WO2005053742A1

WO2005053742A1 - Medicine containing antibody composition

Info

Publication number: WO2005053742A1
Application number: PCT/JP2004/018441
Authority: WO
Inventors: Kenya Shitara; Rinpei Niwa
Original assignee: Kyowa Hakko Kogyo Co., Ltd.
Priority date: 2003-12-04
Filing date: 2004-12-03
Publication date: 2005-06-16
Also published as: JPWO2005053742A1

Abstract

It is intended to provide a medicine which comprises a combination of an antibody composition, wherein the content of sugar chains having no fucose bonded to the N-acetylglucosamine at the sugar chain reducing end amounts to 50% or more in the total N-glycoside bond complex sugar chains bonded to the Fc domain in the antibody composition, with at least one drug.

Description

Specification

Drug containing antibody composition

Technical field

The present invention relates to a total N-glycoside-linked complex type sugar chain that binds to the Fc region contained in the antibody composition, of the sugar chain in which fucose is not bound to N-acetyltylcolasamine at the reducing end of the sugar chain. antibody composition ratio is ^50% or more, relates to a pharmaceutical comprising a combination of at least one agent.

Background art

Antibody-dependent cytotoxic activity (hereinafter abbreviated as ADCC activity) is based on the expression of the Fc region of the antibody on the Fc gamma receptor expressed in a cell population called efector, such as NK cells, monocytes' macrophages, and granulocytes. It happens by combining. NK cells induce strong ADCC activity through FcyRIIIa, a type of Fey receptor on cells. The FcyRIIIa gene has a functional polymorphism at the 158th amino acid, and the 158th amino acid has Valine (hereinafter referred to as Val) FcγRllla more than Phenylalanine (hereinafter referred to as Phe) FcyRIIIa Induces strong ADCC activity [Journal of Clinical Investigation. Clin. Invest.), 100, 1059-1070 (1997)]. In recent years, it has been shown that the clinical effect of Rituxan is significantly higher in patients with Val / Val genotype than in patients with other genotypes [Blood, 99, 754-758 (2002), Arthritis Reum., 48, 455-459 (2003)], suggesting that ADCC activity plays an important role in the clinical efficacy of human IgGl subclass antibody drugs Have been.

The sugar chains of glycoproteins such as antibodies are linked to asparagine (N-glycoside-linked sugar chains) and sugar chains linked to serine, threonine, etc. (0-glycosyl-linked sugars) depending on the mode of binding to the protein part. Chains). N- glycoside-linked sugar chain, the following structural formula have a common core structure underlying shown in (I) [Biochemical Experimental Methods 2 ³ - glycoprotein sugar chain research methods (Gakkai Shuppan Center) Reiko Takahashi Hen (1989 Year) ] .

(Formula 1) ^ ^/ Named (I). In the above structural formula (I), GlcNAc represents N-acetyl dalcosamine, and an represents mannose. 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.

Antibody molecules are composed of tetramers in which two heavy and two light chains are associated. In the IgGl-type human antibody, a sugar chain having the above core structure is bound to asparagine at position 297 from the N-terminus existing in the Fc region of the heavy chain. In addition to this core structure, there is a possibility that fucose may be added to the non-reducing terminal side at the 6-position of N-acetyldarcosamine, galactose, sialic acid, and the reducing terminal N-acetyldarcosamine, respectively. These components are not homogeneous and vary with the antibody-producing cells [Trends Biotechnol., 15, 26-32 (1997)]. Human IgGl ADCC activity of galactose in this sugar chain [Hum Antibodies Hybridomas, 5, 143-151 (1994), Human Antibody's and 'Hypuri Dorma's] Antibodies Hybridomas), 6, 82-88 (1995)], Ν-cetyldarcosamine [Natya Biotechnol.], 17, 176-180, (1999), Biotechnology and Bio Engineering (Biotechnol. Bioeng.), 74, 288-294, (2001)], Fucose [Giannole of Biological 'Chemistry (J. Biol. Chem.), 277, 26733-26740 (2002), Journal It is reported that it is affected by the content of ォ ob ォ biological canole '' chemistry (J. Biol. Chem.), 278, 3466-3473 (2003)]. Among them, fucose has the greatest effect.Human IgGl type antibodies with a small proportion of sugar chains with fucose added to position 6 of 還元 -acetyltylcolsamine at the reducing end markedly enhance ADCC activity. [Jarnal Biolka Nolle 'Chemistry (J. Biol. Chem.), 277, 26733-26740 (2002), Journal' Ob 'Biological' Chemistry (J. Biol. Chem.), 278 , 3466-3473 (20 03)]. However, these reports all show ADCC activity of the antibody alone, and it is not known whether the ADCC activity of a human IgGl antibody having a small amount of fucose is further enhanced by other drugs. On the other hand, it is known that ADCC activity is increased by the site force which stimulates efecta cells. For example, Interleukin (hereinafter referred to as IL) -2 [Cancer 'Immology I' immunotherapy. (Cancer Immunol. Immunother.), 46, 213 (1998), Cancer 'Immology I' immunotherapy. (2002), Blood, 93, 3922 (1999), Japanese journal, research, and cancer research (Jpn. J. Cancer Res.), 87, 497 (1996)], IL-12 [ Blood (Blood), 93, 3922 ( 1999), Japanese Jananore, O blanking-wire carrier capacitors scratch. research _{_{(j pn. j. Cancer R}} es.), 87, 497 (1996)], IL-15 [ Blood ( Blood), 93, 3922 (1999), Jianore Opéperimental Medicine (J. Exp. Med), 180, 1395 (1994)], Interferon (hereinafter IFN)-a [Cancer Immunology · Immunotherapy. (Cancer Immunol. Immunother.), 46, 213 (1998), Cancer 'Imology' (Cancer Immunol. Immunother.), 51, 171 (2002)], IFN-γ [Cancer Immunol. Immunother., 46, 213 (1998), Cancer Immunol. Immunotherapy. (Cancer Immunol. Immunother.), 51, 171 (2002), Japanese jar nanoreop. Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996)], Macropharmaco Mouth knee stimulating factor (hereinafter M-CSF) [Cancer 'Immology' Immunotherapy., 46, 213 (1998), Prad (Blood), 93, 3922 (1999), Japanese 'journal Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996), JAPANESE JANORALEOV QUEAN RESEARCH (Jpn. J. Cancer Res.), 81, 79 (1990)] And stimulate various effector cells in vitro by adding them to effector cells. It is known that increase ADCC activity.

It is not known that low-molecular-weight drugs such as chemotherapeutic agents enhance ADCC activity in vitro. It is known that excellent therapeutic effects can be obtained by using chemotherapy in combination with antibodies. The anti-HER2 / neu humanized antibody rhuMAb HER2 (Herceptin, Roche) is known to have a remarkable effect on breast cancer when combined with taxane-based anticancer drugs [Clinical Therapeutics, 21, 309 ( 1999)]. In addition, the anti-CD20 human chimeric antibody IDEC-C2B8 (Rituxan, IDEC) is available in combination therapy with multidrug therapy. Is known to have a significant effect on B-cell lymphoma [J. Clin. Oncol., 17, 268

(1999)]. ,

Disclosure of the invention

An object of the present invention is to provide a drug using an antibody composition having a high therapeutic effect and at least one drug.

The present invention relates to the following (1) to (14).

