-dextran method [biomanifold] Al Series 4-Gene Transfer and Expression Analysis (Yodo-sha), edited by Takashi Yokota and Kenichi Arai (1994)], the virus vector method [Manipulating the Mouse Embryo, Second Edition], and the like can be mentioned.

  When insect cells are used as the host, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W., et al. H. Proteins can be expressed by methods described in Freeman and Company, New York (1992), Bio / Technology, 6, 47 (1988), and the like. That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can. Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacIII (both from Invitrogen) and the like.

  As the baculovirus, for example, Autographa californica nucleopolyhedrosis virus, which is a virus that infects the night stealing insects, can be used. Examples of insect cells include Sf9, Sf21 [Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, W., which is an ovarian cell of Spodoptera frugiperda. H. Freeman and Company, New York (1992)], Trichoplusian ovary cells, High5 (Invitrogen), and the like can be used.

  Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 227075), the lipofection method [Proceedings of The National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)].

  When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors. Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter and the like. Examples of the host cell include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

  As a method for introducing a recombinant vector into a plant cell, any method can be used as long as it is a method for introducing DNA into a plant cell. For example, a method using Agrobacterium [Japanese Patent Laid-Open No. 59-140885, JP-A-60-70080, WO94 / 00977], electroporation method [JP-A-60-251887], a method using a particle gun (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 2517813] and the like. it can. As a gene expression method, in addition to direct expression, secretory production, expression of a fusion protein between an Fc region and another protein, or the like can be performed according to the method described in Molecular Cloning 2nd edition.

  Thus, a transformant introduced with one or more recombinant vectors can be produced and accumulated in the culture by culturing according to a known method using a medium suitable for the host. it can.

  As a medium for culturing a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host, the medium contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the organism. Any natural or synthetic medium may be used as long as the medium can efficiently be cultured. The carbon source may be anything that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol, Alcohols such as propanol can be used. Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used. Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  Culturing of a transformant using a prokaryote such as E. coli or a eukaryote such as yeast as a host is usually carried out under aerobic conditions such as shaking culture or deep aeration and agitation culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. The pH during the culture is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation. When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like with a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

As a medium for culturing a transformant obtained using an animal cell as a host, a commonly used RPMI 1640 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 [Proceeding Of the Society for the Biological Medicine, 73,1. Of the Society for the Biological Medicine, 73,1 (1950)], Whitten medium [Genetic engineering experiment manual-How to make a transgenic mouse (Kodansha) edited by Motoya Katsuki (1987)], or a medium obtained by adding fetal calf serum or the like to these mediums. 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 . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

  As a medium for culturing a transformant obtained using insect cells as a host, commonly used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCell400, ExCell405 (both are JRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] and the like can be used. The culture is usually performed for 1 to 5 days under conditions of pH 6 to 7, 25 to 30 ° C, and the like. Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.

  A transformant obtained using a plant cell as a host can be cultured as a cell or differentiated into a plant cell or organ. As a medium for culturing the transformant, a commonly used Murashige and Skoog (MS) medium, White medium, or a medium in which plant hormones such as auxin and cytokinin are added to these mediums or the like is used. be able to. The culture is usually performed for 3 to 60 days under conditions of pH 5 to 9, 20 to 40 ° C. Moreover, you may add antibiotics, such as kanamycin and a hygromycin, to a culture medium as needed during culture | cultivation.

  The transformant being cultured can express the antibody naturally or by induction, and can be produced and accumulated in the culture. As an antibody expression method, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed in accordance with the method described in Molecular Cloning, Second Edition. The antibody production method includes a method of producing in the host cell, a method of secreting it outside the host cell, or a method of producing it on the outer membrane of the host cell. This method can be selected.

  When antibodies are produced in or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], Law et al. Methods [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); Gene Development., 4] [Methods of the National Academy of Sciences, Proc. , 1288 (1990)], or by applying the methods described in JP-A-05-336963, JP-A-06-830221 and the like, the antibody can be actively secreted outside the host cell.

  That is, using genetic recombination techniques, an antibody encoding DNA and a DNA encoding a signal peptide appropriate for antibody expression are inserted into an expression vector, and the antibody is introduced after the expression vector is introduced into a host cell. By expressing it, the target antibody can be actively secreted outside the host cell. Further, in accordance with the method described in JP-A-2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the animal or plant into which the gene has been introduced, an animal individual (transgenic non-human animal) or plant individual (transgenic plant) into which the gene has been introduced is constructed, and antibodies are used using these individuals. Can also be manufactured.

  When the transformant is an animal or plant individual, the antibody is produced by breeding or cultivating according to a usual method, producing and accumulating the antibody, and collecting the antibody from the animal or plant individual. Can do. As a method for producing an antibody using an individual animal, for example, a known method [American Journal of Clinical Nutrition (American Journal of Clinical Nutrition), 63, 639S (1996); American Journal of Clinical A target antibody in an animal constructed by introducing a gene according to Nutrition (American Journal of Clinical Nutrition), 63, 627S (1996); Bio / Technology, 9, 830 (1991)] The production method is given.

  In the case of an animal individual, for example, a transgenic non-human animal introduced with DNA encoding the antibody is bred, the antibody is produced and accumulated in the animal, and the antibody is collected from the animal to produce the antibody. can do. Examples of the production / accumulation place in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192), eggs and the like. Any promoter can be used as long as it can be expressed in animals. For example, α-casein promoter, β-casein promoter, β-lactoglobulin promoter, whey acidity that is a mammary cell-specific promoter. A protein promoter or the like is preferably used.

  As a method for producing an antibody using a plant individual, for example, a transgenic plant into which DNA encoding an antibody molecule is introduced is known in the art [tissue culture, 20 (1994); tissue culture, 21 (1995);・ Method of producing antibodies by cultivating according to Trends in Biotechnology, 15, 45 (1997)], generating and accumulating antibodies in the plants, and collecting the antibodies from the plants Can be given. For example, in the case of an antibody produced by a transformant into which a gene encoding an antibody has been introduced, when the antibody is expressed in a dissolved state in the cell, the cell is recovered by centrifugation after completion of the culture, and is added to an aqueous buffer solution. After the suspension, the cells are crushed by an ultrasonic crusher, a French press, a Manton Gaurin homogenizer, a dyno mill 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 a resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Corporation), cation exchange chromatography using a 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. Use alone or in combination to obtain purified antibodies. .

  When the antibody is expressed by forming an insoluble substance in the cell, the cell is similarly collected, disrupted, and centrifuged to recover the antibody insoluble substance as a precipitate fraction. The recovered insoluble body of the antibody is solubilized with a protein denaturant. After the solubilized solution is diluted or dialyzed to return the antibody to a normal three-dimensional structure, a purified preparation of the antibody can be obtained by the same isolation and purification method as described above.

  When the antibody is secreted extracellularly, the antibody or its derivative can be recovered in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation similar to that described above, and a purified antibody preparation is obtained from the soluble fraction by using the same isolation and purification method as described above. Can be obtained.

Examples of the antibodies thus obtained include antibody fragments, fusion proteins having antibody Fc regions (recombinant antibodies capable of inducing antigen-antibody reactions such as chimeric antibodies, humanized antibodies, single chain antibodies), and the like. Can be mentioned.
That is, the present invention includes a step in which the 298th, 333th, 334th and 295th amino acids of Kabat EU index numbers in an antibody having an effector function are replaced with alanine, alanine, alanine and cysteine residues, respectively. Provided is a method for producing an antibody with improved effector function. More specifically, the present invention provides a method for producing an antibody with improved effector function, comprising the following steps.
(A-1) A step of substituting the 298th, 333th, 334th and 295th amino acids of Kabat's EU index number in an antibody having an effector function with alanine, alanine, alanine and cysteine residues, respectively (A-2) DNA encoding the H chain in which the 298th, 333, 334, and 295th amino acids in the Kabat EU index numbers are replaced with alanine, alanine, alanine, and cysteine residues, respectively, and DNA encoding the L chain A step of introducing the DNA into a host cell or host organism and expressing the DNA (A-3) a step of recovering the expression product.
The above (A-1) produces DNA encoding an antibody in which amino acids 298, 333, 334, and 295 in Kabat's EU index are replaced with alanine, alanine, alanine, and cysteine residues, respectively. It may be replaced with the process of.

The present invention also includes a process in which the 239th, 330th, 332th, and 295th amino acids in Kabat EU index numbers in an antibody having an effector function are replaced with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively. And a method for producing an antibody with improved effector function. More specifically, the present invention provides a method for producing an antibody with improved effector function, comprising the following steps.
(B-1) A step of substituting the 239th, 330th, 332th and 295th amino acids of Kabat EU index numbers in antibodies having effector functions with aspartic acid, leucine, glutamic acid and cysteine residues, respectively (B-2 ) DNA encoding the H chain in which the 239th, 330th, 332rd and 295th amino acids in the Kabat EU index numbers are replaced with aspartic acid, leucine, glutamic acid and cysteine residues, respectively, and the L chain A step of introducing the DNA to be introduced into a host cell or host organism and expressing the DNA (B-3) a step of recovering the expression product;
The above (B-1) is a DNA encoding an antibody in which the 239th, 330th, 332th, and 295th amino acids in Kabat EU index numbers are replaced with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively. It may be replaced with a manufacturing process.

That is, the present invention relates to a step of replacing the 295th amino acid residue in the Kabat EU index number with a cysteine residue in an antibody having an effector function, and an enzyme activity for adding fucose to N-acetylglucosamine at the reducing end of the antibody A method for producing an antibody with improved effector function, which comprises a step of reducing or deleting More specifically, the present invention provides a method for producing an antibody with improved effector function, comprising the following steps.
(C-1) replacing the 295th amino acid residue with a Kabat EU index number in an antibody having an effector function, with a cysteine residue;
(C-2) A DNA encoding an H chain in which the 295th amino acid residue in the EU index number of Kabat is replaced with a cysteine residue, and a DNA encoding an L chain as N-acetylglucosamine at the reducing end Introducing into the host cell or host organism in which the activity of the enzyme adding fucose has been reduced or deleted, and expressing the DNA; and (C-3) recovering the expression product.
The above (C-1) may be replaced with a step of producing a DNA encoding an antibody in which the 295th amino acid residue in the Kabat EU index number is replaced with a cysteine residue.

As one embodiment of the present invention, first, an amino acid residue as a substitution site in the above (A) to (C) with the Kabat EU index number in an antibody having an effector function known to those skilled in the art is used. Substitution with the desired amino acid residue in (C).
As another aspect of the present invention, first, an antibody having an effector function is produced, and then the amino acid residue as the substitution site in the above (A) to (C) with the Kabat EU index number in the produced antibody. To the desired amino acid residue. For example, first, an antibody that binds to a desired antigen is obtained by the method described later, and then whether or not the obtained antibody has an effector function is determined by a method well known to those skilled in the art, thereby having an effector function. Antibodies can be produced. Examples of the method for determining the effector function include the methods described in the examples.

  In the present invention, a method for substituting an amino acid residue with a cysteine residue is not particularly limited. For example, a site-directed mutagenesis using M13-derived vector: an efficient and general procedure for the production of point mutations in any fragment of DNA, Mark J. Zoller and Michael Smith, Nucleic Acids Research, Volume 10 Number 20, 6487-6500, 1982; Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis Asako Sawano and Atsushi Miyawaki, Nucleic Acids Research, Volume 28, Number 16, e78, 2000).

  In the present invention, DNA encoding an H chain having an Fc region in which the amino acid residue used as the substitution site in (A) to (C) above is substituted with the target amino acid residue in the Kabat EU index number. And DNA encoding the L chain is introduced into a host cell or host organism by a method well known to those skilled in the art, and the DNA is expressed. The expression product can then be recovered using methods well known to those skilled in the art.

  A DNA encoding an H chain having an Fc region in which an amino acid residue as a substitution site in the above (A) to (C) is substituted with a target amino acid residue using the Kabat EU index number is divided into partial DNAs. Can be manufactured. Examples of the combination of partial DNAs include DNA encoding a variable region and DNA encoding a constant region, or DNA encoding a Fab region and DNA encoding an Fc region, but are not limited to these combinations. Absent. Similarly, the DNA encoding the L chain can also be produced by dividing it into partial DNAs.

A method for obtaining an antibody that binds to a desired antigen is described below.
A known sequence can be used as the gene encoding the H chain or L chain of the antibody, and it can also be obtained by methods known to those skilled in the art. For example, it can be obtained from an antibody library, or can be obtained by cloning a gene encoding an antibody from a hybridoma producing a monoclonal antibody.
Many antibody libraries have already been known for antibody libraries, and methods for preparing antibody libraries are also known, and those skilled in the art can appropriately obtain antibody libraries. For example, for antibody phage libraries, Clackson et al., Nature 1991, 352: 624-8, Marks et al., J. Mol. Biol. 1991, 222: 581-97, Waterhouses et al., Nucleic Acids Res 1993, 21: 2265-6, Griffiths et al., EMBO J. 1994, 13: 3245-60, Vaughan et al., Nature Biotechnology 1996, 14: 309-14, and Japanese National Publication No. 20-504970 Can be referred to. In addition, a known method such as a method using eukaryotic cells as a library (WO95 / 15393 pamphlet) or a ribosome display method can be used. Furthermore, a technique for obtaining a human antibody by panning using a human antibody library is also known. For example, the variable region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method, and a phage that binds to the antigen can be selected. By analyzing the gene of the selected phage, the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined. If the DNA sequence of scFv that binds to the antigen is clarified, a suitable expression vector can be prepared based on the sequence to obtain a human antibody. These methods are already well known, and WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, and WO95 / 15388 can be referred to.

  A method for obtaining a gene encoding an antibody from a hybridoma basically uses a known technique, and uses a desired antigen or a cell that expresses a desired antigen as a sensitizing antigen. Therefore, the immune cells obtained are immunized, and the obtained immune cells are fused with known parental cells by ordinary cell fusion methods, and monoclonal antibody-producing cells (hybridomas) are screened by ordinary screening methods, and reverse transcription is performed from the resulting hybridoma mRNA. It can be obtained by synthesizing cDNA of an antibody variable region (V region) using an enzyme and ligating it with DNA encoding a desired antibody constant region (C region).