(1) Of the total ダ -daricoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not linked to 還元 -acetyltylcolasamine at the reducing end of the sugar chain an antibody composition is proportion ^50% or more, a pharmaceutical including a combination of at least one agent.

(2) Of the total Ν-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not bound to 還元 -acetyltylcolasamine at the reducing end of the sugar chain. an antibody composition is proportion ^50% or more, a medicament for administration in combination with at least one agent.

(3) Of all Ν-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not linked to 還元 -acetyltylcolasamine at the reducing end of the sugar chain medical for proportion and antibody compositions is ^50% or more, the administration of at least one agent at the same time or sequentially.

(4) Of the total Ν-glycosidic bond complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not bound to 還元 -acetyltylcolasamine at the reducing end of the sugar chain. ratio than when the antibody composition is ^50% or more was administered alone, a high therapeutic effect and wherein the indicating the above (1) to (3) the medicament according to any one of.

(5) The medicament according to (4), wherein exhibiting high therapeutic effect means exhibiting high antibody-dependent cytotoxicity.

(6) A cell in which the antibody composition is resistant to a lectin recognizing a sugar chain structure in which the 6-position of _-glycoside-linked complex-type sugar chain reducing terminal and the 1-position of fucose are α-linked. The medicament according to any one of the above (1) to (5), which is an antibody composition produced from:

(7) Fucose binds to Ν-acetyl dalcosamine at the reducing end of the sugar chain in the total Ν-daricoside bond complex type sugar chain in which the antibody composition binds to the Fc region contained in the antibody composition. The medicament according to any one of the above (1) to (6), which is an antibody thread that is not a sugar chain.

(8) 'The sugar chain to which fucose is not bonded is a sugar chain in which the 1-position of the fucose is not bonded to the 6-position of 還元 -glycidylcosamine at the reducing end of Ν-glycoside-linked complex type sugar chain. The medicament according to any one of the above (1) to (7).

(9) The genomic gene of an enzyme involved in sugar chain modification in which fucose is bonded to position 6 of fucose at the reducing end of 還元 -glycoside-linked complex type glycan at the reducing end is knocked out. The medicament according to the above (7) or (8), which is an antibody composition produced from the obtained cells.

(10) An enzyme involved in glycosylation, in which the first position of fucose is a-linked to the sixth position of チル -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex-type sugar chain, is α1,6-fucosyltransferase. The medicament according to the above (9), which is perase.

(11) The medicament according to any one of the above (1) to (10), wherein the drug is a substance selected from the group consisting of a protein, a small molecule drug, and a biological response modifier. (12) The medicament according to the above (11), wherein the protein is a cytokine or an antibody.

(13) The medicament according to the above (12), wherein the cytokine is a cytokine selected from IFN-V, IL-2 and IL-15.

(14) The medicament according to any one of the above (1) to (13), wherein the medicament is a remedy for a disease associated with a tumor. As the form of the medicament of the present invention, of the total ダ -daricoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, fucose is bound to Ν-acetylethyldarcosamine at the reducing end of the sugar chain. A drug comprising a combination of an antibody composition having a sugar chain content of not less than 50% and at least one drug, a total 、 -daricoside binding complex that binds to the Fc region contained in the antibody composition A combination of an antibody composition in which the percentage of sugar chains in which fucose is not bound to glycine glucosamine at the reducing end of sugar chains is 50% or more, and at least one drug in combination Sugars in which fucose is not bound to glycine-linked glucosamine at the reducing end of all glycan-linked glycosides that bind to the Fc region contained in the pharmaceutical or antibody composition for administration. antibodies sets the proportion of the chain is ^50% or more Pharmaceuticals for the simultaneous or sequential administration of the composition and at least one agent. Here, the drug in combination refers to fucose at the reducing end of glycine-linked glycoside of glycine-linked glycoside, which is bound to the Fc region contained in the antibody composition. Even if a drug is prepared by separately preparing an antibody composition having an unbound sugar chain ratio of 50% or more and at least one drug, and simultaneously or sequentially administering these drugs in combination. Or a mixture of the respective drug components. In the mixture prepared by mixing the respective drug components, among the total シド -glycoside-linked complex-type sugar chains binding to the Fc region contained in the antibody composition, に -acetylmethylcolasamine at the reducing end of the sugar chain was added. A fusion antibody in which at least one drug is bound to an antibody composition in which the ratio of sugar chains to which fucose is not bound is <<)% or more is also included.

In the present invention, the ratio of sugar chains in which fucose is not linked to N-acetyltyl glucosamine at the sugar chain reducing end, of all N-daricoside-linked complex type sugar chains that bind to the Fc region contained in the antibody composition Refers to that the fucose is not bound to N-acetyldarcosamine at the reducing end of the sugar chain with respect to the total number of all N-glycoside-linked complex type sugar chains that bind to the Fc region contained in the composition. Refers to the ratio occupied by the number of sugar chains. The ratio of the sugar chain is preferably such that the 1-position of fucose is not a-bonded to the 6-position of N-acetyldarcosamine at the reducing end of the sugar chain.

In the present invention, a sugar chain in which fucose is not bonded to N-acetyldarcosamine at the N-glycoside-linked complex type sugar chain reducing terminal is defined as the N-glycoside-linked complex type sugar chain reducing terminal at the N-glycoside-linked complex type sugar chain reducing end. -Acetyl danolecosamine refers to a sugar chain that is not _α- linked. Specifically, position 1 of the fucose is _α- linked to position 6 of Ν-acetyldarcosamine of the Ν-glycoside-linked complex type sugar chain. If not linked, sugar chains can be mentioned. The antibody composition of the present invention includes any composition as long as it contains an antibody molecule having an Ν-daricoside-linked complex type sugar chain in the Fc region.

An antibody molecule is a tetramer in which two types of heavy chains and light chains (hereinafter, referred to as H chains and L chains, respectively) are associated with two molecules of each. About one-fourth of the N-terminal side of the H chain and about one-half of the N-terminal side of the L chain (each more than 100 amino acids) are called V regions, and are rich in diversity and can bind to antigens. Directly involved. Most of the part other than the V region is called the C region. Antibody molecule is C region IgG by _{homology, I g M, IgA, IgD} , are BunYasushi to each class of IgE.

The IgG class by homology C region, are further classified into subclasses IgGl~I _g G4. The H chain is divided into four immunoglobulin domains, VH, CH1, CH2, and CH3, from the N-terminal side.There is a highly flexible peptide region called the hinge region between CH1 and CH2, which separates CH1 and CH2. Can be Structural unit comprising CH2 and CH ³ after the hinge region is called Fc region, N- glycoside-linked sugar chain is attached. This region is the region where Fc receptors, traps, etc. bind (Immunology Illustrated, Original 5th Edition, published February 10, 2000, Nankodo Edition, Introduction to Antibody Engineering, January 25, 1994) First edition, Citizen Library).

The sugar chains of glycoproteins such as antibodies can be linked to asparagine (N-glycoside-linked sugar chains) and sugar chains linked to serine, threonine, etc. (0-glycosyl-glycoside), depending on the mode of binding to the protein part. ) Are roughly divided into two types. The N-glycoside-linked sugar chain has a basic common core structure represented by the following structural formula (I) [Biochemical Experimental Method 23—Glycoprotein Glycan Research Method (Society Press Center), edited by Reiko Takahashi ( 1989)]. .

In the above structural formula (i), the terminal of the sugar chain binding to asparagine is called a reducing end, and the opposite side is called a non-reducing terminal.

As the N-glycoside-linked sugar chain, any one having a core structure of the above structural formula (I) may be used, but a high mannose type in which only mannose is bonded to the non-reducing end of the core structure, and a non-core structure having a core structure It has one or more parallel branches of galactose-N-acetyldarcosamine (hereinafter referred to as Gal-GlcNAc) at the reducing end, and sialic acid and piecing at the non-reducing end of Gal-GlcNAc. Complex-type (complex type) having a structure such as N-acetyldarcosamine, and a hybrid type having both high-mannose type and complex type branches on the non-reducing end side of the core structure.

Since the Fc region of the antibody molecule has a region in which N-daricoside-linked sugar chains bind one by one, two sugar chains are bound per antibody molecule. Since the N-darcoside-linked sugar chain that binds to the antibody molecule includes any sugar chain containing the core structure represented by the structural formula (I), two N-darcoside-linked sugar chains that bind to the antibody are included. Has many combinations of sugar chains.

Therefore, the antibody composition according to the present invention may be composed of an antibody molecule having a single sugar chain structure or an antibody having a plurality of different sugar chain structures as long as the effects of the present invention can be obtained. It may be composed of molecules.

Antibody molecules include any molecules that contain the Fc region of the antibody. Specific examples include an antibody, an antibody fragment, and a fusion protein containing an Fc region.

Antibodies include hybridoma cells prepared from spleen cells of immunized animals by immunizing animals with the antigen. And an antibody produced by a gene recombination technique, that is, an antibody obtained by introducing an antibody expression vector containing an antibody gene into a host cell. Specific examples include antibodies produced by humans and hybridomas, humanized antibodies, human antibodies, and the like. Hybridomas have a desired antigen specificity, obtained by cell fusion of B cells obtained by immunizing a mammal other than human with an antigen and myeloma cells derived from mice, rats, and the like. A cell that produces a monoclonal antibody.

Examples of the humanized antibody include a human chimeric antibody and a human CDR-grafted antibody.

The human chimeric antibody is composed of a non-human animal H chain V region (hereinafter also referred to as HV or VH) and an antibody L chain V region (hereinafter also referred to as LV or VL) and a human antibody H chain C region (hereinafter referred to as LV or VL). Hereafter, it refers to an antibody consisting of a human antibody L chain C region (hereinafter, also referred to as CL). As a non-human animal, any animal such as a mouse, a rat, a hamster, and a rabbit can be used as long as it can produce a hybridoma.

The human chimeric antibody is obtained by obtaining cDNAs encoding VH and VL from a hybridoma producing a monoclonal antibody, and inserting the cDNAs into host cell expression vectors having genes encoding human antibodies CH and CL, respectively. The expression vector can be produced by constructing a human-type chimeric antibody expression vector and introducing it into a host cell.

As the CH of the human chimeric antibody, a human immunoglobulin (hereinafter referred to as hlg) may be any so long as it belongs to, but it is preferable that the hlgG class, hI _g Gl, hIgG2 belonging more hlgG class, Any of the subclasses hIgG3 and hIgG4 can be used. As the CL of the human chimeric antibody, any CL belonging to hlg may be used, and a κ class or L class can be used. .

The human CDR-grafted antibody refers to an antibody obtained by grafting the amino acid sequences of CDRs of VH and VL of a non-human animal to the human body at appropriate positions of VH and VL of a human antibody.

The human CDR-grafted antibody is constructed by constructing a cDNA encoding a V region obtained by grafting the VH and VL CDR sequences of a non-human animal antibody to the VH and VL CDR sequences of any human antibody, A human CDR-grafted antibody expression vector is constructed by inserting each into a host cell expression vector having a gene encoding the CL of a human antibody, and the expression vector is introduced into a host cell to obtain the human CDR. The transplant antibody can be expressed and manufactured.

The CH of human CDR-grafted antibody may be any so long as it belongs to the hlg but it is preferable that the hlgG class, further hI _g belonging to hlgG class Gl, hIgG2, MgG3, any of subclasses such MgG4 Can be used. The CL of the human CDR-grafted antibody may be any CL as long as it belongs to hlg, and a κ class or え class can be used.

Human antibody originally refers to an antibody that naturally exists in a human body, but human antibody phage libraries and human antibodies produced by recent advances in genetic engineering, cytology, and developmental engineering technologies Also included are antibodies obtained from transgenic non-human animals or human antibody transgenic plants. .

Antibodies present in the human body can be obtained, for example, by isolating human peripheral blood lymphocytes, infecting them with an EB virus, etc., immortalizing them, and cloning the cells to produce the antibody-producing lymphocytes. The antibody can be purified. The human antibody phage library 1 is a library in which antibody fragments such as Fab and single-chain antibodies are expressed on the phage surface by introducing an antibody gene prepared from human B cells into the phage gene. From the library, a phage expressing an antibody fragment having a desired antigen-binding activity can be recovered using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted to a human antibody molecule consisting of two complete L chains and two complete L chains by genetic engineering techniques.

A human antibody transgenic non-human animal refers to an animal in which a human antibody gene has been integrated into cells. Specifically, it is possible to produce a human antibody transgenic non-human animal by introducing a mouse antibody gene from a mouse embryonic stem cell, transplanting the embryonic stem cell into the early embryo of another mouse, and then developing the embryonic stem cell. it can. In addition, a human antibody transgenic non-human animal can be prepared by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. Human antibody transgenic methodA method for producing human antibodies from non-human animals is to obtain human antibody hybridomas by the normal hybridoma production method used in mammals other than humans, and to culture human antibodies in culture. Antibodies can be accumulated.

Transgenic non-human animals include porcupines, sheep, goats, pigs, porcupines, mice, rats, chickens, monkeys, and egrets.

The antibody fragment refers to a fragment containing at least a part of the Fc region of the antibody. The Fc region refers to a region on the C-terminal side of the whole H chain, a CH2 region and a CH3 region. At least a part of the Fc region preferably refers to a fragment containing the CH2 region, more preferably a region containing the first aspartic acid present in the CH2 region. Fc region of IgG class, power bat (Kabat) et al., EU Index [sequencing's O Breakfast, Protein's O Breakfast, Imunorojikaru '· Intaresuto (Sequences of Proteins of Immunological Interest) , 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)] means from the 226th Cys to the C-terminus or the 230th Pro to the C-terminus. Antibody fragments include H chain monomers, H chain dimers, and the like. The Fc region in the present invention includes a natural type and its mutant type.

A fusion protein having a part of the Fc region is a substance obtained by fusing an antibody or a fragment of the antibody containing a part of the Fc region of an antibody with a protein such as an enzyme or a cytokine (hereinafter referred to as an Fc fusion protein). Say.

Of the total N-daricoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition of the present invention, the proportion of sugar chains in which fucose is not linked to N-acetyldarcosamine at the sugar chain reducing end is low. 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, and binds to the Fc region contained in the antibody composition. It is most preferred that all of the N-glycoside-linked complex-type sugar chains to be used are sugar chains in which fucose is not linked to N-acetyltyldarcosamine at the reducing end of the sugar chain.

As described above, of the total N-daricoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not linked to N-acetyldarcosamine at the reducing end of the sugar chain The higher the, the higher the antibody composition has the higher antibody-dependent cytotoxic activity.