  More specifically, although not particularly limited to the following examples, the sensitizing antigen for obtaining the antibody gene encoding the H chain and L chain of the present invention includes a complete antigen having immunogenicity, It includes both incomplete antigens including haptens that do not exhibit immunogenicity. For example, a full-length protein of the target protein or a partial peptide can be used. Antigens can be prepared by methods known to those skilled in the art, for example, according to methods using baculovirus (for example, WO98 / 46777). The hybridoma can be produced, for example, according to the method of Milstein et al. (G. Kohler and C. Milstein, Methods Enzymol. 1981, 73: 3-46). When the immunogenicity of the antigen is low, immunization may be performed by binding to an immunogenic macromolecule such as albumin. Moreover, it can also be made a soluble antigen by combining an antigen with another molecule as required. When a transmembrane molecule such as a receptor is used as an antigen, the extracellular region of the receptor can be used as a fragment, or a cell expressing the transmembrane molecule on the cell surface can be used as an immunogen.

  Antibody-producing cells can be obtained by immunizing an animal with the appropriate sensitizing antigen described above. Alternatively, antibody-producing cells can be obtained by immunizing lymphocytes capable of producing antibodies in vitro. Various mammals can be used as the animal to be immunized, and rodents, rabbits, and primates are generally used. Examples include rodents such as mice, rats and hamsters, rabbits such as rabbits, and primates such as monkeys such as cynomolgus monkeys, rhesus monkeys, baboons and chimpanzees. In addition, transgenic animals having a repertoire of human antibody genes are also known, and human antibodies can be obtained by using such animals (WO96 / 34096; Mendez et al., Nat. Genet. 1997, 15: 146-56). Instead of using such transgenic animals, for example, human lymphocytes are sensitized in vitro with the desired antigen or cells expressing the desired antigen, and the sensitized lymphocytes are fused with human myeloma cells, such as U266. Thus, a desired human antibody having an antigen-binding activity can also be obtained (see Japanese Patent Publication No. 1-59878). Further, a desired human antibody can be obtained by immunizing a transgenic animal having all repertoires of human antibody genes with a desired antigen (WO93 / 12227, WO92 / 03918, WO94 / 02602, WO96 / 34096, (See WO96 / 33735).

  For immunization of animals, for example, sensitized antigens are appropriately diluted and suspended in Phosphate-Buffered Saline (PBS) or physiological saline, etc., and mixed with an adjuvant as necessary, and then emulsified intraperitoneally or subcutaneously. This is done by injection. Thereafter, preferably, the sensitizing antigen mixed with Freund's incomplete adjuvant is administered several times every 4 to 21 days. Confirmation of antibody production can be performed by measuring the desired antibody titer in the serum of animals by a conventional method.

  Hybridomas can be made by fusing antibody-producing cells obtained from animals or lymphocytes immunized with the desired antigen with myeloma cells using conventional fusion agents (e.g., polyethylene glycol) (Goding , Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, 59-103). If necessary, hybridoma cells are cultured and expanded, and the binding specificity of the antibody produced from the hybridoma is measured by a known analysis method such as immunoprecipitation, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), etc. . Thereafter, if necessary, the hybridoma producing the antibody whose target specificity, affinity or activity is measured can be subcloned by a technique such as limiting dilution.

  Subsequently, a probe encoding a gene encoding the selected antibody from a hybridoma or an antibody-producing cell (sensitized lymphocyte, etc.) (for example, an oligo complementary to a sequence encoding an antibody constant region). For example, nucleotides). It is also possible to clone from mRNA by RT-PCR.

  An antibody can be obtained by expressing the constructed antibody gene by a known method. In the case of mammalian cells, the antibody gene is expressed in an expression vector containing a useful promoter / enhancer that is commonly used, the antibody gene to be expressed, and DNA in which a poly A signal is operably linked to the 3 ′ downstream thereof. Can do. For example, the promoter / enhancer includes human cytomegalovirus early promoter / enhancer. In addition, viral promoters / enhancers such as retrovirus, polyomavirus, adenovirus, and simian virus 40 (SV40) and promoters / enhancers derived from mammalian cells such as human elongation factor-1α can be used. . For example, when the SV40 promoter / enhancer is used, the antibody gene can be easily expressed according to the method of Mulling et al. (Mulling RC et al., Nature (1979) 277: 108-14). When human elongation factor-1α is used, an antibody gene can be expressed easily according to the method of Mizushima (Mizushima, Nucleic Acids Res (1990) 18: 5322). In the case of Escherichia coli, an antibody gene can be expressed by an expression vector containing a commonly used useful promoter, a signal sequence for antibody secretion, and a DNA to which an antibody gene to be expressed is functionally linked. Examples of the promoter include LacZ promoter and araB promoter.

  Antibodies obtained by hybridoma culture / growth or genetic recombination can be purified until they are homogeneous. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, if the chromatography column such as affinity chromatography, filter, ultrafiltration, salting out with ammonium sulfate or sodium sulfate, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. are appropriately selected and combined, the antibody Can be isolated and purified (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. Examples of the column used for affinity chromatography include a protein A column, a protein G column, and a protein L column. Whether or not the antibody thus obtained has an effector function can be determined by a method well known to those skilled in the art, and can be determined, for example, by the method described in the Examples.

  As a host cell or animal individual or plant individual for producing an antibody having an N-glycoside-linked sugar chain in which fucose does not bind to N-acetylglucosamine or is reduced in binding, for example, it binds to the Fc region of the antibody. Any cell or individual that has low enzyme activity or does not have enzyme activity for adding fucose to N-acetylglucosamine can be used. Cells with low or no enzyme activity that add fucose to N-acetylglucosamine that binds to the Fc region of the antibody include rat myeloma YB2 / 3HL.P2.G11.16Ag.20 cells (YB2 / 0 cells and (Abbreviated) (conserved as ATCC CRL 1662), FTVIII knockout CHO cells (WO 02/31140), Lec13 cells (WO03 / 035835), fucose transporter deficient cells (WO2006 / 067847, WO2006 / 067913), etc. Can be mentioned.

  In addition, those skilled in the art can delete a gene of an enzyme involved in α1,6 binding in a host cell, an animal individual or a plant individual by a known method, or give a mutation to the gene to reduce or delete the enzyme activity. Thus, a cell or individual having little or no enzyme activity involved in α1,6 binding can be produced and used as a host cell, animal individual or plant individual. The enzyme involved in α1,6 binding includes fucosyltransferase, preferably α1,6-fucosyltransferase (hereinafter sometimes referred to as FUT8). As the host cell, any cell, yeast, animal cell, insect cell, plant cell, etc. can be used so long as it can express the target gene.

  Sugar chains can be analyzed by methods known to those skilled in the art. For example, N-Glycosidase F (Roche) or the like is allowed to act on the antibody to release the sugar chain from the antibody. Then, after desalting by solid phase extraction using a cellulose cartridge (Shimizu Y. et al., Carbohydrate Research 332 (2001), 381-388), the solution is concentrated to dryness and fluorescently labeled with 2-aminopyridine (Kondo A et al., Agricultural and Biological Chemistry 54: 8 (1990), 2169-2170). The obtained PA sugar chain is removed by a solid phase extraction using a cellulose cartridge and then concentrated by centrifugation to obtain a purified PA sugar chain. Then, it can measure by performing reverse phase HPLC analysis with an ODS column. In addition, it is also possible to carry out two-dimensional mapping, which is a combination of reverse-phase HPLC analysis using an ODS column and normal-phase HPLC analysis using an amine column after the preparation of PA sugar chain.

  To produce a host cell or host organism in which the activity of the enzyme that adds fucose is reduced or eliminated, antisense RNA complementary to the transcript of the DNA encoding the enzyme can be used.

  There are several factors as described below for the action of an antisense nucleic acid to suppress the expression of a target gene. That is, transcription initiation inhibition by triplex formation, transcription inhibition by hybridization with a site where an open loop structure is locally created by RNA polymerase, transcription inhibition by hybridization with RNA that is undergoing synthesis, intron and exon Of splicing by hybrid formation at the junction with the protein, suppression of splicing by hybridization with the spliceosome formation site, suppression of transition from the nucleus to the cytoplasm by hybridization with mRNA, hybridization with capping sites and poly (A) addition sites Splicing suppression by formation, translation initiation suppression by hybridization with the translation initiation factor binding site, translation suppression by hybridization with the ribosome binding site near the initiation codon, peptization by hybridization with the mRNA translation region and polysome binding site For example, inhibition of gene chain elongation is prevented, and hybridization between nucleic acid and protein interaction sites is performed. They inhibit the expression of target genes by inhibiting transcription, splicing, or translation processes.

The antisense sequence used in the present invention may suppress the expression of the target gene by any of the actions described above. In one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene is designed, it will be effective for inhibiting translation of the gene. However, sequences complementary to the coding region or the 3 ′ untranslated region can also be used. As described above, a DNA containing an antisense sequence of a non-translated region as well as a translated region of a gene is also included in the antisense DNA used in the present invention. The antisense DNA to be used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The DNA thus prepared can be transformed into a desired plant by a known method. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene or a part thereof possessed by the plant to be transformed, but may be not completely complementary as long as the gene expression can be effectively inhibited. Good. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcription product of the target gene. In order to effectively inhibit the expression of a target gene using an antisense sequence, the length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, more preferably 500 bases or more. is there. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb.
Derivatives or modified products of antisense oligonucleotides can also be used as antisense oligonucleotides. Examples of such modified products include lower alkyl phosphonate modifications such as methyl phosphonate or ethyl phosphonate types, phosphorothioate modifications, and phosphoramidate modifications.

  To produce a host cell or host organism in which the activity of the enzyme that adds fucose is reduced or deleted, dsRNA complementary to the transcript of the DNA encoding the enzyme can be used. Also includes RNAi technology. RNAi is a phenomenon in which, when a double-stranded RNA (hereinafter referred to as dsRNA) having the same or similar sequence as a target gene sequence is introduced into a cell, the expression of the introduced foreign gene and target endogenous gene are both suppressed. When dsRNA of about 40 to several hundred base pairs is introduced into a cell, a RNaseIII-like nuclease called Dicer with a helicase domain is present in the presence of ATP, and dsRNA is about 21 to 23 base pairs from the 3 ′ end. Cut out and produce siRNA (short interference RNA). A protein specific to this siRNA binds to form a nuclease complex (RISC: RNA-induced silencing complex). This complex recognizes and binds to the same sequence as siRNA, and cleaves the mRNA of the target gene at the center of siRNA by RNaseIII-like enzyme activity. In addition to this pathway, the antisense strand of siRNA binds to mRNA and acts as a primer for RNA-dependent RNA polymerase (RsRP) to synthesize dsRNA. There is also a possibility that this dsRNA becomes Dicer's substrate again to generate new siRNA and amplify the action.

  To produce a host cell or host organism in which the activity of an enzyme that adds fucose is reduced or deleted, an antisense coding DNA that encodes an antisense RNA against any region of the mRNA of the target enzyme, It can be expressed from a sense code DNA encoding a sense RNA in any region of the mRNA of the enzyme to be used. Moreover, dsRNA can also be used from these antisense RNA and sense RNA.

  When the antisense RNA and dsRNA are retained in a vector, the antisense RNA and sense RNA may be expressed from the same vector, and the antisense RNA and sense RNA may be expressed from different vectors, respectively. . For example, antisense RNA and sense RNA can be expressed from the same vector as an antisense RNA in which a promoter capable of expressing a short RNA such as polIII is linked upstream of the antisense code DNA and the sense code DNA. It can be constructed by constructing an expression cassette and a sense RNA expression cassette, respectively, and inserting these cassettes into the vector in the same direction or in the opposite direction. It is also possible to construct an expression system in which antisense code DNA and sense code DNA are arranged in opposite directions so as to face each other on different strands. In this configuration, one double-stranded DNA (siRNA-encoding DNA) in which an antisense RNA coding strand and a sense RNA coding strand are paired is provided, and antisense RNA and sense RNA from each strand are provided on both sides thereof. A promoter is provided oppositely so that it can be expressed. In this case, in order to avoid adding extra sequences downstream of the sense RNA and antisense RNA, a terminator is added to the 3 ′ end of each strand (antisense RNA coding strand, sense RNA coding strand). It is preferable to provide. As this terminator, a sequence in which four or more A (adenine) bases are continued can be used. Moreover, in this palindromic style expression system, it is preferable that the types of the two promoters are different.

  In addition, as a configuration for expressing antisense RNA and sense RNA from different vectors, for example, antisense coding DNA and an antisense in which a promoter capable of expressing a short RNA such as polIII is linked upstream of sense coding DNA. An RNA expression cassette and a sense RNA expression cassette can be constructed, and these cassettes can be held in different vectors.

  SiRNA may be used as a dsRNA for producing a host cell or host organism in which the activity of an enzyme that adds fucose is reduced or deleted. The term “siRNA” refers to a double-stranded RNA molecule that prevents translation of a target mRNA. siRNA refers to a double-stranded RNA molecule that inhibits translation of a target mRNA. Standard techniques for introducing siRNA into cells are used, including DNA as a template for RNA transcription. The siRNA includes a sense nucleic acid sequence, an antisense nucleic acid sequence, or both. The siRNA may comprise two complementary molecules, or so that a single transcript has both sense and complementary antisense sequences from the target gene, i.e. in some embodiments microRNA (miRNA ) May be constructed to be a hairpin that leads to production.

  Standard techniques are used to introduce siRNA into cells, including using DNA as a template to transcribe RNA. The binding of siRNA to the target gene results in decreased protein production by the cell. The length of the oligonucleotide is at least 10 nucleotides and may be the same length as the naturally occurring transcript. Preferably, the oligonucleotide is about 19 to about 25 nucleotides in length. Most preferably, the oligonucleotide is less than about 75, about 50, about 25 nucleotides in length. In addition, nucleotide “u” can be added to the 3 ′ end of the antisense strand of the target sequence to enhance the inhibitory activity of the siRNA. The number of “u” added is at least 2, generally from about 2 to about 10, preferably from about 2 to about 5. The added “u” forms a single strand at the 3 ′ end of the antisense strand of the siRNA. The siRNA is introduced directly into the cell in a form that can bind to the mRNA transcript. In these embodiments, the siRNA molecules of the invention are typically modified as previously described for antisense molecules. Other modifications are possible, for example, siRNA conjugated to cholesterol showed improved pharmacological properties (Song et al. Nature Med. 9: 347-351 (2003)). Alternatively, the DNA encoding siRNA is present in a vector.