The ratio of sugar chains in which fucose is not bound to N-acetyltylcolasamine at the reducing end of sugar chains contained in a composition comprising an antibody molecule having an N-glycoside-linked complex type sugar chain in the Fc region is determined by the ratio of the antibody From the molecule Using known methods such as hydrazinolysis and enzymatic digestion [Biochemical Experimental Method 23—Glycoprotein Glycosylation Research Method (Academic Press Center), edited by Reiko Takahashi (1989)], the sugar chains are released and the released sugar chains are released. Can be determined by fluorescent labeling or isotope labeling, and separating the labeled sugar chains by one of the methods of mouth chromatography. In addition, the released sugar chains can be determined by analysis by the HPAED-PAD method [Journal 'Op' Liquid 'Chromatography (J. Liq. Chromatogr.), 6, 1577 (1983)]. Can be.

In the present invention, high therapeutic effects include, for example, a high level of cytotoxic activity 1 ". Examples of the cytotoxic activity of the antibody composition include antibody-dependent cytotoxic activity (hereinafter abbreviated as ADCC), Nematode-dependent cytotoxicity (monoclonal antibodies), activity to suppress the growth of antigen-expressing cells by binding to antigens, etc. The growth-suppressing activity includes inducing apoptosis and inducing differentiation of target cells. Also include those that promote cell growth [Cancer Research 60, 7170 (2000), Nature's Medicine (Nature Medicine) 1, 644 (1995), Cell Growth Differ. 3, 401 (1992)].

ADCC activity means that in vivo, an antibody bound to a cell surface antigen such as a tumor cell activates an effector cell through the binding of an antibody Fc region to an Fc receptor present on the effector cell surface. , Which refers to the activity of damaging tumor cells, etc. [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Capter 2.1 ( 1995)]. Effector cells include immune cells such as natural killer cells, macrophages, monocytes, dendritic cells, and granulocytes. Fc receptor is Fco; receptor I, Fc _E receptor I,? (: _£ receptor II, FCT receptor) :, Fc _V receptor IIa, Fey receptor IIb, Fcy receptor IIc, FCT / receptor IIIa, Fcy receptor IIIb, Fc receptor n, etc. Among them, Fcy receptor Ilia is mainly expressed on natural killer cells and is one of the important Fc receptors for ADCC activity [monoclonal 'Antibodies: Principles'and' practice ( Monoclonal Antibodies: principles and practice), Thirdiidition, Acad. Press, 1996 (hereinafter abbreviated as "monoclonal 'Antibodies')".

Therefore, in the present invention, the proportion of sugar chains in which fucose is not bound to N-acetylglycosamine at the reducing end of sugar chains is 50% of all complex N-glycoside-linked sugar chains that bind to the Fc region of the antibody molecule. % Of the total N-glycoside-linked complex type glycan that binds to the Fc region of the antibody molecule, indicating that the antibody composition exhibits a higher therapeutic effect than when the antibody composition alone is administered alone. The combination of an antibody composition in which the proportion of sugar chains in which fucose is not bound to N-acetylsyllucosamine is 50% or more and at least one or more drugs results in a case where the antibody composition is administered alone. A drug that exerts a therapeutic effect that is higher than the therapeutic effect that is exhibited.

In addition, the medicament of the present invention is characterized in that, among all N-glycoside-linked complex-type sugar chains that bind to the Fc region of an antibody molecule, a sugar chain in which fucose is not linked to N-acetyltyldarcosamine at the reducing end of the sugar chain. It shows a higher therapeutic effect than a drug comprising a combination of an antibody composition having a ratio of less than 50% and at least one drug.

The medicament of the present invention can have a smaller amount of drug than when the drug is used alone, and can reduce the side effects of concern when a high dose of the drug alone is administered to a patient.

N-glycoside-linked complex type sugar chain 6-position of N-acetyldarcosamine at the reducing end and 1 of fucose Cells that are resistant to lectins that recognize an α-linked sugar chain structure (hereinafter referred to as a 1,6-fucoccin-resistant ^ cells) include antibody compositions such as yeast, animal cells, insect cells, and plant cells. A cell capable of producing a product, which has lectin resistance that recognizes a sugar chain structure in which the 6-position of 還元 -glycidyl-linked glycan-reducing terminal 鎖 -acetyldarcosamine and the 1-position of fucose are α-linked. Any cells can be used.

Specifically, a hybridoma resistant to a lectin that recognizes a sugar chain structure in which the 6-position of の -glycidyl-linked glycan at the reducing end of 複合 -glycidyl-linked glycan and the 1-position of fucose is α-linked Cells, host cells for producing human antibodies and humanized antibodies, transgenic for producing human antibodies, embryonic stem cells and fertilized egg cells for producing non-human animals, transgenic for producing human antibodies Examples include plant callus cells, myeloma cells, and cells derived from transgenic non-human animals for producing plants. Myeloma cells can be used as fusion cells when producing hybridoma cells. Alternatively, a transgenic non-human animal can be immunized with an antigen, and the spleen cells of the animal can be removed and used to produce hybridoma cells.

Lectin I A cell having this resistance is a cell whose growth is not inhibited even when cell culture is performed by giving an effective concentration of lectin to a culture medium.

In the present invention, lectin effective concentration that does not inhibit growth, yo be appropriately determined according to each cell line bur normally _{_{10 μ § / ηι1~10. 0πι ε}} Αι1, preferably 0. 5~2. 0m _g / ml. The effective concentration of lectin when a mutation is introduced into the parent cell is not less than the concentration at which the parent cell cannot grow normally, preferably the same concentration as that at which the parent cell cannot grow normally, more preferably 2%. The concentration refers to a concentration of up to 5 times, more preferably 10 times, and preferably 20 times or more.

The parent cell is a cell before any treatment, that is, a cell before performing the step of selecting a1,6-fucose / lectin-resistant cells used in the present invention, because the above-mentioned enzyme activity is reduced or deleted. Refers to cells before being subjected to genetic engineering treatment.

The parent cell is not particularly limited, but specific examples of the parent cell of various cell lines include the following cells.

Parent cells of NS0 cells are described in the literature such as BIO / TECHNOLOGY, 10, 169 (1992), and Biotechnology 'Bioengineering (Biotechnol. Bioeng.), 73, 261, (2001). NS0 cells. Also, there are ^ 0-9 cells registered with the RIKEN Cell Development Bank (RCB0213), or substrains obtained by adapting these strains to a growth medium. ·

Parent cells of SP2 / 0-Agl4 cells include, for example, Journal of Immunology (J. Immunol '.), 126, 317 (1981), Nature, 276, 269 (1978), and Human Antibody. SP2 / 0-Agl4 cells described in the literature such as Ibodies and Hypridomas (Human Antibodies and Hybridomas), 3, 129 (1992). In addition, SP2 / 0-Agl4 cells registered with the ATCC (ATCC CRL-1581), or a substrain (ATCC CRL-15S1.1.1) obtained by adapting these strains to a medium in which they can grow, may also be mentioned.

As parental cells of CH0 cells derived from chick hamster ovary fibrous tissue, Journal of Experimental Medicine, 108, 945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics, 55, 513 (1968), Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA Natl.Acad.Sci. 60, 1275 (1968), Cell, 6, 121 (1975), Molecular Cell Genetics, Appendix 1, 11 (p883-900). CHO cells and the like. In addition, CHO-K1 strain (ATCC CCL-61), DUXB11 strain (ATCCCRL-9096) and Pro-5 strain (ATCC CRL-1781) registered with ATCC, and commercially available CHO-S strain (Lif etechnologies Cat # ll ⁶ l ^9), or the like sub-cell lines obtained by naturalizing these cell lines to viable medium may be mentioned.

Parent cells of the rat myeloma cell line YB2 / 3HL. P2. GIL 16Ag. 20 cells include cell lines established from Y3 / Agl. 2.3 cells (ATCC CRL-1631). Specific examples thereof include YB2 / 3HL. Ρ2.Gil. 16Ag described in documents such as J. Cell. Biol., 93, 576 (1982) and Methods Enzymol., 73Β1 (1981). 20 cells. Also, there are YB2 / 3HL. P2.Gil. 16Ag. 20 cells (ATCC CRL-1662) registered with the ATCC, or substrains obtained by adapting these strains to a medium capable of growing.

As a lectin that recognizes a sugar chain structure in which the 6-position of N-acetyldarcosamine at the reducing end of the N-glycoside-linked complex type sugar chain and the 1-position of fucose are c-bonded, any lectin that can recognize the sugar chain structure can be used. All lectins are included. Specific examples thereof include lentil lectin LCA (Lenti l Agglutinin from Lens Cul inari s), enduma lectin PSA (Pea Lectin from Pi sum sativum), fava bean lectin VFA (Agglutinin from Vicia f aba), phyllo Chawantakelectin ML (Lectin from Aleuria aurantia) and other products are also being promoted.

In the present invention, al, 6-fucose / lectin-resistant cells may be any cells as long as their growth is not inhibited in the presence of a certain effective concentration of lectin. For example, at least one of the following proteins may be used. A cell whose activity is lower or deleted than that of the parent cell line is exemplified.

(a) Intracellular sugar nucleotide GDP-an enzyme protein involved in the synthesis of fucose (hereinafter referred to as "GDP-fucose synthase");

(b) An enzyme protein involved in glycosylation in which the first position of fucose binds to position 6 of N-acetyldarcosamine at the reducing end of N-glycoside-linked complex-type sugar chain (hereinafter referred to as “hi 6-fucose modifying enzyme”). Notation);

(c) Intracellular sugar nucleotide GDP-a protein involved in the transport of fucose to the Golgi apparatus (hereinafter referred to as "GDP-fucose transport protein").

As a GDP-fucose synthase, T includes any enzyme involved in the synthesis of sugar nucleotide GDP-fucose, which is a source of fucose to sugar chains in the cell. Enzymes that affect synthesis and the like.

GDP-fucose, an intracellular sugar nucleotide, is supplied by the de novo synthetic pathway or the salvage synthetic pathway. Therefore, all enzymes involved in these synthetic pathways are included in GDP-fucose synthase.

GDP-fucose synthase involved in the de novo synthesis pathway includes GDP-mannose 4-dehydratase (GDP-mannose 4-dehydratase; hereinafter referred to as GMD), GDP-keto-6-deoxymannose 3, 5-epimerase , 4-reductase (GDP-keto-deoxymannose 3,5-epimerase, 4-reductase; hereinafter referred to as Fx). GDP-beta-L-fucose pyrophosphorylase (GDP-beta-L-fucose-pyrophosphorylase; hereinafter referred to as GFPP), Fucokinase (fucokinase) and the like are involved in the GDP-fucose synthase involved in the Salvage synthesis pathway. can give. Examples of GMD include GMD having the amino acid sequence represented by SEQ ID NO: 1, 2, or 3.

Examples of the enzyme that affects the synthesis of the intracellular sugar nucleotide GDP-fucose include those that affect the activity of the enzyme involved in the above-described synthesis pathway of the intracellular sugar nucleotide GDP-fucose and the substances that serve as substrates for the enzyme. Enzymes that affect structure are also included.

a 1,6-Fucose-modifying enzyme includes any enzyme that participates in the reaction in which the 6-position of N-glycidyl-linked complex-type sugar chain reducing terminal N-acetyldarcosamine and the 1-position of fucose are α-linked. Included. The enzyme involved in the reaction in which the 6-position of 還元 -glycidyl-linked glycan at the reducing end of 複合 -acetyldarcosamine and the 1-position of fucose are α-linked is the Ν-glycoside-linked glycan at the reducing end of glycan. Any enzyme that affects the reaction in which the 6-position of acetyl gnorecosamine and the 1-position of fucose are α-linked is included. Specific examples include al, 6-fucosyltransferase and Hi-L-fucosidase. The αΐ, 6-fucosyltransferase is represented by SEQ ID NO: 4 or 5: α1,6-fucosyltransferase having a amino acid sequence is exemplified. In addition, the enzyme which affects the reaction in which the 6-position of 還元 -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex type sugar chain and the 1-position of fucose are α-linked includes the Ν-glycoside-linked complex type sugar described above. Enzyme that affects the activity of the enzyme involved in the reaction in which the position 1 of fucose binds to position 6 of fucose at the 6-position of Ν-acetyldarcosamine at the chain reduction terminal, or that affects the structure of the substrate substance of the enzyme Are also included.

The GDP-fucose transport protein includes any protein involved in the transport of the intracellular sugar nucleotide GDP-fucose to the Golgi apparatus, and specifically includes the GDP-fucose transporter.

In addition, proteins that influence the reaction of transporting the intracellular sugar nucleotide GDP-fucose into the gonoresi are also included in the GDP-fucose transport protein, and specifically, the above-mentioned intracellular sugar nucleotide GDP-fucose Golgi ίφ: Proteins that affect the activity of proteins involved in transport to the cell or that affect expression.

As a method for obtaining cells used in the production method of the present invention, any method can be used as long as α1,6-fucose lectin-resistant cells can be selected. Specific examples include the above-described techniques for reducing or deleting the activity of a protein. Techniques for deleting the above-mentioned proteins that reduce their activity include:

(a) gene disruption techniques targeting protein genes;

(b) a method for introducing a dominant negative body of a protein gene;

(c) a technique for introducing a mutation in a protein;

(d) a method for suppressing transcription or translation of a protein gene;

Nadogaa de are ^{^{^{(W00 2/3 11 4 0}}} , 003/085107, W003 / 085118).

In the present invention, as a cell in which a genomic gene of an enzyme involved in sugar chain modification in which fucose is linked to α-position at position 6 of N-acetyldarcosamine at the reducing end of N-glycoside-linked complex type sugar chain is knocked out. Completely inhibits the expression or function of the genomic gene of an enzyme involved in glycosylation, in which fucose binds to position 6 of 還元 -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex type glycan Cells and the like. However, in the present invention, the Itoda vesicle in which the genomic gene has been knocked out has a fucose at position 6 of N-acetylacetylcosamine at the reducing end of the N-glycoside-linked complex type sugar chain using the method described below. Cells in which the genomic gene of an enzyme involved in sugar chain modification in which position 1 is _α- linked are knocked out.

Specific examples of cells in which a genomic gene has been knocked out include cells in which all or part of a target gene has been deleted from the genome. As a method for obtaining such cells, any method can be used, as long as the desired genome can be modified, but a genetic engineering method is preferable. As a specific method, there is a method of reducing or deleting the activity of the above-mentioned enzyme.

In addition, a method for selecting a lectin-resistant cell line that recognizes a sugar chain structure in which the 6-position of 還元 -acetyltilcosamine at the reducing end of Ν-glycoside-linked glycan and the 1-position of fucos, as described above, is used. As a result, cells in which the genomic gene of an enzyme involved in glycosylation, in which fucose is linked to position 6 of の -acetyldarcosamine at the reducing end of Ν-daricoside-linked complex type glycan, are knocked out are selected. can do.