  The vector can be obtained, for example, by cloning the target sequence into an expression vector so that the functionally linked control sequences are adjacent to the target sequence in a manner that allows expression of both strands (by transcription of the DNA molecule). (Lee, NS, Dohjima, T., Bauer, G., Li, H., Li, M.-J., Ehsani, A., Salvaterra, P., and Rossi, J. (2002) Human Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells (Nature Biotechnology 20: 500-505). An RNA molecule that is antisense to the target mRNA is transcribed by a first promoter (eg, a promoter sequence at the 3 ′ end of the cloned DNA), and an RNA molecule that is the sense strand for the target mRNA is a second promoter Transcribed by (for example, the promoter sequence at the 5 ′ end of the cloned DNA). The sense and antisense strands hybridize in vivo to generate an siRNA construct for silencing the target gene. Alternatively, two constructs are utilized to create the sense and antisense strands of the siRNA construct. A cloned target gene can encode a construct having a secondary structure, eg, a hairpin, in which a single transcript has both sense and complementary antisense sequences from the target gene.

  For example, the loop sequence in a preferred siRNA having a hairpin structure of the present invention can be selected from the group consisting of CCC, UUCG, CCACC, CCACACC, and UUCAAGAGA.

  Expression can be regulated independently in time or space by the same or different regulatory sequences adjacent to the target sequence. The siRNA is transcribed in the cell, for example, by cloning the target gene template into a vector containing the RNA polymerase III transcription unit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter. Transfection enhancing agents can be used to introduce the vector into the cell. FuGENE (Rochediagnostices), Lipofectamine 2000 (Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure Chemical) are useful as transfection enhancers.

  The siRNA nucleotide sequence can be designed using a siRNA design computer program available from the Ambion website (http://www.ambion.com/techlib/misc/siRNA_finder.html). it can. The nucleotide sequence for siRNA is selected by a computer program based on the following protocol.

siRNA target site selection:
1． Starting downstream from the AUG start codon of the transcript of interest, it is scanned downstream for the AA dinucleotide sequence. Record the occurrence of each AA and the 19 nucleotides adjacent to the 3 ′ end as potential siRNA target sites. Tuschl et al. ((Targeted mRNA degradation by double-stranded RNA in vitro. Genes Dev 13 (24): 3191-7 (1999)) reported that siRNA 5 'and 3' untranslated regions (UTR) and near the start codon (75 It is recommended not to design for (intrabase) regions, since these regions may contain many regulatory protein binding sites, UTR binding proteins and / or translations. The initiation complex may interfere with the binding of the siRNA endonuclease complex.
2． The potential target sites are compared to the human genome database and any target regions with apparent homology with other coding sequences are excluded from consideration. Homology searches can be performed using BLAST, which is on the NCBI server: www.ncbi.nlm.nih.gov/BLAST/.
3． Select a qualified target sequence for synthesis. Ambion can select several target sequences for evaluation along the entire length of the gene.

  In order to produce a host cell or host organism in which the activity of the enzyme that adds fucose is reduced or deleted, it is also possible to use a DNA encoding a ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities. Among them, research on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes for site-specific cleavage of RNA. Some ribozymes have a group I intron type and a size of 400 nucleotides or more like M1RNA contained in RNaseP, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type.

  For example, the self-cleaving domain of hammerhead ribozyme cleaves on the 3 ′ side of C15 of G13U14C15, but it is important for U14 to form a base pair with A at position 9, and the base at position 15 is In addition to C, A or U is also shown to be cleaved. If the ribozyme substrate binding site is designed to be complementary to the RNA sequence near the target site, it is possible to create a restriction enzyme-like RNA-cleaving ribozyme that recognizes the UC, UU, or UA sequence in the target RNA. It is. For example, there are a plurality of sites that can be targets in the coding region of the enzyme of the present invention that serves as an inhibition target.

  Hairpin ribozymes are also useful for producing host cells or host organisms in which the activity of the enzyme that adds fucose is reduced or deleted. Hairpin ribozymes are found, for example, in the minus strand of tobacco ring spot virus satellite RNA (J. M. Buzayan Nature 323: 349, 1986). This ribozyme has also been shown to be designed to cause target-specific RNA cleavage.

  A ribozyme designed to cleave the target is linked to a promoter such as the cauliflower mosaic virus 35S promoter and a transcription termination sequence so that it is transcribed in plant cells. However, at that time, if an extra sequence is added to the 5 ′ end or 3 ′ end of the transcribed RNA, the ribozyme activity may be lost. In such a case, in order to accurately cut out only the ribozyme part from the RNA containing the transcribed ribozyme, another trimming ribozyme that acts in cis for trimming is placed on the 5 'side or 3' side of the ribozyme part. (K. Taira et al. (1990) Protein Eng. 3: 733, AMDzianottand JJ Bujarski (1989) Proc. Natl. Acad. Sci. USA. 86: 4823, CAGrosshansand RTCech (1991) Nucleic Acids Res. 19: 3875, K. Taira et al. (1991) Nucleic Acids Res. 19: 5125). In addition, such structural units can be arranged in tandem so that multiple sites in the target gene can be cleaved, thereby further enhancing the effect (N. Yuyama et al. Biochem. Biophys. Res. Commun. 186: 1271,1992). Such a ribozyme can be used to specifically cleave the transcription product of the target gene in the present invention, thereby suppressing the expression of the gene.

  Hereinafter, as a more specific example of obtaining the antibody of the present invention, a method for producing a humanized antibody will be described, but other antibodies can be obtained in the same manner as the method.

(1) Construction of a humanized antibody expression vector A humanized antibody expression vector is a Kabat EU index number in which the amino acid residue used as the substitution site in (A) to (D) above is the target amino acid residue. An animal cell expression vector in which a gene encoding the substituted human antibody heavy chain (H chain) C region and human antibody light chain (L chain) C region is incorporated. Genes encoding the H chain C region of a human antibody and the L chain C region of a human antibody in which the amino acid residue that is the substitution site in the above (A) to (D) is substituted with the target amino acid residue by the EU index number Can be constructed by cloning each.

The C region of the human antibody can be the H chain and L chain C region of any human antibody. And the C region of the κ class of the L chain (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, pAGE 107 [Cytotechnology, 3, 133 (1990)], pAGE 103 [Journal of Biochemistry, 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)], pSG1βd2-4 [Site Technology (Cytotechnology), 4, 173 (1990)]. Examples of 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 [biochemicals]. 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.

  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, the ease of introduction into animal cells, and the balance of the expression levels of antibody H and L chains in animal cells. Vectors are preferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandem humanized antibody expression vector include pKANTEX93 [Molecular Immunology (Mol. Immunol.), 37, 1035 (2000)], pEE18 [Hybridoma (Hybridoma), 17,559 (1998)] and the like.

  The constructed humanized antibody expression vector can be used for expression of human chimeric antibody and human CDR-grafted antibody 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 H chain and L chain V region of mouse antibody as follows. Can do.

  MRNA is extracted from hybridoma cells producing the desired mouse antibody, and cDNA is synthesized. The synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library. From the 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 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. The entire base sequences of the H chain and L chain V regions of the target mouse antibody on the recombinant phage or recombinant plasmid are determined, and the entire amino acid sequences of the H chain and L chain V regions are deduced from the base sequences.

  As animals other than humans, any mouse, rat, hamster, rabbit, etc. can be used as long as hybridoma cells can be produced.

  Methods for preparing total RNA from hybridoma cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], and mRNA from total RNA. As a preparation method, an oligo (dT) -immobilized cellulose column method [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989]. Examples of kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like. As a method for synthesizing cDNA and preparing a cDNA library, conventional methods [Molecular Cloning: A Laboratory Manual], Cold Spring Harbor Lab. Press New York, 1989; Current Protocols in Molecular Biology, Supplement 1-3B, or a commercially available kit, eg, Super ScriptTM Plasmid I System Science P And a method using a ZAP-cDNA Synthetic Kit (manufactured by Stratagene).

  When preparing a cDNA library, any vector can be used as the vector into which cDNA synthesized using mRNA extracted from hybridoma cells as a template is incorporated. For example, ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)], λzap II Ne (Strategies). ), Λgt10, λgt11 [DN cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (Clontech), λExCell, pT7T3hP ), PcD2 [Mole. Cell. Biol., 3, 28 0 (1983)] and pUC18 [Gene, 33, 103 (1985)] and the like 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-Blue MRF ′ [Stratesies, 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.

  The selection of cDNA clones encoding the H chain and L chain V regions of non-human animal antibodies from a cDNA library includes colony hybridization or plaque hybridization using isotopes or fluorescently labeled probes. [Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press New York, 1989]. In addition, primers were prepared and cDNA or cDNA library synthesized from mRNA was used as a template. Polymerase Chain Reaction (hereinafter referred to as PCR method; Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Lab. Press New York, 1989; Current Protocols in Molecular Biology, Supplement 1-34] 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 cloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used nucleotide sequence analysis method such as Sanger et al. Of the dideoxy method [Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci., USA), 74, 5463 (1977)] Automatic sequence analyzers such as A.I. L. F. The base sequence of the cDNA can be determined by analysis using a DNA sequencer (Pharmacia) or the like.

  The entire amino acid sequences of the H chain and L chain V regions are deduced from the determined base sequences, and all the amino acid sequences of known antibody H chain and L chain V regions [Sequences of Proteins of Immunological Interest ( Sequences of Proteins of Immunological Interest), US Dept. Compared with Health and Human Services, 1991], it is possible to confirm whether the obtained cDNA encodes the complete amino acid sequences of the H chain and L chain V regions of the antibody including the secretory signal sequence.

  Furthermore, when the amino acid sequence of the antibody variable region or the base sequence of DNA encoding the variable region is already known, it can be produced using the following method.

  If the amino acid sequence is known, the amino acid sequence can be determined according to the frequency of codon usage [Sequences of Proteins of Immunological Interest, US Dept. In consideration of Health and Human Services, 1991], and based on the designed DNA sequence, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR is performed using them. To obtain DNA. When the base sequence is known, DNA can be obtained by synthesizing several synthetic DNAs having a length of about 100 bases based on the information and performing PCR using them.

(3) Analysis of amino acid sequence of V region of antibody of non-human animal antibody Regarding the complete amino acid sequence of H chain and L chain V region of antibody including secretory signal sequence, known antibody H chain and L chain V region The complete amino acid sequence of [Sequences of Proteins of Immunological Interest, US Dept. Compared with Health and Human Services, 1991], the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and further, 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 amino acid sequence of the known antibody H chain and L chain V region [Sequences of Proteins of Sequences of Proteins. of Immunological Interest), US Dept. Health and Human Services, 1991].

(4) Construction of human chimeric antibody expression vector The humanized antibody expression vector described in (1) is upstream of the gene encoding the H chain and L chain C region of the human antibody. A cDNA encoding the chain and L chain V regions can be cloned to construct a human chimeric antibody expression vector. For example, a cDNA encoding the H chain and L chain V region of a non-human animal antibody, the nucleotide sequence at 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 chain antibody expression vector according to (1), which is composed of a base sequence on the 5 ′ end side of the L chain C region and linked to a synthetic DNA having an appropriate restriction enzyme recognition sequence at both ends. A human chimeric antibody expression vector can be constructed by cloning the gene encoding the H chain and L chain C region of the human 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, the amino acid sequence of the framework (hereinafter referred to as FR) of the H chain and L chain V region 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 transplanted is selected. . 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 derived from a human antibody can be used. For example, FR amino acid sequences of human antibody H chain and L chain V regions, and human antibody H chain and L chain V region FR subgroups registered in databases such as Protein Data Bank [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991] and the like. Among them, in order to produce a human CDR-grafted antibody having sufficient activity, the H chain and L chain V regions of the antibody of the target non-human animal are used. 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.

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 of designed amino acid sequence in antibody gene base sequence [Sequences of Proteins of Immunological Interest, US Dept. In consideration of Health and Human Services, 1991], it is converted into 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, several synthetic DNAs having a length of about 100 bases are synthesized, and PCR is performed using them. In this case, it is preferable to design 4 to 6 synthetic DNAs for both the H chain and the L chain from the reaction efficiency in PCR and the length of DNA that can be synthesized.
In addition, by introducing an appropriate restriction enzyme recognition sequence into the 5 ′ end of the synthetic DNA located at both ends, it can be easily cloned into the humanized antibody expression vector constructed in (1). After PCR, the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the base sequence is determined by the method described in (2), and the H chain and L of the desired human CDR-grafted antibody are determined. A plasmid having a DNA sequence encoding the amino acid sequence of the chain V region is obtained.

(6) Construction of human CDR-grafted antibody expression vector Human constructed in (5) upstream of the gene encoding the human antibody H chain and L chain C regions of the humanized antibody expression vector described in (1) A cDNA encoding the H chain and L chain V regions of a type CDR-grafted antibody can be cloned 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), an appropriate restriction enzyme recognition sequence is introduced at the 5 ′ end of the synthetic DNA located at both ends. Then, the humanized antibody expression vector described in (1) is cloned upstream of the gene encoding the H chain and L chain C region of the human antibody so that they are expressed in an appropriate form, and the human CDR A transplanted antibody expression vector can be constructed.