Examples of the antibody composition in the present invention include an antigen expressed on a cell associated with a disease, or an antibody composition against an antigen associated with pathogenesis such as proliferation or metastasis of a cell associated with a disease. Specifically, GD2, GD3, GM2, HER2, CD20, CD22, CD25, CD33, CD52, MAGE, HMl. 24, CC chemokine receptor 4 (CCR4), parathyroid hormone-related protein (PTHrP), basic fibroblast Cell growth factor, fibroblast growth factor 8, basic fibroblast growth factor receptor, fibroblast growth factor 8 receptor, epithelial fibroblast growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCam ), Insulin-like growth factor, insulin-like growth factor receptor, PMSA, vascular endothelial cell growth factor, vascular endothelial cell growth factor receptor, respiratory syncytial virus (RSV), IL-5 receptor ο; chain, etc. Antibody compositions are included.

Specific examples of the above-mentioned antibody composition include anti-GD2 antibody [Anti 'Cancer' Research (Anticancer Res.), 13, 331 (1993)], anti-GD3 antibody [Cancer 'Imology Immunotherapy (Cancer Immunol. Immunother. ), 36, 260 (1993), W001 / 23432, US6437098], anti-GM2 antibody [Cancer Res. (Cancer Res.), 54, 1511 (1994), US6423511], anti-HER2 antibody [Proceedings Op. The National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 89, 4285 (1992)], anti-CD20 antibody [Blood], 83, 435 (1994), W003 / 55993 ], Anti-CD22 antibody [Seminars. Oncol., 30, 253 (2003)], anti-CD25 antibody [Human Antibodies, 10, 127-142 (2001) )], Anti-CD33 antibody [J. Clin. Oncol., 19, 3244 (2001)], anti-CD33 antibody CD52 antibody [Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 89, 4285 (1992)], anti-MAGE antibody [British 'Journal' ob. Cancer (British J. Cancer), 83, 493 (2000)], anti-HMl. 24 antibody [Molecular Immunol., 36, 387 (1999)], anti-CCR4 antibody [W001 / 64754, W003 / 18635, W00O / 42O74, W099 / 15666, US6245332], anti-parathyroid hormone-related protein (PTHrP) antibody [Cancer, 88, 2909 (2000)], anti-fibroblast growth factor 8 antibody [Proceedindus Prob. Natl. Acad. Sci. USA, 86, 9911 (1989), 003/002608], anti-fibroblast growth factor 8 receptor antibody [Journal 'Ob' biology Cal * chemistry (J. Biol. Chem.), 265, 16455 (1990)], anti-epidermal growth factor receptor antibody [Canner. Research (Cancer Res.), 59, 1236 (1999) 3, anti-epithelial activity Cell adhesion molecule antibody

[Proceedings 'Op' The 'National' Academy 'Ob' Science (Proc. Natl. Acad. Sci. USA), 76, 1438 (1979)], Anti-insulin-like growth factor antibody [Journal 'Op' Neuro Science .. Research (J. Neurosci. Res.), 40, 647 (1995), W003 / 93317], Anti-insulin-like growth factor receptor antibody [Journal 'ob' Neuroscience 'Research (J. Neurosci. Res.) ), 40, 647 (1995)], anti-PMSA antibody [J. Urology, 160, 2396 (1998)], anti-vascular endothelial cell growth factor antibody [Cancer Res. , 57, 4593 (1997)], anti-vascular endothelial cell growth factor receptor antibody [Oncogene, 19, 2138 (2000)], anti-RSV antibody [Antiviral Res., 47, 57- 77 (2000)], anti-IL-5 receptor ct chain antibody (US6O 18032, US6538111). Specific antibody names include Herceptin, Rituxan, Campus, Avast in, Bexar, LytnphoCi de, Mylotarg, and Panorex. (Panorex), Zevalin (Nat. Rev. Cancer, 1, 118 (2001)), Zenapax, Simulect, Remicade, Synagis ), ReoPro (R _eo p _ro) [Hiyuman 'ante body's (Human Antibodies), 10, 127-142 (2001)], Enburenore (Enbrel) [expert opinion on Pharmacology Therapy (expert opinion on Pharmacotherapy) , 2, 1137-1148 (2001)], Zolair [Lesviratory Medicine.

(Respiratory Medicine), 97, 123-129 (2003)].

In the present invention, ADCC activity can be measured by an in vitro measurement system such as ⁵¹ Cr release method, lactate dehydrogenase (LDH) release method, flow cytometry, or an in vivo evaluation system using an animal model. .

In the present invention, among all N-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not bound to N-acetyldarcosamine at the sugar chain reducing end, Drugs used in combination with the antibody composition having a ratio of 50% or more include cytokines, proteins such as antibodies, small molecule drugs, biological response modifiers (hereinafter referred to as BRMs), and the like. Cytokines include interleukins (ILs), colony stimulating factors (CSFs), interferons (IFNs), tumor necrosis factors (TFs), chemokines, or combinations thereof. Specifically, IFN-c¾, IFN-β, IFN--y, TF-a, TNF- / 3, IL-1a, IL-1; 3, IL-2, IL-13, IL-4 , IL-6, IL-7, IL-9, IL-12, IL_15, IL_17, IL-18, IL-21, fractalkine, GM-CSF ヽ G-CSF, M-CSF, SCF, or a combination of these can give. Preferably, IL-2, IL-15, IFN-a and IFN-2γ are mentioned.

Low molecular drugs include amifostine (ethyol), cisplatin (ci spl at in), dacanolevadine (DTIC) [dacarbazine (DTIC)], and dactinomycin

(dactinomyc in), mechlorethamine (nitroshen master) [mechlorethamine ^ ni trogen mustard), ■ streptozocin streptozocm), cyclophosphamide, cyclophosphamide, carnotine (BCNU) [carmustine (BCNU)], mouth mucin (CCNU) ) [lomustine (CCNU)], doxorubicin (adriamycin) [doxorubi cin (adriamycin)], doxorubicin lipo (doxorubi cin lipo (doxilj J, gumcitabine Jemcitabine, gemzar)], Daunorubicin (daunorubicin), daunorubicin lipo (daunoxome), daunorubicin lipo (daunoxome), procarinazine (procarbazine), my I, mycin (mitomycin), cytarabine ^ cytarab ine, ethoside etoposide Methotrexate, 5-fluoroenocil, 5-fluorouracil, vinblastine, pinklistin, bleomycin, paclitaxel (taxol), docetaxel (taxotea) ) [docetaxel (taxotere)], anoledesleukin (aldesleukin), asno laginase (asparaginase), busnolephane (busulfan), canolepoplatin (carboplatin), cladribine (cladribine), camptothecin, CPT 10-Hydroxy-7-ethyl-camptothecin (SN3) 8)], floxaridine, fludarabine, hydrourea, hydroxyurea, ifosfamide, idarubicin, mesna, irinotecan (mitoxantrone), topotecan (topotecan), leuprolide (leuprol ide), megestol d-nore (megestrol), menolephalan (melpharan), menole; epupurin (mercaptopurine), plicamycin (plicamycin), mitotane ), Sword snofuga ¹ i pegaspargase ヽヽスタスタ pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent pent ), Thiotepa, ゥ uracil mustard, vinoreno Vinorelbine, chlorambucil, prednisolone, vindesine, nimstine, semsten semustinli, capecitabine, FT, atomizexin (oxaloplatin), gefitinib (Iressa), imatinib (STJ571) [imatinib (STI571)], amsacrine, 11-trans retinoic acid, salidomide thalidomi de), bexarotene (targretin), dexamethasone, dexamethasone, anastrozole (Alimidex), leuplin or a combination thereof. Preferably, pinklistin, cyclophosphamide, etoposide, methotrexate or a combination thereof is used.