(7) Stable production of humanized antibody By introducing the humanized antibody expression vector described in (4) and (6) into an appropriate animal cell, a human chimeric antibody and a human CDR-grafted antibody (hereinafter, collectively) A transformant that stably produces a humanized antibody) can be obtained. Examples of the method for introducing a humanized antibody expression vector into animal cells include electroporation (Japanese Patent Laid-Open No. 2-257891; Cytotechnology, 3, 133 (1990)). As the animal cell into which the humanized antibody expression vector is introduced, any cell can be used as long as it is an animal cell capable of producing a humanized antibody.
Specifically, mouse myeloma cells NSO cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr- cells, CHO / DG44 cells, rat myeloma YB2 / 0 cells, IR983F cells, BHK derived from Syrian hamster kidney Examples thereof include Namalva cells, which are human myeloma cells, and preferably CHO / DG44 cells, rat myeloma YB2 / 0 cells, which are Chinese hamster ovary cells, and the like.

  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 these drugs. 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 fetal calf serum (hereinafter referred to as FCS) to these mediums can be used. 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 the enzyme immunoantibody method [hereinafter referred to as ELISA method; Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory]. , Chapter 14, 1998, Monoclonal Antibodies: Principles and Practices, Academic Press Limited, 1996], and the like. 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-2-257891.

  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 Practices, Academic Press Limited, 1996]. In addition, a purification method usually used in protein purification can be used. For example, it can be purified by a combination of gel filtration, ion exchange chromatography, ultrafiltration and the like. 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 Laboratories, Chapter 12, 1988, Monoclonal Antibodies: Principles and Practices (Monoclonals Principals and Practices) Press Limited, 1996].

  As described above, the method for producing an antibody using an animal cell as a host has been described. As described above, an antibody can also be produced in a yeast, insect cell, plant cell, animal individual, or plant individual by a method similar to that for animal cells. it can.

  When the host cell already has an ability to express an antibody, the cell for expressing the antibody in the present invention is prepared using a known method, and then the cell is cultured, and the target antibody is obtained from the culture. By purifying the antibody, the antibody of the present invention can be produced.

  As a method of measuring the protein amount, antigen binding activity or effector function of the purified antibody, Monoclonal Antibodies: principals and practice, Third Edition, Acad.Press, 1993, or Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Known methods described in 1988 and the like can be used. As a specific example, when the antibody is a humanized antibody, the binding activity to the antigen and the binding activity to the antigen-positive cell line are determined by ELISA method and fluorescent antibody method (Cancer Immunol. Immunother.). , 36, 373 (1993)]. Cytotoxic activity against an antigen-positive cultured cell line can be evaluated by measuring CDC activity, ADCC activity, etc. [Cancer Immunol. Immunother., 36, 373 (1993)]. . In addition, the safety and therapeutic effect of antibodies in humans can be evaluated using an appropriate model of animal species relatively close to humans such as cynomolgus monkeys.

  The antibody of the present invention is improved in terms of ADCC activity measured by the above method, compared to amino acid substitution, or a polypeptide before amino acid substitution and fucose reduction. For this reason, when using for antibody therapy, since the usage-amount of a chemical | medical agent can be made low, it is economical and can reduce a side effect.

  Since the antibody of the present invention has an extremely improved effector function as described above, it can be used for a wide range of applications such as treatment, diagnosis, and evaluation.

  The therapeutic composition of the present invention contains the antibody of the present invention. For this reason, ADCC activity is improved, a sufficient pharmacological effect can be obtained with a small dose, and it is extremely advantageous for an excellent antibody therapeutic use. In addition to the above-described antibodies, for example, nucleic acid, amino acid, peptide, protein, other organic compounds, inorganic compounds, excipients such as excipients and combinations thereof may be added to the therapeutic composition. Good. The therapeutic composition of the present invention can be formulated using molding means as necessary and can be administered orally or parenterally. The therapeutic composition of the present invention can be suitably used particularly by methods such as parenteral administration using injections, drops, and transdermal absorption agents.

  More specifically, the present invention provides a therapeutic composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier. The present invention also provides a method for treating a mammal, comprising administering the antibody of the present invention. Mammals include humans and non-human mammals (eg, mice, rats, monkeys, etc.).

  The therapeutic composition of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the antibody. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of active ingredient in these preparations is such that an appropriate volume within the indicated range can be obtained.

Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as You may use together with alcohol, specifically ethanol, polyalcohol, for example, propylene glycol, polyethylene glycol, nonionic surfactant, for example, polysorbate 80 (TM), HCO-50.

  Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent. Moreover, you may mix | blend with buffer, for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant. The prepared injection solution is usually filled into a suitable ampoule.

  Administration is preferably parenteral administration, and specific examples include injection, nasal administration, pulmonary administration, and transdermal administration. As an example of the injection form, it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

  The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the antibody of the present invention is, for example, in the range of about 1 μg to 100 mg (for example, about 1 μg to 15 mg, about 10 μg to 20 mg, about 0.1 mg to 20 mg) per kg body weight at a time. It is possible to choose. The pharmaceutical composition of the present invention can be administered to a single site or to multiple separate sites. The pharmaceutical composition of the present invention can be continuously administered. Repeated administration over several days is continued, depending on the condition, until suppression of the desired disease state occurs. The dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The therapeutic composition of the present invention can be used to treat a wide range of diseases to which antibody therapy can be applied. Diseases and symptoms to which antibody therapy is applied include, for example, metastatic breast cancer (anti-HER2 antibody), CD20 positive B cell non-Hodgkin's lymphoma (anti-CD20 antibody), rheumatoid arthritis and Crohn's disease (anti-TNFα antibody), after kidney transplantation And acute rejection (anti-CD3 antibody, anti-CD25 antibody) and the like. The parentheses indicate antibodies used for treatment. For these diseases and the like, by using the therapeutic composition of the present invention instead of the conventional antibody, the therapeutic effect can be remarkably improved, and the economical and physical burden on the patient can be reduced. Furthermore, it can be suitably used as an antibody drug that can be applied to antibody therapy in the future.
The antibody of the present invention also relates to a therapeutic method using the above therapeutic composition. Alternatively, the present invention relates to use in the manufacture of the above therapeutic agent.

The present invention also includes a step of substituting the 298th, 333, 334, and 295th amino acids of Kabat EU index numbers in antibodies having effector functions with alanine, alanine, alanine, and cysteine residues, respectively. Provided is a method for improving the effector function.
Further, the present invention includes a step of replacing the 239th, 330th, 332th, and 295th amino acids of Kabat EU index numbers in antibodies having effector functions with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively. A method for improving the effector function of an antibody is provided.
The present invention also provides a method for improving the effector function of an antibody comprising the following steps (1) to (3).
(1) a step of replacing the 295th amino acid residue in the Kabat EU index number in an antibody having an effector function with a cysteine residue;
(2) DNA encoding the H chain in which the 295th amino acid residue is replaced by a cysteine residue in the EU index number of Kabat, and DNA encoding the L chain are converted to N-acetylglucosamine at the reducing end with fucose. Introducing into the host cell or host organism in which the activity of the enzyme to be added has been reduced or deleted, and expressing the DNA; and (3) recovering the expression product.
The present invention also provides a method for improving the effector function of an antibody, comprising the step of replacing the 295th amino acid in the Kabat EU index number in an antibody having an effector function with an aspartic acid, serine or alanine residue.
In the above method, the step of substituting amino acids and the step of reducing fucose can be performed by the above-described methods.
In addition, all prior art documents cited in the present specification are incorporated herein by reference.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to the aspect described in these Examples.
[Example 1]
Anti-CD20 chimeric antibodies of 298Ala / 333Ala / 334Ala type and 298Ala / 333Ala / 334Ala / 295Cys type were prepared, and ADCC activity of these Anti-CD20 chimeric antibodies was evaluated. As a result, compared with the ADCC activity of the 298Ala / 333Ala / 334Ala type Anti-CD20 chimeric antibody, 298Ala / 333Ala / 334Ala type added 295Cys, the 298Ala / 333Ala / 334Ala / 295Cys type Anti-CD20 chimeric antibody was It showed high ADCC activity.
In the following, a method for preparing Anti-CD20 chimeric antibody of 298Ala / 333Ala / 334Ala type and 298Ala / 333Ala / 334Ala / 295Cys type, ADCC activity measurement and reactivity to CD20 molecule are described.

1) Preparation of Anti-CD20 chimeric antibody A chimeric antibody is obtained as a purified chimeric antibody through the following steps A) to F).
A) Cloning of genes necessary for producing chimeric antibodies
B) Mutation introduction of the cloned gene,
C) Construction of a chimeric antibody expression vector combining the cloned gene and its mutated gene,
D) Gene introduction of chimeric antibody expression vector into CHO cells and screening of CHO cells that highly express chimeric antibodies,
E) culturing CHO cells that highly express chimeric antibodies,
F) Column purification from the culture supernatant of cells expressing high chimeric antibodies.
The steps A) to F) are described in order below.

A) Cloning of genes required for production of chimeric antibodies
In order to produce one kind of Anti-CD20 chimeric antibody, four kinds of genes are required. These four genes are the Anti-CD20 mouse L chain variable region gene, the Anti-CD20 mouse H chain variable region gene, the Human IgG1 L chain constant region gene, and the Human IgG1 H chain constant region gene. Examples of cloning of this gene are described below.

(Anti-CD20 mouse L chain variable region gene)
MRNA was obtained from the hybridoma cells producing Anti-CD20 mouse monoclonal antibody possessed by the applicant using QuickPrep micro mRNA purification kit (Amersham Biosciences, code 27-9255-01). The mRNA was converted to cDNA using First-Strand cDNA Synthesis kit (Amersham Biosciences, code 27-9261-01). Genes were amplified by PCR using this cDNA as a template, and PCR reaction was carried out using combinations of 11 patterns of Primers shown below.
PCR reaction conditions are
CDNA from mouse hybridoma 4 μL
2.5 mM dNTPs 4 μL
One of 11 types of MKV1 to MKV11 primer (20 μM) 2.5 μL
MKC primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 28.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4min
4 ℃ unlimited time
See below for Primer DNA sequences.
MKV1 primer: ATGAAGTTGCCTGTTAGGCTGTTGGTGCTG (SEQ ID NO: 35)
MKV2 primer: ATGGAGWCAGACACACTCCTGYTATGGGTG (SEQ ID NO: 36)
MKV3 primer: ATGAGTGTGCTCACTCAGGTCCTGGSGTTG (SEQ ID NO: 37)
MKV4 primer: ATGAGGRCCCCTGCTCAGWTTYTTGGMWTCTTG (SEQ ID NO: 38)
MKV5 primer: ATGGATTTWCAGGTGCAGATTWTCAGCTTC (SEQ ID NO: 39)
MKV6 primer: ATGAGGTKCYYTGYTSAGYTYCTGRGG (SEQ ID NO: 40)
MKV7 primer: ATGGGCWTCAAGATGGAGTCACAKWYYCWGG (SEQ ID NO: 41)
MKV8 primer: ATGTGGGGAYCTKTTTYCMMTTTTTCAATTG (SEQ ID NO: 42)
MKV9 primer: ATGGTRTCCWCASCTCAGTTCCTTG (SEQ ID NO: 43)
MKV10 primer: ATGTATATATGTTTGTTGTCTATTTCT (SEQ ID NO: 44)
MKV11 primer: ATGGAAGCCCCAGCTCAGCTTCTCTTCC (SEQ ID NO: 45)
MKC primer: ACTGGATGGTGGGAAGATGG (SEQ ID NO: 46)
(M = A or C, R = A orG, W = A orT, S = C or G, Y = C or T, K = G orT)

In the PCR reaction, the anti-CD20 mouse L chain variable region gene was amplified by the combination of MKV5 and MKC primer, and this gene was temporarily inserted into the pCR2.1 vector (Invitogen) and stored. In addition, this gene named the inserted vector pCR2.1-MLV. The DNA sequence of the gene-cloned Anti-CD20 mouse L chain variable region is attached in FIG.
In addition, the nucleotide sequences of CDR1, CDR2, and CDR3 of the Anti-CD20 mouse L chain variable region are shown in SEQ ID NOs: 7, 9, and 11, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the Anti-CD20 mouse L chain variable region are shown in SEQ ID NOs: 8, 10, and 12, respectively.

(Anti-CD20 mouse heavy chain variable region gene)
Similar to the cloning of the Anti-CD20 mouse L chain variable region gene, the following 12 patterns of PCR amplification were performed using cDNA prepared from a hybridoma cell producing an Anti-CD20 mouse monoclonal antibody as a template.
CDNA from mouse hybridoma 4 μL
2.5 mM dNTPs 4 μL
One of 12 types of MHV1 to MHV12 primer (20 μM) 2.5 μL
MHCG2b primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 28.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
MHV1 primer: ATGAAATGCAGCTGGGGCATSTTCTTC (SEQ ID NO: 47)
MHV2 primer: ATGGGATGGAGCTRTATCATSYTCTT (SEQ ID NO: 48)
MHV3 primer: ATGAAGWTGTGGTTAAACTGGGTTTTT (SEQ ID NO: 49)
MHV4 primer: ATGRACTTTGGGYTCAGCTTGRTTT (SEQ ID NO: 50)
MHV5 primer: ATGGACTCCAGGCTCAATTTAGTTTTCCTT (SEQ ID NO: 51)
MHV6 primer: ATGGCTGTCYTRGSGCTRCTCTTCTGC (SEQ ID NO: 52)
MHV7 primer: ATGGRATGGAGCKGGRTCTTTMTCTT (SEQ ID NO: 53)
MHV8 primer: ATGAGAGTGCTGATTCTTTTGTG (SEQ ID NO: 54)
MHV9 primer: ATGGMTTGGGTGTGGAMCTTGCTATTCCTG (SEQ ID NO: 55)
MHV10 primer: ATGGGCAGACTTACATTCTCATTCCTG (SEQ ID NO: 56)
MHV11 primer: ATGGATTTTGGGCTGATTTTTTTTATTG (SEQ ID NO: 57)
MHV12 primer: ATGATGGTGTTAAGTCTTCTGTACCTG (SEQ ID NO: 58)
MHCG2b primer: CAGTGGATAGACTGATGGGGG (SEQ ID NO: 59)
(M = A or C, R = A orG, W = A orT, S = C or G, Y = C or T, K = G orT)

In the PCR reaction, the Anti-CD20 mouse H chain variable region gene was amplified by the combination of MHV7 and MHCG2b primer, and this gene was temporarily inserted into the pCR2.1 vector (Invitogen) and stored. The vector into which this gene was inserted was named pCR2.1-MHV. The DNA sequence of the gene-cloned Anti-CD20 mouse heavy chain variable region is attached in FIG.
The nucleotide sequences of CDR1, CDR2, and CDR3 of the Anti-CD20 mouse H chain variable region are shown in SEQ ID NOs: 1, 3, and 5, respectively. In addition, the amino acid sequences of CDR1, CDR2, and CDR3 of the Anti-CD20 mouse heavy chain variable region are shown in SEQ ID NOs: 2, 4, and 6, respectively.

(Human IgG1 L chain constant region gene)
Lymphocytes were removed from human blood using Lymphoprep (Axis Shield). From this lymphocyte, mRNA was obtained using QuickPrep micro mRNA purification kit (Amersham Biosciences, code 27-9255-01). The mRNA was converted to cDNA using First-Strand cDNA Synthesis kit (Amersham Biosciences, code 27-9261-01). Using the cDNA from this human lymphocyte as a template, the following PCR reaction was performed to obtain a human IgG1 L chain constant region gene. This gene was also temporarily inserted into the pCR2.1 vector (Invitogen) and stored. The vector into which this gene was inserted was named pCR2.1-LC.
PCR reaction conditions are as follows.
Human lymphocyte cDNA 4 μL
2.5 mM dNTPs 4 μL
hIgG1 LCF primer (20 μM) 2.5 μL
hIgG1 LCR primer (20 μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 28.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
hIgG1 LCF primer: ACTGTGGCTGCACCATCTGTCTTC (SEQ ID NO: 60)
hIgG1 LCR primer: TTAACACTCTCCCCTGTTGAAGCTCTT (SEQ ID NO: 61)

(Human IgG1 H chain constant region gene)
In the same manner as the cloning of the human IgG1 L chain constant region gene, the following PCR reaction was performed using cDNA from human lymphocytes as a template to obtain a Human IgG1 H chain constant region gene. This gene was also temporarily inserted and stored in the pCR2.1 vector (Invitrogen). This vector was named pCR2.1-HC (wild type).
PCR reaction conditions are as follows.
Human lymphocyte cDNA 4 μL
2.5 mM dNTPs 4 μL
hIgG1 HCF primer (20 μM) 2.5 μL
hIgG1 HCR primer (20 μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 28.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (30 cycles)
72 ℃ 4 min
4 ℃ unlimited time
See below for Primer DNA sequences.
hIgG1 HCF primer: GCCTCCACCAAGGGCCCATCGGTC (SEQ ID NO: 62)
hIgG1 HCR primer: TTATTTACCCGGAGACAGGGAGAGGCT (SEQ ID NO: 63)

B) Mutation introduction of cloned genes
In order to obtain a 298Ala / 333Ala / 334Ala chimeric antibody or a 298Ala / 333Ala / 334Ala / 295Cys chimeric antibody, a mutation must be introduced into a specific portion of the cloned human IgG1 heavy chain constant region. According to the number of the human IgG1 heavy chain constant region shown in Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991) by Elvin A. Kabat et al., Ser # 298 is Ala and Glu # 333 is Ala In order to change Lys at position 334 to Ala and Glu at position 295 to Cys, the pCR2.1 vector into which the cloned Hunan IgG1 heavy chain constant region was inserted: pCR2.1-HC (wild type) Mutations were sequentially introduced by PCR as a template. Details of this mutagenesis method are described below.

298 (Ser → Ala) mutation
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
298 Ala1 primer (20 μM) 1.25 μL
298 Ala2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
298 Ala1 primer: GAGCAGTACAACGCTACGTACCGTGTG (SEQ ID NO: 64)
298 Ala2 primer: CACACGGTACGTAGCGTTGTACTGCTC (SEQ ID NO: 65)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (298Ala) into which the human IgG1 heavy chain constant region into which 298 (Ser → Ala) mutation was introduced was inserted.

333 (Glu → Ala) mutation
pCR2.1-HC (298Ala) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
333 Ala1 primer (20 μM) 1.25 μL
333 Ala2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
333 Ala1 primer: CCAGCCCCCATCGCTAAAACCATCTCC (SEQ ID NO: 66)
333 Ala2 primer: GGAGATGGTTTTAGCGATGGGGGCTGG (SEQ ID NO: 67)

After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (298Ala / 333Ala) into which the human IgG1 heavy chain constant region into which 333 (Glu → Ala) mutation was introduced was inserted.

334 (Lys → Ala) mutation
pCR2.1-HC (298Ala / 333Ala) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
334 Ala1 primer (20 μM) 1.25 μL
334 Ala2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
334 Ala1 primer: GCCCCCATCGCTGCTACCATCTCCAAA (SEQ ID NO: 68)
334 Ala2 primer: TTTGGAGATGGTAGCAGCGATGGGGGC (SEQ ID NO: 69)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, we named it the pCR2.1 vector pCR2.1-HC (298Ala / 333Ala / 334Ala) into which the 334 (Lys → Ala) mutant Human IgG1 heavy chain constant region was inserted. It was.

295 (Glu → Cys) mutation
pCR2.1-HC (298Ala / 333Ala / 334Ala) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
295 Cys1 primer (20 μM) 1.25 μL
295 Cys2 primer (20 μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
295 Cys1 primer: ACAAAGCCGCGTGAGGAGTGCTACAACGCTACGTACCGT (SEQ ID NO: 70)
295 Cys2 primer: ACGGTACGTAGCGTTGTAGCACTCCTCACGCGGCTTTGT (SEQ ID NO: 71)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector pCR2.1-HC (298Ala / 333Ala / 334Ala / 295Cys) inserted with a human IgG1 heavy chain constant region 295 (Glu → Cys) mutated Was attached.

C) Construction of chimeric antibody expression vector combining cloned gene and mutated gene
In order to express one type of Anti-CD20 chimeric antibody, this can be achieved by transfecting CHO cells with two types of expression vectors of the L chain and H chain of the Anti-CD20 chimeric antibody. Here, the L-chain expression vector of the Anti-CD20 chimeric antibody is an expression vector in which an anti-CD20 mouse L chain variable region gene + human IgG1 L chain constant region gene is combined, The expression vector for the H chain of the -CD20 chimeric antibody is obtained by incorporating an anti-CD20 mouse H chain variable region gene + human IgG1 H chain constant region gene into the expression vector. When creating anti-CD20 chimeric antibodies of wild type, 298Ala / 333Ala / 334Ala type or 298Ala / 333Ala / 334Ala / 295Cys type, the anti-CD20 chimeric antibody L chain expression vector expresses these three chimeric antibodies. The anti-CD20 chimeric antibody H chain expression vector is used for wild type, 298Ala / 333Ala / 334Ala type or 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody, respectively. A special heavy chain expression vector is required. Here, we describe in detail the construction of an anti-CD20 chimeric antibody L chain expression vector that can be commonly used for the expression of each chimeric antibody, and then 298Ala / 333Ala / 334Ala type or 298Ala / 333Ala / 334Ala / 295Cys type chimera. The construction of a dedicated anti-CD20 chimeric antibody heavy chain expression vector required for antibody expression is described.

Construction of an expression vector for the Anti-CD20 chimeric antibody L chain The BCMG-neo vector is used as the expression vector, and the Anti-CD20 mouse L chain variable region gene + human IgG1 L chain constant region gene is inserted into the XhoI and NotI sites of this vector. To insert. A fragment of the Anti-CD20 mouse L chain variable region gene was obtained by performing the following PCR reaction using the previously prepared pCR2.1-MLV as a template. In addition, a fragment of the human IgG1 L chain constant region gene was obtained by the following PCR reaction using the previously prepared pCR2.1-LC as a template.

(Anti-CD20 mouse L chain variable region gene amplification reaction)
pCR2.1-MLV (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
L1 primer (20μM) 2.5 μL
L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4min
4 ℃ unlimited time
L1 primer: ACCGCTCGAGATGGATTTTCAGGTGCAGATTATCAGC (SEQ ID NO: 72)
L2 primer: TTTCAGCTCCAGCTTGGTCCCAGCACC (5'-phosphorylated) (SEQ ID NO: 73)

(Human IgG1 L chain constant region gene amplification reaction)
pCR2.1-LC (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
L3 primer (20μM) 2.5 μL
L4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
L3 primer: ACTGTGGCTGCACCATCTGTCTTCATC (5'-phosphorylated) (SEQ ID NO: 74)
L4 primer: ATAGTTTAGCGGCCGCTTAACACTCTCCCCTGTTGAAGCTCTTTGT (SEQ ID NO: 75)

  The Anti-CD20 mouse L chain variable region gene fragment obtained by the PCR reaction was purified after being cleaved with the restriction enzyme XhoI (Takara). Further, the human IgG1 L chain constant region gene fragment obtained by PCR reaction was cleaved with restriction enzyme NotI (Takara) and then purified. The fragment purified by cutting the BCMG-neo vector with XhoI and NotI was mixed with the purified Anti-CD20 mouse L chain variable region gene fragment and the purified Human IgG1 L chain constant region gene fragment for ligation. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-CD20 chimeric antibody L chain expression vector was named pchimeric LC.

Construction of Anti-CD20 chimeric antibody H chain expression vector BCMG-neo vector is used as the expression vector, and Anti-CD20 mouse H chain variable region gene + Human IgG1 H chain constant region gene is used at the XhoI and NotI sites of this vector. Insert it. The anti-CD20 mouse heavy chain variable region gene was obtained by performing the following PCR reaction using the previously prepared pCR2.1-MHV as a template. The H chain expression vector dedicated to each of the wild type, 298Ala / 333Ala / 334Ala type or 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody is a plasmid that serves as a template for PCR amplification of the human IgG1 H chain constant region gene. However, the operation itself was carried out in the same manner to obtain various human IgG1 heavy chain gene fragments. Details including the PCR reaction are described below.

(Anti-CD20 mouse heavy chain variable region gene amplification reaction)
pCR2.1-MHV (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H1 primer (20μM) 2.5 μL
H2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H1 primer: ACCGCTCGAGATGGGATGGAGCTGGGTCTTTCTCTTC (SEQ ID NO: 76)
H2 primer: TGAGGAGACGGTGACCGTGGTCCC (5'-phosphorylated) (SEQ ID NO: 77)

(Human IgG1 H chain constant region gene amplification reaction)
# Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H4 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 78)
H4 primer: ATAGTTTAGCGGCCGCTTATTTACCCGGAGACAGGGAGAGGCTCTT (SEQ ID NO: 79)

#: PCR2.1-HC (wild type) was used as a template to prepare a gene fragment for a wild type Anti-CD20 chimeric antibody. PCR2.1-HC (298Ala / 333Ala / 334Ala) was used as a template to prepare a gene fragment for the 298Ala / 333Ala / 334Ala type Anti-CD20 chimeric antibody. In order to prepare a gene fragment for the 298Ala / 333Ala / 334Ala / 295Cys type Anti-CD20 chimeric antibody, pCR2.1-HC (298Ala / 333Ala / 334Ala / 295Cys) was used as a template.

  The Anti-CD20 mouse H chain variable region gene fragment obtained by the PCR reaction was purified after being cleaved with the restriction enzyme XhoI (Takara). In addition, Human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after digestion with restriction enzyme NotI (Takara). The BCMG-neo vector was cleaved with XhoI and NotI and purified, and the purified Anti-CD20 mouse heavy chain variable region gene fragment and purified human IgG1 heavy chain constant region gene fragment were mixed and ligated. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-CD20 chimeric antibody H chain expression vector is named p-chimeric HC (wild type), p-chimeric HC (298Ala / 333Ala / 334Ala type) or p-chimeric HC (298Ala / 333Ala / 334Ala / 295Cys type), respectively. It was attached.

D) Introduction of chimeric antibody expression vectors into CHO cells and screening of CHO cells that highly express chimeric antibodies The various combinations of the expression vectors used in the following combinations use the TransIT-CHO Transfection kit (Mirus, MIR2170) The gene was introduced into CHO cells.
Wild type chimeric antibody: p chimera LC + p chimera HC (wild type)
298Ala / 333Ala / 334Ala type chimeric antibody: p chimera LC + p chimera HC (298Ala / 333Ala / 334Ala type)
298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody: p chimera LC + p chimera HC (298Ala / 333Ala / 334Ala / 295Cys)

Transfected CHO cells were Dulbecco's Modified Eagle's Medium (Sigma, D5796) supplemented with 10% Fetal Bovine Serum (EQITEC-BIO) and 1 mg / mL Geneticin (GIBCO, 10131-035) at 37 ° C, 5% CO2. It grew by culturing under carbon conditions. CHO cells that highly express the chimeric antibody are selected from these CHO cells that have grown and formed colonies. First, a portion of each colony was transplanted into a 96-well plate and cultured for another 10 days. . A part of the culture supernatant was taken out and subjected to ELISA measurement shown below to determine a highly expressing CHO cell clone.
(ELISA measurement)
1) The culture supernatant of Tansfectioned CHO cells was added to a 96-well plate coated with Anti-Human IgG (γ-chain) (MBL, 103AG) at 100 μL / well and left at room temperature for 1 hour.
2) The culture supernatant was discarded from the 96-well plate, and wells were sufficiently washed with PBS containing 0.05% Tween 20 (PBS-0.05% Tween 20).
3) A solution obtained by diluting Anti-Human IgG (γ-chain) Peroxidase conjugated (MBL, 208) 2000-fold with PBS-0.05% Tween20 was added at 100 μL / well and left at room temperature for 1 hour.
4) The solution was discarded from the 96-well plate, and wells were sufficiently washed with PBS-0.05% Tween20.
5) 100 μL / well of TMB Peroxidase Substrate (Moss, TMBE-1000s) was added to cause a color reaction.
6) After 10 to 15 minutes of color reaction, 1N sulfuric acid was added at 100 μL / well to stop the color reaction.
7) The absorbance at 450 nm was measured.
A colony showing a high color development value in this ELISA measurement is a clone that highly expresses the chimeric antibody.