Examples of the antibody include the above-mentioned antibodies.

BRMs in the present invention include BCG, anaerobic corynebacterium, bacterial preparations such as muramyl dipeptide, trehalose dimycolic acid, pyran copolymer, MVE, poly I: C, pyrimidine, thymosin, thymulin, thymopoesin, picibanil, fucoidan. , Krestin and the like.

The effect of the medicament of the present invention can be examined, for example, by using an ilim cytotoxicity assay system. As an example of the system for measuring the activity of the itoda cyst of ii i ^, there is a system for measuring ADCC activity. ADCC activity was impaired by contacting antigen-expressing target cells with effector cells such as peripheral blood mononuclear cells, monocytes, macrophages, and granulocytes from humans or other animals in the presence of antibodies. It can be measured by detecting the degree of the target cell and quantifying it. The degree of impaired target cells, ⁵¹ Cr release assay, a method of detecting the enzymatic activity of the target cells can be detected Te detection methods such as [Koyo' by flow cytometer. The immunocompetent cells of the present invention in the ADCC activity measurement system The effect of the substance to be activated or the substance having antitumor activity can be determined by adding these substances to the ADCC activity measurement system or by preliminarily preserving these substances in target cells, effector cells, or both. It can be measured by observing the effect of a period of exposure on ADCC activity.

The effect of the medicament of the present invention can also be examined by measuring iiLJd ^ antitumor activity using an animal model.

As animal models, xenograft models in which cultured cell lines derived from human cancer tissues are transplanted into immunodeficient mice such as nude mice, and syngeneic transplant models in which cultured mouse cancer cell lines are transplanted into wild-type mice having a normal immune system And so on.

Xenograft models can be prepared by implanting human cancer cell lines into various sites such as subcutaneous, intradermal, intraperitoneal, and intravenous sites of immunodeficient mice such as nude mice.

By comparing the effects of the single administration of an antibody, the single administration of a substance that activates immunocompetent cells or a substance having antitumor activity with the above-described animal model, and the effects of the medicament of the present invention, The antitumor effect of the drug can be evaluated.

Although the medicament of the present invention can be administered alone, it is usually mixed with one or more pharmacologically acceptable carriers, and any of the well-known drugs well known in the technical field of pharmaceutics is used. It is desirable to provide it as a pharmaceutical preparation produced by the method.

It is desirable to use the most effective route for treatment, including oral administration or parenteral administration such as buccal, respiratory, rectal, subcutaneous, intramuscular and intravenous administration. In the case of a protein preparation, intravenous administration can be preferably used.

Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. ,

Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, p-hydroxybenzoic acid. It can be produced by using preservatives such as acid esters, flavors such as stomach berry, and flavors such as permint as additives.

Capsules, tablets, powders, granules, etc. are excipients such as lactose, pudose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate, talc, polyvinyl It can be produced using a binder such as alcohol, hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.

Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.

The injection is prepared using a carrier comprising a salt solution, a pudose solution, or a mixture of the above.

Suppositories are prepared using carriers such as lactic acid, hydrogenated fats or carboxylic acids.

Sprays are also prepared using the drug itself or a carrier that does not irritate the oral and respiratory mucosa of the recipient and disperses the drug as fine particles to facilitate absorption.

Specific examples of the carrier include lactose and glycerin. Depending on the nature of the drug and the carrier used Thus, preparations such as aerosols and dry powders are possible. In addition, in these parenteral preparations, the components exemplified as carotenoids in oral preparations can also be added.

Dose or frequency of administration is 0. 1~20m _{_g} / k _g the desired therapeutic effect, administration method, treating period, age, as different forces through adult dose of antibody per amount by weight, or the like. The dose of the drug used in combination with the antibody is the same or lower than that used alone for clinical use. Brief Description of Drawings

FIG. 1 shows the enhancing effect of various cytokines on ADCC activity of KM2760-1 and KM3060. The vertical axis represents cytotoxic activity (%). The medulla shows no cytotoxicity, the mouth shows the addition of IL-2, and the hatched lines show the cytotoxic activity when IL-15 is added.

Figure 2 illustrates the enhancement effects of various cytokines on the ADCC activity of KM ³ 0 ⁶⁵ and Rituxan. The vertical axis represents cytotoxic activity (%). In the figure, the garden shows no cytokine addition, the mouth shows IL-2 addition, and the shaded lines show the Itoda cyst damage activity when IL-15 is added.

Figure 3 shows the enhancing effect of various cytokines on the ADCC activity of KM ³ 0 ⁶⁵ and Rituxan. The vertical axis represents cytotoxic activity (%). In the figure, the country indicates the absence of cytokines, the mouth indicates IFN-addition force [1, and the hatched lines indicate the cytotoxic activity when IFN-7 was added.

The following is a detailed description of the S month. This application claims the priority of Japanese Patent Application No. 2003-406589 filed on December 4, 2003, which includes the content of the description and / or drawings of the patent application. .

Best shape bear for carrying out the invention

Example 1 Combined effect of anti-CCR4 antibody and cytokine on in vitro cytotoxicity

The effect of the combination of cytokines with the CCR4 human chimeric antibodies KM2760-1 and KM3060 (002/31140) on the in vitro activity against intestinal cysts was measured by the following method.

KM2760-1 is an anti-CCR4 chimeric antibody produced from the cell line KM2760-1 # 58-35-16 obtained by introducing an anti-CCR4 chimeric antibody-expressing strain into the rat myeloma cell line YB2 / 0 cells. The KM2760-1 # 58-35-16 strain has lectin resistance, and the content of sugar chains to which fucose does not bind in the total sugar chains of KM2760-1 is 87%. On the other hand, KM306O is an anti-CCR4 chimeric antibody produced from cell line 5-03 obtained by introducing an anti-CCR4 chimeric antibody-expressing strain into the CH0 / DG44 cell line. {Δ} has no lectin resistance, and the content of sugar chains to which fucose is not bound in the total sugar chains of {3060} is 8%. KM2760-1 and ΚΜ3060 have the same amino acid sequence, and have the same antigen binding activity.

(a) Preparation of effector-cell solution

A healthy person's venous rainbow (50 mL) was collected, and heparin sodium (Shimizu Pharmaceutical Co., Ltd.) (0.5 mL) was added and mixed gently. This was centrifuged (400 g, 20 minutes) using a MONO-POLY separation solution (manufactured by Dainippon Pharmaceutical Co., Ltd.) according to the instruction manual to separate a mononuclear cell layer. After washing by centrifugation three times with RPMI1640-FCS (5) medium, the cells were resuspended in the same medium at a concentration of 3 × 10 ⁶ cells / mL to obtain an effector cell solution.