E) Chimera antibody high expression CHO cell culture
CHO cells with high expression of chimeric antibody selected by ELISA were grown by culturing in Dulbecco's Modified Eagle's Medium supplemented with 10% Fetal Bovine Serum and 0.1 mg / mL Geneticin under conditions of 5% carbon dioxide at 37 ° C. It was. Fully expanded chimeric antibody-expressing CHO cells were replaced with culture in CHO-S-SFMII (GIBCO, 12052-098), a serum-free medium supplemented with 0.1 mg / mL Geneticin. It was cultured to some extent.

F) The culture supernatant of the chimeric antibody-highly expressing CHO cells cultured in CHO-S-SFMII, which is a column-purified serum-free medium, from the culture supernatant of the chimeric antibody-highly expressing cells was collected. Purification of the wild-type chimeric antibody was performed by Protein A column purification, which is usually used for antibody purification, because no change was made to its Fc region structure. However, the 298Ala / 333Ala / 334Ala type chimeric antibody and the 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody are mutated in their Fc region structure, so that the structure of the Fc region is changed and the normal protein A column is used. Is hardly adsorbed. For this reason, these mutant chimeric antibodies were purified using a Protein L column. The anti-CD20 chimeric antibody prepared this time has a variable region of κ chain. This Protein L is a protein that can recognize and bind to this variable region κ chain. Details of protein A column purification of the wild type chimeric antibody and protein L column purification of the mutant type chimeric antibody are described below.
Protein A column purification of wild type chimeric antibody
1) The culture supernatant obtained by culturing wild-type chimeric antibody highly expressing CHO cells with 1000 mL of CHO-S-SFMII was obtained by using Amicon Stirred Uitrafiltration Cell (Millipore, Millipore, Code YM10) with Amicon Ultrafiltration Membranes (Millipore, Code YM10). Using Model 8400), it was concentrated from 1000 mL to 100 mL.
2) Add 8 mL of Protein A-Sepharose rProteinA Sepharose Fast Flow (Amersham Bioscience, 17-1279-03) to the culture supernatant concentrate, and stir at 4 ° C for 2 days to adsorb the wild-type chimeric antibody to ProteinA I let you.
3) Protein A-Sepharose adsorbed with the wild type chimeric antibody was packed in a column having a diameter of 1.5 cm and a length of 8 cm.
4) The column packed with Protein A-Sepharose was washed with 100 mL of PBS, and the wild-type chimeric antibody was eluted from the column with Elution Buffer (0.17M Glycine-HCl, pH 2.3).
5) The eluted wild type chimeric antibody solution was immediately neutralized with pH by adding an appropriate amount of 1M Tris-HCl pH8.5.
6) The wild type chimeric antibody eluate was dialyzed into 5 L of PBS at 4 ° C. in Cell Sep T2 (Membrane filtration products Inc, 8030-23) as a dialysis tube. The wild-type chimeric antibody collected after dialysis was used as a purified product.

Protein L column purification of mutant chimeric antibody
1) Culture supernatant obtained by culturing CY cells with high expression of Cys-type chimeric antibody in 1000 mL of CHO-S-SFMII was obtained from Amicon Stirred Uitrafiltration Cell (Millipore, Model 8400) was concentrated from 1000 mL to 100 mL.
2) 100 mL of the culture supernatant concentrate was mixed with 100 mL of Protein L Binding Buffer (0.1 M Phosphate, 0.15 M NaCl, pH 7.2).
3) A column having a diameter of 1.5 cm and a length of 8 cm was packed with 2 mL of ImmunoPure Immobilized Protein L Plus (PIERCE, 20250) as Protein L-Sepharose.
4) The liquid prepared in 2) was added from the top of the column and slowly poured out from the bottom of the column. By this operation, the mutant chimeric antibody was adsorbed to Protein L-Sepharose. Thereafter, the column was washed with 50 mL of Protein L Binding Buffer to remove excess protein adsorbed on the column.
5) The mutant chimeric antibody was eluted from the column with Elution Buffer (0.17M Glycine-HCl, pH 2.3). The eluted mutant chimeric antibody solution was immediately neutralized by adding an appropriate amount of 1M Tris-HCl pH8.5.
6) The mutant chimeric antibody eluate was put into Cell Sep T2 (Membrane filtration products Inc, 8030-23), which was a dialysis tube, and dialyzed against 5 L of PBS at 4 ° C. The mutant chimeric antibody collected after dialysis was used as a purified product.
As described above, the purified wild-type chimeric antibody or mutant-type chimeric antibody was subjected to 12.5% SDS-PAGE and then stained with silver. This electrophoresis image is attached to FIG.

<Evaluation of ADCC activity of various chimeric antibodies>
The antibody-antibody-dependent cellular cytotoxicity (ADCC) activity of various chimeric antibodies was evaluated by a lactate dehydrogenase release assay. In this assay, Daudi cells having CD20 molecules on the cell membrane surface were used as target cells, and human peripheral blood mononuclear cells (PBMC) were used as effector cells. This Human PBMC was prepared from human blood using Lymphoprep (Axis Shield).
Daudi cells (1 × 10 ⁴ cells / 50 μL) are placed in each well of a 96-well U-bottomed plate, and Human PBMC, which is an Effector cell, is 2 × 10 2 so that the E / T ratio is 20/1. ⁵ were added. Furthermore, various anti-CD20 chimeric antibodies were added to this cell solution in a serial dilution series, and incubated at 37 ° C. for 20 hours. After incubation, the 96-well U-bottomed plate was centrifuged and the lactate dehydrogenase activity in the supernatant was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). Antibody-dependent and specific Cytotoxicity% was calculated using the following formula:
% Cytotoxicity = 100 × (E−S _E −S _T ) / (M−S _T )

Here, E represents Experimental release, and is a lactate dehydrogenase activity relaese from Target cells when the antibody and Effctor cells are incubated with Target cells. S _E is lactate dehydrogenase activity released spontaneously from Effector cells. The S _T, is spontaneously released lactate dehydrogenase activity from Target cells. M represents the lactate dehydrogenase activity released to the maximum of Target cells, which is the lactate dehydrogenase activity released from Target cells by addition of Lysis Solution (9% Triton X-100).
The ADCC activity results of the anti-CD20 chimeric antibody of wild type, 298Ala / 333Ala / 334Ala, 298Ala / 333Ala / 334Ala / 295Cys by the above evaluation method are attached in FIG. FIG. 4 shows that the 298Ala / 333Ala / 334Ala type chimeric antibody and the 298Ala / 333Ala / 334Ala / 295Cys type under the high antibody concentration conditions (0.001 to 10 μg / mL) compared with the ADCC activity of the wild type chimeric antibody. The chimeric antibody showed very high ADCC activity. 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody, which is 298Ala / 333Ala / 334Ala type chimeric antibody plus 295Cys, has a higher ADCC activity than 298Ala / 333Ala / 334Ala type chimeric antibody at low concentrations (0.001 μg / mL or less) showed that.

Furthermore, the ratio of cytotoxicity between the 298Ala / 333Ala / 334Ala type chimeric antibody and the 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody was compared by a lactate dehydrogenase release assay. As a result, the percentage of cytotoxicity when the concentration of the added antibody is 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL is 5.8%, 10.3% for the 298Ala / 333Ala / 334Ala type chimeric antibody, 20.7%, 41.4%, 52.2%, 52.6%, 55.3%, 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody is 10%, 21.8%, 26.6%, 36.6%, 51.1%, 51.4%, 58.6% respectively there were. Therefore, the ratio of ADCC activity (ratio of the ratio of cytotoxicity of the 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody to the 298Ala / 333Ala / 334Ala type chimeric antibody between the two points where the wild type antibody and the mutant antibody have the same concentration) ) Were 1.7 times, 2.1 times, 1.3 times, 0.9 times, 1 time, 1 time, and 1.1 times, respectively (Table 1).

In addition, from Table 1, two points with the same rate of cytotoxicity were selected between the 298Ala / 333Ala / 334Ala type chimeric antibody and the 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody, and comparison of ADCC activity was attempted. . For example, 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody shows about 20% cytotoxicity at 0.0001 μg / mL, and 298Ala / 333Ala / 334Ala type chimeric antibody shows about 20% cytotoxicity at 0.001 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced by a factor of 10. From this, the ADCC activity increased about 10 times (the 298Ala / 333Ala / 334Ala type chimeric antibody and the 298Ala / 333Ala / 334Ala / 295Cys type chimeric antibody were compared with the wild type antibody between the two points showing the same rate of cytotoxicity. It can be determined that the ratio of the concentration of the mutant antibody has increased 10 times).
Such an increase in ADCC activity (about 10 times) was confirmed in other concentrations. Those skilled in the art can calculate the increase in ADCC activity from FIG. 4 and Table 1 following the above method.

[Example 2]
In the same manner as in Example 1, 239Asp / 330Leu / 332Glu type and 239Asp / 330Leu / 332Glu / 295Cys type Anti-CD20 chimeric antibodies were prepared, and ADCC activities of these various variants of Anti-CD20 chimeric antibodies were evaluated. . As a result, the 239Asp / 330Leu / 332Glu / 295Cys anti-CD20 chimeric antibody is higher compared to the ADCC activity of the 239Asp / 330Leu / 332Glu type Anti-CD20 chimeric antibody. ADCC activity was shown.
In the following, the preparation method of Anti-CD20 chimeric antibody of 239Asp / 330Leu / 332Glu type and 239Asp / 330Leu / 332Glu / 295Cys type, its ADCC activity measurement and reactivity to CD20 molecule are described.

1) Preparation of Anti-CD20 chimeric antibody A chimeric antibody is obtained as a purified chimeric antibody through the following steps A) to F).
A) Cloning of genes necessary for producing chimeric antibodies
B) Mutation introduction of the cloned gene,
C) Construction of a chimeric antibody expression vector combining the cloned gene and its mutated gene,
D) Gene introduction of chimeric antibody expression vector into CHO cells and screening of CHO cells that highly express chimeric antibodies,
E) culturing CHO cells that highly express chimeric antibodies,
F) Column purification from the culture supernatant of cells expressing high chimeric antibodies.
Among these steps, A), C) to F) are the same as those in Example 1. Therefore, in this embodiment, step B) will be described.

B) Mutation introduction of cloned genes
In order to obtain a 239Asp / 330Leu / 332Glu type chimeric antibody or a 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody, a mutation must be introduced into a specific part of the cloned human IgG1 heavy chain constant region. According to the number of human IgG1 heavy chain constant region shown in Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991) by Elvin A. Kabat et al., Ser 239 is Asp and 330 Ala is Leu Next, the pCR2.1 vector with the cloned Hunan IgG1 heavy chain constant region inserted: pCR2.1-HC (wild type) was used as a template to change the 332nd Ile to Glu and the 295th Glu to Cys. As described above, mutation was introduced by PCR. Details of this mutagenesis method are described below.

(1) Mutation introduction to 239 (Ser → Asp)
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
239 Asp1 primer (20μM) 1.25 μL
239 Asp2 primer (20μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
239 Asp1 primer: GAACTCCTGGGGGGACCGGATGTCTTCCTCTTCCCCCCA (SEQ ID NO: 80)
239 Asp2 primer: TGGGGGGAAGAGGAAGACATCCGGTCCCCCCAGGAGTTC (SEQ ID NO: 81)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector (239Asp) into which the human IgG1 heavy chain constant region introduced with mutation 239 (Ser → Asp) was inserted.

(2) Mutation introduction to 332 (Ile → Glu)
pCR2.1-HC (239Asp) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
332 Glu1 primer (20μM) 1.25 μL
332 Glu2 primer (20μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
332 Glu1 primer: AAAGCCCTCCCAGCCCCCGAAGAGAAAACCATCTCCAAA (SEQ ID NO: 82)
332 Glu2 primer: TTTGGAGATGGTTTTCTCTTCGGGGGCTGGGAGGGCTTT (SEQ ID NO: 83)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, a pCR2.1 vector (239Asp / 332Glu) into which a human IgG1 heavy chain constant region introduced with mutation 332 (Ile → Glu) was obtained was obtained.

(3) Mutation introduction to 330 (Ala → Leu)
pCR2.1-HC (239Asp / 332Glu) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
332 Glu1 primer (20μM) 1.25 μL
332 Glu2 primer (20μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
330 Leu1 primer: TCCAACAAAGCCCTCCCACTCCCCGAAGAGAAAACCATC (SEQ ID NO: 84)
330 Leu2 primer: GATGGTTTTCTCTTCGGGGAGTGGGAGGGCTTTGTTGGA (SEQ ID NO: 85)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, a pCR2.1 vector (239Asp / 330Leu / 332Glu) into which a human IgG1 heavy chain constant region introduced with mutation 330 (Ala → Leu) was obtained was obtained.

(4) Mutation introduction into 295 (Glu → Cys)
pCR2.1-HC (239Asp / 330Leu / 332Glu) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
295 Cys3 primer (20μM) 1.25 μL
295 Cys4 primer (20μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
295 Cys3 primer: ACAAAGCCGCGTGAGGAGTGCTACAACAGCACGTACCGT (SEQ ID NO: 86)
295 Cys4 primer: ACGGTACGTGCTGTTGTAGCACTCCTCACGCGGCTTTGT (SEQ ID NO: 87)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector (239Asp / 330Leu / 332Glu / 295Cys) into which the human IgG1 heavy chain constant region introduced with mutation 295 (Glu → Cys) was inserted.

<Silver staining analysis of various purified chimeric antibodies>
The silver staining analysis of the purified various chimeric antibodies was performed according to the same method as described in Example 1 (FIG. 5).

<Evaluation of ADCC activity of various chimeric antibodies>
Antibody-dependent cellular cytotoxicity (ADCC) activity evaluation of various chimeric antibodies was also performed according to the same method as described in Example 1. FIG. 6 shows the ADCC activity results of the wild-type, 239Asp / 330Leu / 332Glu, 239Asp / 330Leu / 332Glu / 295Cys Anti-CD20 chimeric antibody. From the figure, 239Asp / 330Leu / 332Glu type chimeric antibody is higher in concentration (0.001-10 μg / mL) than wild type, and 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody Compared with, it showed high ADCC activity. In addition, 239Asp / 330Leu / 332Glu / 295Cys showed higher ADCC activity at a lower concentration (a concentration of 0.001 μg / mL or less) than 239Asp / 330Leu / 332Glu.