(b) Stimulation by effector cells

The fefecta single cell solution obtained in the above (a) was dispensed at 50 / i L into a 96-well U-bottom plate (Falcon). Further RPMIIMO - FCS diluted following solutions (5) was added in 50 / L, and allowed to stand 5% C0 in ₂ Inki Yubeta ί trowel 3 days. (1) 1¾¾11640—Fuji 5 (5) only (control with no added site force)

(2) IL-2 (protech) 2ng / mL

(3) IL-15 (protech) 2ng / mL

(c) Preparation of target cell solution

G418 (manufactured by Nacalai Tesque) containing at ^{0. 5 mg / mL RPMIIMO- FCS (} 10) medium (manufactured by RPMI1640 medium (GIBCO BRL, Inc. containing 10% FCS)) and CCR4 / EL4 cells (CCR4 gene cultured in the introduction Mouse thymoma cells, W001 / 64754) were washed with RPMIIMO-FCS (5) medium (RPI1640 medium containing 5% FCS (GIBCO BRL)) by centrifugation and suspension, and then RPMI1640-FCS (5 ) 2 x 10 ⁵ cells AnL was prepared with the medium and used as the target cell solution.

(d) Measurement of cytotoxic activity

To each well of the 96-well U-bottom plate prepared in (b), 50 / x L (1 × 10 ⁴ cells / well) of the target cell solution prepared in (c) was dispensed. At this time, the ratio of effector cells to target cells is 15: 1. Further, KM2760-1 or KM3060 was added to a final concentration of 1 ng / mL or 100 ng / mL to make the total volume 200 / zL, and the reaction was carried out at 37 ° C for 4 hours. After the reaction, the plate is centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant is measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to the attached instructions. Was measured. Absorbance data for spontaneous release of target cells uses only medium instead of effector cell solution and antibody solution, and absorbance data for spontaneous release of effector cells uses only medium instead of target cell solution and antibody solution Were obtained by performing the same operation as above. For the absorbance data of total release of target cells, use the medium instead of the antibody solution and effector cell solution, add 9% Triton X-100 solution 45 minutes before the completion of the reaction, and perform the same operation as above. It was determined by measuring the LDH activity. ADCC activity was determined by the following equation. (Number 1)

Cytotoxic activity (%) = [(Absorbance of specimen) spontaneous release of effector cells 1 (Absorbance of spontaneous release of target cells and cells)] / [(Absorbance of total release of target cells) 1 (Spontaneous release of target cells) Absorbance)] X 100

Figure 1 shows the results. ADCC activity of KM2760-1 without cytokine was higher than that of KM3060, but was further enhanced by cytokine addition. This result indicates that, among all N-daricoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, of the sugar chains in which fucose is not bound to N-acetyldarcosamine at the reducing end of the sugar chain. The high ADCC activity of the antibody composition with a proportion of 50% or more indicates that the cytokine further increases.

Example 2 Combined effect of anti-CD20 antibody and cytokine on iiL ^ iii ^ cytotoxicity

The effect of the combination of the cytokine CD20 human chimeric pile KM3065 (W003 / 55993) and the commercially available anti-CD20 human chimeric antibody Rituxan (Genentech) and cytokine on in vitro cytotoxic activity was described in Example 1. Was measured according to the method described above. .

Cytokines are IL-2, IL-15 (all manufactured by Peprotech, final concentration Ing / mL), IFN-α (Peprotech, final concentration lOOng / mL), IFN-γ (R & D Systems, final concentration lOOng) / mL) was used. KM ³ 0 ⁶⁵ is an anti-CD20 chimeric antibody produced from the cell line KM3065 obtained by introducing the anti-CD20 chimeric antibody expression strain rat myeloma cell line YB2 / 0 cells. KM3065 strain has lectin resistance, KM3065 The content of sugar chains to which fucose is not bound in the total sugar chains is 96%. On the other hand, the content of sugar chains that do not bind to fucose in all sugar chains of Rituxan is 6%. KM3065 and Rituxan have the same amino acid sequence, and have the same antigen binding activity.

Figures 2 and 3 show the results. The ADCC activity of KM3065 without cytokine was higher than that of Rituxan, but was further enhanced by the addition of cytokine. This result indicates that, among all N-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, sugar chains in which fucose is not linked to N-acetyldarcosamine at the sugar chain reducing end The high ADCC activity of the antibody composition having a ratio of 50% or more indicates that the cytokine further increases.

Industrial applicability

Of all N-daricoside-linked complex type sugar chains that bind to the Fc region contained in the antibody composition, the ratio of sugar chains in which fucose is not linked to N-acetyldarcosamine at the reducing end of the sugar chain is 50%. A medicament using the antibody composition and at least one drug is expected to have a high therapeutic effect.

Claims

The scope of the claims

I. Of the total N-glycoside-linked complex type sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not linked to N-acetyldarcosamine at the reducing end of the sugar chain is A medicament comprising a combination of at least 50% of an antibody composition and at least one drug.

2. Among the total N-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not linked to N-acetyldarcosamine at the reducing end of the sugar chain is low. A medicament for administering a combination of at least 50% of an antibody composition and at least one drug.

3. Of the total N-glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not linked to N-acetyldarcosamine at the reducing end of the sugar chain is low. A medicament for simultaneous or sequential administration of 50% or more of the antibody composition and at least one drug.

4. Among all N-glycoside-linked complex type sugar chains that bind to the Fc region contained in the antibody composition, the ratio of sugar chains in which fucose is not linked to N-acetylglucosamine at the reducing end of the sugar chain is 50%. %. The medicament according to any one of claims 1 to 3, wherein the medicament exhibits a higher therapeutic effect than when the antibody composition is administered alone at a ratio of not less than 1%.

5. The medicament according to claim 4, wherein exhibiting a high therapeutic effect is exhibiting a high antibody-dependent cytotoxic activity.

6. The antibody composition is produced from a cell resistant to a lectin that recognizes a sugar chain structure in which the 6-position of N-acetyldarcosamine at the reducing end of N-daricoside-linked complex type sugar chain and the 1-position of fucose are _α- linked. The medicament according to any one of claims 1 to 5, which is an antibody composition to be used.

7 .. Among all Ν-glycoside-linked complex glycosides in which the antibody composition binds to the Fc region contained in the antibody composition, fucose is bound to Ν-acetylacetylchocosamine at the reducing end of the sugar chain. Claims: !! is an antibody composition that has no sugar chains. 7. The medicament according to any one of claims 6 to 6.

8. The sugar chain to which fucose is not bonded is a sugar chain in which the 1-position of the fucose is not α-linked to the 6-position of Ν-glycidol-linked complex type sugar chain at the reduced end of チル -acetyldarcosamine. The medicament according to any one of claims 1 to 7.

9. The antibody composition knocked out the gesom gene of an enzyme involved in glycosylation in which the 1-position of fucose is α-linked to the 6-position of 還元 -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex-type sugar chain 9. The medicament according to claim 7, which is an antibody composition produced from cells.

10. Α-, 6-fucosyltransferase is an enzyme involved in glycosylation in which α-linked fucose at position 6 of 還元 -acetyldarcosamine at the reducing end of Ν-glycoside-linked complex type glycan The medicament according to claim 9.

I I. The medicament according to any one of claims 1 to 10, wherein the drug is a substance selected from the group consisting of a protein, a small molecule drug, and a biological response modifier.

12. The medicament according to claim 11, wherein the protein is a cytokine or an antibody.

13. The medicament according to claim 12, wherein the cytokine is a cytokine selected from IFN-γ, IL-2 and IL-15.

1.4. The medicament according to any one of claims 1 to 13, wherein the medicament is a therapeutic agent for a disease involving a tumor.