Furthermore, the ratio of cytotoxicity between the 239Asp / 330Leu / 332Glu type chimeric antibody and the 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody was compared by a lactate dehydrogenase release assay. As a result, when the concentration of the added antibody was 0.0000001, 0.000001, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10 μg / mL, the percentage of cytotoxicity was 7.2% for the 239Asp / 330Leu / 332Glu type chimeric antibody, respectively. , 7.3%, 9.5%, 15.1%, 26%, 39.5%, 56.4%, 52.5%, 53.3%, 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody 23.4%, 19.6%, 22.9%, 26.3%, respectively 30.1%, 39.5%, 53.2%, 54.3%, 52.5%. Therefore, the ratio of ADCC activity (239Asp / 330Leu / 332Glu type chimeric antibody and 239Asp / 330Leu / 332Glu type chimeric antibody at the same concentration of 239Asp / 330Leu / 332Glu type chimeric antibody to 239Asp / 330Leu / 332Glu type chimeric antibody, The ratio of cytotoxicity of / 295Cys type chimeric antibody was found to be 3.3 times, 2.7 times, 2.4 times, 1.7 times, 1.2 times, 1 times, 0.9 times, 1 times and 1 times, respectively ( Table 2).

Further, from the table, two points having the same rate of cytotoxicity were selected between the 239Asp / 330Leu / 332Glu type chimeric antibody and the 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody, and comparison of ADCC activity was attempted. For example, the 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody shows about 40% cytotoxicity at 0.01 μg / mL, and the 239Asp / 330Leu / 332Glu type chimeric antibody shows about 40% cytotoxicity at 1 μg / mL. When compared using these two points, the antibody concentration required to obtain the same ADCC activity was reduced to about 1/100. From this, ADCC activity was increased about 100 times (239Asp / 330Leu / 332Glu type chimeric antibody and 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody between 239Asp / 330Leu / The ratio of the concentration of the 239Asp / 330Leu / 332Glu / 295Cys type chimeric antibody to the 332Glu type chimeric antibody was 100 times).
Those skilled in the art can calculate the increase in ADCC activity from FIG. 6 and Table 2 following the above method.

Example 3
Anti-CD20 chimeric antibodies of fucose-reduced wild type and fucose-reduced 295 Cys were prepared in the same manner as in Example 1, and the ADCC activity of these various anti-CD20 chimeric antibodies was evaluated.
In the following, methods for producing anti-CD20 chimeric antibody fucose-reduced wild type and fucose-reduced 295 Cys type, ADCC activity measurement and reactivity to CD20 molecule are described.

1) Preparation of Anti-CD20 chimeric antibody A chimeric antibody is obtained as a purified chimeric antibody through the following steps A) to G).
A) Cloning of genes necessary for producing chimeric antibodies
B) Mutation introduction of the cloned gene,
C) Construction of a chimeric antibody expression vector combining the cloned gene and its mutated gene,
D) Preparation of FUT8 Knock Down CHO cells,
E) Gene transfer of chimeric antibody expression vector into fucose Knock Down CHO cells and screening of fucose Knock Down CHO cells that highly express chimeric antibodies,
F) Culture of chimeric antibody high-expressing fucose Knock Down CHO cells,
G) Column purification from the culture supernatant of cells expressing high chimeric antibodies.
Of these steps, A) is the same as Example 1. Therefore, in this embodiment, steps B), C), D), E), F), and G) will be described.

B) Mutation of cloned gene In order to obtain a fucose-reduced 295Cys type chimeric antibody, mutation must be introduced into a specific part of the cloned human IgG1 heavy chain constant region. According to the number of the human IgG1 heavy chain constant region shown in Sequence of proteins of Immunological Interest (NIH Publication No.91-3242, 1991) by Elvin A. Kabat et al. Mutation was carried out by PCR using pCR2.1 vector: pCR2.1-HC (wild type) in which the Hunan IgG1 heavy chain constant region was inserted as a template. Details of this mutagenesis method are described below.

(1) Mutation introduction into 295 (Glu → Cys)
pCR2.1-HC (wild type) (25 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
295 Cys3 primer (20μM) 1.25 μL
295 Cys4 primer (20μM) 1.25 μL
× 10 pfu polymerase Buffer 5 μL
pfu DNA polymerase 1 μL
Sterile water 35.5 μL
total 50 μL
(PCR amplification reaction)
95 ℃ 30 sec
95 ° C 30 sec 55 ° C 1 min 68 ° C 13 min (12 cycles)
68 ℃ 4 min
4 ℃ unlimited time
The base sequence of Primer is as follows.
295 Cys3 primer: ACAAAGCCGCGTGAGGAGTGCTACAACAGCACGTACCGT (SEQ ID NO: 86)
295 Cys4 primer: ACGGTACGTGCTGTTGTAGCACTCCTCACGCGGCTTTGT (SEQ ID NO: 87)
After completion of the PCR reaction, 1 μL of DpnI (New England BioLabs) was added and incubated at 37 ° C. for 2 hours. Using the solution after the incubation, E. coli JM109 was transformed, and the plasmid was purified from the transformed E. coli. By confirming the DNA sequence of the purified plasmid, it was named pCR2.1 vector (295Cys) into which the human IgG1 heavy chain constant region introduced with mutation 295 (Glu → Cys) was inserted.

C) Construction of chimeric antibody expression vector combining cloned gene and mutated gene
In order to express one type of Anti-CD20 chimeric antibody, this can be achieved by transfecting CHO cells with two types of expression vectors of the L chain and H chain of the Anti-CD20 chimeric antibody. Here, the L-chain expression vector of the Anti-CD20 chimeric antibody is an expression vector in which an anti-CD20 mouse L chain variable region gene + human IgG1 L chain constant region gene is combined, The expression vector for the H chain of the -CD20 chimeric antibody is obtained by incorporating an anti-CD20 mouse H chain variable region gene + human IgG1 H chain constant region gene into the expression vector. In producing wild-type and 295 Cys-type Anti-CD20 chimeric antibodies, the anti-CD20 chimeric antibody L chain expression vector is commonly used to express these two chimeric antibodies. The H chain expression vector of the CD20 chimeric antibody requires a dedicated H chain expression vector for each of the wild type and 295 Cys type chimeric antibodies. Here, the construction of the anti-CD20 chimeric antibody L chain expression vector that can be commonly used for the expression of each fucose-reducing chimeric antibody is described in detail, and then the dedicated Anti-CD20 is required for the expression of the 295Cys type chimeric antibody. The construction of the chimeric antibody heavy chain expression vector is described.

Construction of the expression vector of the Anti-CD20 chimeric antibody L chain The pQCXIH (Clontech) vector is used as the expression vector, and the Anti-CD20 mouse L chain variable region gene + Human IgG1 L chain constant region is placed on the NotI and EcoRI sites of this vector. Insert a gene. A fragment of the Anti-CD20 mouse L chain variable region gene was obtained by performing the following PCR reaction using the previously prepared pCR2.1-MLV as a template. In addition, a fragment of the human IgG1 L chain constant region gene was obtained by the following PCR reaction using the previously prepared pCR2.1-LC as a template.

(Anti-CD20 mouse L chain variable region gene amplification reaction)
pCR2.1-MLV (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
L5 primer (20μM) 2.5 μL
L2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4min
4 ℃ unlimited time
L5 primer: AATAGCGGCCGCATGGATTTTCAGGTGCAGATTATCA (SEQ ID NO: 88)
L2 primer: TTTCAGCTCCAGCTTGGTCCCAGCACC (5'-phosphorylated) (SEQ ID NO: 73)

(Human IgG1 L chain constant region gene amplification reaction)
pCR2.1-LC (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
L3 primer (20μM) 2.5 μL
L6 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
L3 primer: ACTGTGGCTGCACCATCTGTCTTCATC (5'-phosphorylated) (SEQ ID NO: 74)
L6 primer: CGGTAGAATTCCTAACACTCTCCCCTGTTGAAGCTC (SEQ ID NO: 89)

  The Anti-CD20 mouse L chain variable region gene fragment obtained by the PCR reaction was purified after being cleaved with the restriction enzyme NotI (Takara). Further, the human IgG1 L chain constant region gene fragment obtained by PCR reaction was cleaved with the restriction enzyme EcoRI (Takara) and then purified. The fragment obtained by cleaving the pQCXIH vector with NotI and EcoRI and purified was mixed with the purified Anti-CD20 mouse L chain variable region gene fragment and the purified Human IgG1 L chain constant region gene fragment. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. This anti-CD20 chimeric antibody light chain expression vector was named pKD chimeric LC.

Construction of Anti-CD20 chimeric antibody heavy chain expression vector The pQCXIP vector (Clontech) is used as the expression vector, and the Anti-CD20 mouse heavy chain variable region gene + Human IgG1 heavy chain constant region gene at the NotI and EcoRI sites of this vector. To insert. The anti-CD20 mouse heavy chain variable region gene was obtained by performing the following PCR reaction using the previously prepared pCR2.1-MHV as a template. The dedicated H chain expression vector for each of the wild type and 295 Cys type chimeric antibodies is different in plasmid as a template for PCR amplification of the human IgG1 H chain constant region gene, but the operation itself is carried out in the same manner. Various human IgG1 heavy chain gene fragments were obtained. Details including the PCR reaction are described below.

(Anti-CD20 mouse heavy chain variable region gene amplification reaction)
pCR2.1-MHV (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H5 primer (20μM) 2.5 μL
H2 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H5 primer: AATAGCGGCCGCATGGGATGGAGCTGGGTCTTTCTC (SEQ ID NO: 90)
H2 primer: TGAGGAGACGGTGACCGTGGTCCC (5'-phosphorylated) (SEQ ID NO: 77)

(Human IgG1 H chain constant region gene amplification reaction)
# Various template plasmids (20 ng / μL) 2 μL
2.5 mM dNTPs 4 μL
H3 primer (20μM) 2.5 μL
H6 primer (20μM) 2.5 μL
DMSO 2.5 μL
× 10 pfu polymerase Buffer 5 μL
pfu polymerase 1 μL
Sterile water 30.5 μL
total 50 μL
94 ℃ 2 min
94 ℃ 1 min 55 ℃ 2min 72 ℃ 2min (20 cycles)
72 ℃ 4 min
4 ℃ unlimited time
H3 primer: GCCTCCACCAAGGGCCCATCGGTC (5'-phosphorylated) (SEQ ID NO: 78)
H6 primer: CGGTAGAATTCTTATTTACCCGGAGACAGGGAGAGGC (SEQ ID NO: 91)

#: PCR2.1-HC (wild type) was used as a template to prepare a gene fragment for a wild type Anti-CD20 chimeric antibody. In order to prepare a gene fragment for 295 Cys-type Anti-CD20 chimeric antibody, pCR2.1-HC (295cys) was used as a template.

  The Anti-CD20 mouse H chain variable region gene fragment obtained by the PCR reaction was purified after being cleaved with a restriction enzyme NotI (Takara). In addition, Human IgG1 H chain constant region gene fragments obtained by various PCR reactions with different templates were purified after digestion with restriction enzyme EcoRI (Takara). The purified fragment obtained by cleaving the pQCXIP vector with NotI and EcoRI was mixed with the purified Anti-CD20 mouse heavy chain variable region gene fragment and the purified human IgG1 heavy chain constant region gene fragment for ligation. In the vector obtained by this ligation, it was confirmed by sequencing that the desired fragment was inserted. The anti-CD20 chimeric antibody H chain expression vectors were named pKD chimeric HC (wild type) and pKD chimeric HC (295 Cys).

D) Preparation of FUT8 Knock Down CHO cells The fucose-reducing chimeric antibody is a CHO cell knocked down with α1.6 fucose transferase (FUT8). It can be prepared by transfection (the base sequence of FUT8 is shown in SEQ ID NO: 96 and the amino acid sequence is shown in SEQ ID NO: 97). Here, a method for producing FUT8 Knock Down CHO cells necessary for producing a fucose-reducing chimeric antibody is described.
FUT8 siRNA Sense primer (5'-gatccccgctgagtctctccgaatacttcaagagagtattcggagagactcagcttttta-3 'SEQ ID NO: 92) and FUT8 siRNA Anti-Sense primer (5'-tcgataaaaagctgactcttgaggactcggctctggagactctggtctgagag Was annealed by heating from 99 ° C. to 4 ° C. over 2 hours after heating at 99 ° C. for 2 minutes in the presence of NaCl according to a conventional method. The obtained DNA fragment was inserted into pSuper gfp + neo (Oligoengine). The inserted DNA base sequence was confirmed using ABI3100-Avant (Applied Biosystems), and a FUT-8 siRNA expression vector FUT-8 SiRNA / pSuper gfp + neo was constructed.
The resulting FUT-8 siRNA expression vector FUT-8 siRNA / pSuper gfp + neo was introduced into CHO-K1 cells (ATCC Catalog No. CCL-61) using FuGENE (Roche) according to the instruction manual. The transfected CHO-K1 cells are cultured in RPMI-1640 medium (SIGMA) containing 10% FCS (Hyclone) -penicillin-streptomycin (SIGMA), and then G418 (Wako Pure Chemicals) Drug selection was performed by adding 0.6 mg / ml. Cells that acquired drug resistance were sorted using a cell sorter (EPICS ALTRA, manufactured by Beckman Coulter) using the expression level of GFP as an index. Cells after sorting were subjected to limiting dilution to obtain cells that highly express GFP, that is, cells that highly express FUT-8 siRNA. The expression level of FUT-8 mRNA in the same cells was determined using the sense primer for FUT-8 expression analysis and the anti-sense primer for FUT-8 expression analysis (5'-ATTGACCAGGGGACAGCTACA-3). 'SEQ ID NO: 95) and real time PCR using ABI PRISM 7000 (Applied Biosystems) quantified, mRNA expression was suppressed 22.8% of cells before gene transfer and FUT-8 expression I confirmed. The obtained cells with a high FUT-8 siRNA expression level were used as FUT-8 knockdown CHO-K1 cells in the experiment.

E) Gene expression of chimeric antibody expression vectors into FUT8 Knock Down CHO cells and screening of CHO cells that highly express chimeric antibodies. Used to transfect FUT8 Knock Down CHO cells.
Wild type chimeric antibody: pKD chimera LC + pKD chimera HC (wild type)
295Cys type chimeric antibody: pKD chimera LC + pKD chimera HC (295Cys type)

FUT8 Knock Down CHO cells transfected with 10% Fetal Bovine Serum (EQITEC-BIO), 0.2 mg / mL Geneticin (GIBCO, 10131-035), 0.1 mg / mL Hygromycin (Invitrogen, 10687-010), 2 μg / mL The cells were grown by culturing in RPMI1640 (Sigma, R8758) supplemented with mL Puromycin (Merck Japan, 540411-25MG) at 37 ° C under 5% carbon dioxide. The FUT8 Knock Down CHO cells that highly express the chimeric antibody are selected from these expanded FUT8 Knock Down CHO cells. First, a part of each colony is transplanted into a 96-well plate. The cells were further cultured for 10 days. A part of the culture supernatant was taken out and subjected to ELISA measurement shown below to determine a high-expression FUT8 Knock Down CHO cell clone.
(ELISA measurement)
1) The culture supernatant of Tansfectioned CHO cells was added to a 96-well plate coated with Anti-Human IgG (γ-chain) (MBL, 103AG) at 100 μL / well and left at room temperature for 1 hour.
2) The culture supernatant was discarded from the 96-well plate, and wells were sufficiently washed with PBS containing 0.05% Tween 20 (PBS-0.05% Tween 20).
3) A solution obtained by diluting Anti-Human IgG (γ-chain) Peroxidase conjugated (MBL, 208) 2000-fold with PBS-0.05% Tween20 was added at 100 μL / well and left at room temperature for 1 hour.
4) The solution was discarded from the 96-well plate, and wells were sufficiently washed with PBS-0.05% Tween20.
5) 100 μL / well of TMB Peroxidase Substrate (Moss, TMBE-1000s) was added to cause a color reaction.
6) After 10 to 15 minutes of color reaction, 1N sulfuric acid was added at 100 μL / well to stop the color reaction.
7) The absorbance at 450 nm was measured.
A colony showing a high color development value in this ELISA measurement is a clone that highly expresses the chimeric antibody.

E) Chimera antibody high expression FUT8 Knock Down CHO cell culture
CHO cells with high chimeric antibody expression selected by ELISA were RPMI1640 supplemented with 10% Fetal Bovine Serum, 0.2 mg / mL Geneticin, 0.1 mg / mL Hygromycin, and 2 μg / mL Puromycin, at 37 ° C and 5% carbon dioxide. It was made to proliferate by culture | cultivating. A well-expressed chimeric antibody high-expression FUT8 Knock Down CHO cell is CHO-S-SFMII (GIBCO, 12052-), a serum-free medium supplemented with 0.2 mg / mL Geneticin, 0.1 mg / mL Hygromycin, and 2 μg / mL Puromycin. 098), and about 1000 mL was continuously cultured.

F) Column supernatant from chimera antibody high expression cell culture supernatant The culture supernatant of chimera antibody high expression FUT8 Knock Down CHO cells cultured in CHO-S-SFMII, which is a serum-free medium, was collected. Purification of the wild-type chimeric antibody was performed by Protein A column purification, which is usually used for antibody purification, because no change was made to its Fc region structure. However, since the 295 Cys type chimeric antibody is mutated in its Fc region structure, it is accompanied by a change in the structure of the Fc region and hardly adsorbs to a normal Protein A column. For this reason, these mutant chimeric antibodies were purified using a Protein L column. The anti-CD20 chimeric antibody prepared this time has a variable region of κ chain. This Protein L is a protein that can recognize and bind to this variable region κ chain. Details of protein A column purification of the wild type chimeric antibody and protein L column purification of the mutant type chimeric antibody are described below.
Protein A column purification of wild type chimeric antibody
1) Culture supernatant obtained by culturing FUT8 Knock Down CHO cells with high expression of wild-type chimeric antibody in 1000 mL of CHO-S-SFMII is Amicon Stirred Uitrafiltration Cell with Amicon Ultrafiltration Membranes (Millipore, Code YM10) (Millipore, Model 8400) and concentrated from 1000 mL to 100 mL.
2) Add 8 mL of Protein A-Sepharose rProteinA Sepharose Fast Flow (Amersham Bioscience, 17-1279-03) to the culture supernatant concentrate, and stir at 4 ° C for 2 days to adsorb the wild-type chimeric antibody to ProteinA I let you.
3) Protein A-Sepharose adsorbed with the wild type chimeric antibody was packed in a column having a diameter of 1.5 cm and a length of 8 cm.
4) The column packed with Protein A-Sepharose was washed with 100 mL of PBS, and the wild-type chimeric antibody was eluted from the column with Elution Buffer (0.17M Glycine-HCl, pH 2.3).
5) The eluted wild type chimeric antibody solution was immediately neutralized with pH by adding an appropriate amount of 1M Tris-HCl pH8.5.
6) The wild type chimeric antibody eluate was dialyzed into 5 L of PBS at 4 ° C. in Cell Sep T2 (Membrane filtration products Inc, 8030-23) as a dialysis tube. The wild-type chimeric antibody collected after dialysis was used as a purified product.

Protein L column purification of mutant chimeric antibody
1) Culture supernatant obtained by culturing FUT8 Knock Down CHO cells with high expression of 295Cys type chimeric antibody in 1000 mL of CHO-S-SFMII was obtained from Amicon Stirred Uitrafiltration Cell with Amicon Ultrafiltration Membranes (Millipore, Code YM10). (Millipore, Model 8400) was used to concentrate from 1000 mL to 100 mL.
2) 100 mL of the culture supernatant concentrate was mixed with 100 mL of Protein L Binding Buffer (0.1 M Phosphate, 0.15 M NaCl, pH 7.2).
3) A column having a diameter of 1.5 cm and a length of 8 cm was packed with 2 mL of ImmunoPure Immobilized Protein L Plus (PIERCE, 20250) as Protein L-Sepharose.
4) The liquid prepared in 2) was added from the top of the column and slowly poured out from the bottom of the column. By this operation, the mutant chimeric antibody was adsorbed to Protein L-Sepharose. Thereafter, the column was washed with 50 mL of Protein L Binding Buffer to remove excess protein adsorbed on the column.
5) The mutant chimeric antibody was eluted from the column with Elution Buffer (0.17M Glycine-HCl, pH 2.3). The eluted mutant chimeric antibody solution was immediately neutralized by adding an appropriate amount of 1M Tris-HCl pH8.5.
6) The mutant chimeric antibody eluate was put into Cell Sep T2 (Membrane filtration products Inc, 8030-23), which was a dialysis tube, and dialyzed against 5 L of PBS at 4 ° C. The mutant chimeric antibody collected after dialysis was used as a purified product.
As described above, the purified wild-type chimeric antibody or mutant-type chimeric antibody was subjected to 12.5% SDS-PAGE and then stained with silver. This electrophoresis image is attached to the figure (FIG. 7).

  According to the antibody of the present invention, the effector function can be remarkably improved by adding a mutation to a specific amino acid residue in the Fc region of the antibody or reducing fucose of a sugar chain bound to the antibody having the mutation. it can. A therapeutic composition comprising such an antibody is extremely useful as an antibody drug because an excellent pharmacological effect can be obtained in a small amount. In addition, the cost can be reduced by reducing the amount of use, and the specificity and the side effects are few, so that the patient's economic and physical burden can be greatly reduced. In addition, other treatment methods such as chemotherapy and radioisotope-labeled antibodies that have been used in combination with antibody treatment can be avoided, and side effects due to these treatment methods can be avoided.

It is a figure which shows the DNA sequence of an Anti-CD20 mouse | mouth L chain variable region. It is a figure which shows the DNA sequence of Anti-CD20 mouse | mouth heavy chain variable region. 2 is a photograph showing electrophoresis results of purified wild-type anti-CD20 chimeric antibody, 298Ala / 333Ala / 334Ala-type anti-CD20 chimeric antibody, and 298Ala / 333Ala / 334Ala / 295Cys-type anti-CD20 chimeric antibody. In Example 1, it is a graph which shows the evaluation result which measured ADCC activity of the wild type anti-CD20 chimeric antibody, the 298Ala / 333Ala / 334Ala type anti-CD20 chimeric antibody, and the 298Ala / 333Ala / 334Ala / 295Cys type anti-CD20 chimeric antibody. 2 is a photograph showing electrophoresis results of purified wild-type CD20 chimeric antibody, 239Asp / 330Leu / 332Glu-type CD20 chimeric antibody, and 239Asp / 330Leu / 332Glu / 295Cys-type CD20 chimeric antibody. In Example 2, it is a graph which shows the evaluation result which measured ADCC activity of the wild type CD20 chimeric antibody, the 239Asp / 330Leu / 332Glu type CD20 chimeric antibody, and the 239Asp / 330Leu / 332Glu / 295Cys type CD20 chimeric antibody. It is a photograph showing the electrophoresis results of purified wild type anti-CD20 chimeric antibody, fucose reduced wild type chimeric antibody, and fucose reduced 295 Cys type chimeric antibody.

Claims (18)

An IgG1 antibody wherein the amino acids at positions 298, 333, 334, and 295 in Kabat EU index numbers are replaced with alanine, alanine, alanine, and cysteine residues, respectively. An IgG1 antibody characterized in that the amino acids at the 239th, 330th, 332th and 295th positions in the Kabat EU index number are replaced with aspartic acid, leucine, glutamic acid and cysteine residues, respectively. An IgG1 antibody in which the 295th amino acid in the Kabat EU index number is replaced with a cysteine residue, and fucose binds to N-acetylglucosamine at the reducing end in the N-glycoside-linked sugar chain bound to the Fc region Not IgG1 antibody. The IgG1 antibody according to any one of claims 1 to 3 , which has a site that recognizes a human cell surface molecule. Cytokine receptors, cell adhesion molecules, cancer cell surface molecules, cancer stem cell surface molecules, blood cell surface molecules, which recognizes at least one selected from the group consisting of virus-infected cell surface molecules, of claims 1 to 4 The IgG1 antibody according to any one of the items. Antigen CD3, CD11a, CD20, CD22, CD25, CD28, CD33, CD52, Her2 / neu, EGF receptor, EpCAM, MUC1, GD3, CEA, CA125, HLA-DR, TNFalpha receptor, VEGF receptor, CTLA-4 The IgG1 antibody according to any one of claims 1 to 5 , which recognizes at least one selected from the group consisting of AILIM / ICOS and integrin molecules. An isolated nucleic acid encoding the IgG1 antibody of any one of claims 1-6 . A vector comprising the nucleic acid according to claim 7 . A host cell or non-human host organism comprising the vector of claim 8 . A method for producing an IgG1 antibody, comprising culturing the host cell or non-human host organism according to claim 9 so as to express an IgG1 antibody encoded by a nucleic acid. A therapeutic composition comprising the IgG1 antibody according to any one of claims 1 to 6 . A cell that expresses an IgG1 antibody having an effector function in which the 295th amino acid residue in the EU index number of Kabat is replaced with a cysteine residue, and has an activity of an enzyme that adds fucose to N-acetylglucosamine at the reducing end Reduced or deleted cells. A method for producing an IgG1 antibody with improved effector function, comprising the following steps (1) to (3):
(1) replacing the 298th, 333, 334, and 295th amino acids of Kabat in the IgG1 antibody having an effector function with alanine, alanine, alanine, and cysteine residues, respectively;
(2) A DNA encoding an H chain in which the 298th, 333, 334, and 295th amino acids in the EU index number of Kabat are substituted with alanine, alanine, alanine, and cysteine residues, respectively, and an L chain Introducing the encoded DNA into a host cell or non-human host organism and expressing the DNA; and (3) recovering the expression product. A method for producing an IgG1 antibody with improved effector function, comprising the following steps (1) to (3):
(1) A step of replacing the 239th, 330th, 332th, and 295th amino acids of Kabat in the IgG1 antibody having an effector function with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively.
(2) DNA encoding the H chain in which the 239th, 330th, 332th and 295th amino acids in the Kabat EU index numbers are replaced with aspartic acid, leucine, glutamic acid and cysteine residues, respectively, and the L chain A step of introducing a DNA encoding a host cell or a non-human host organism to express the DNA, and (3) a step of recovering the expression product. A method for producing an IgG1 antibody with improved effector function, comprising the following steps (1) to (3):
(1) replacing the 295th amino acid residue with a Kabat EU index number in IgG1 antibody having an effector function, with a cysteine residue;
(2) DNA encoding the H chain in which the 295th amino acid residue is replaced with a cysteine residue in the EU index number of Kabat, and DNA encoding the L chain are converted to N-acetylglucosamine at the reducing end with fucose. Introducing into a host cell or non-human host organism in which the activity of the enzyme to be added has been reduced or deleted, and expressing the DNA; and (3) recovering the expression product. The effector function of IgG1 antibody, comprising the steps of replacing the 298th, 333, 334, and 295th amino acids of Kabat EU index numbers in IgG1 antibody having effector function with alanine, alanine, alanine, and cysteine residues, respectively. How to improve. The effector function of IgG1 antibody, comprising the steps of substituting amino acids 239, 330, 332, and 295 in Kabat EU index numbers of IgG1 antibody having effector function with aspartic acid, leucine, glutamic acid, and cysteine residues, respectively. How to improve. A method for improving the effector function of an IgG1 antibody comprising the following steps (1) to (3);
(1) replacing the 295th amino acid residue with a Kabat EU index number in IgG1 antibody having an effector function, with a cysteine residue;
(2) DNA encoding the H chain in which the 295th amino acid residue is replaced with a cysteine residue in the EU index number of Kabat, and DNA encoding the L chain are converted to N-acetylglucosamine at the reducing end with fucose. Introducing into a host cell or non-human host organism in which the activity of the enzyme to be added has been reduced or deleted, and expressing the DNA; and (3) recovering the expression product.