JPWO2005053742A1 - Medicament containing antibody composition - Google Patents

Abstract

In the present invention, among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50%. Provided is a medicament comprising a combination of the above antibody composition and at least one kind of drug.

Description

  In the present invention, among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50%. The present invention relates to a pharmaceutical comprising a combination of the above antibody composition and at least one kind of drug.

上記構造式（Ｉ）において、ＧｌｃＮＡｃはＮ−アセチルグルコサミン、Ｍａｎはマンノースを示す。またアスパラギンと結合する糖鎖の末端を還元末端、反対側を非還元末端という。
抗体分子は重鎖と軽鎖が２分子ずつ会合した４量体として構成される。ＩｇＧ１型ヒト抗体は、重鎖のＦｃ領域中に存在するＮ末端から２９７番目のアスパラギンに上記のコア構造を有する糖鎖が結合している。このコア構造にさらに非還元末端側にＮ−アセチルグルコサミン、ガラクトース、シアル酸、還元末端のＮ−アセチルグルコサミンの６位にフコースがそれぞれ付加する可能性があるが、通常これらの成分は均一ではなく、抗体を生産する細胞によって変化する［トレンズ・イン・バイオテクノロジー（Ｔｒｅｎｄｓ Ｂｉｏｔｅｃｈｎｏｌ．），１５，２６−３２（ｌ９９７）］。ヒトＩｇＧ１のＡＤＣＣ活性は、この糖鎖中のガラクトース［ヒューマン・アンティボディズ・アンド・ハイブリドーマズ（Ｈｕｍ Ａｎｔｉｂｏｄｉｅｓ Ｈｙｂｒｉｄｏｍａｓ），５，１４３−１５１（１９９４）、ヒューマン・アンティボディズ・アンド・ハイブリドーマズ（Ｈｕｍ Ａｎｔｉｂｏｄｉｅｓ Ｈｙｂｒｉｄｏｍａｓ），６，８２−８８（１９９５）］、Ｎ−アセチルグルコサミン［ネイチャー・バイオテクノロジー（Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．），１７，１７６−１８０，（１９９９）、バイオテクノロジー・アンド・バイオエンジニアリング（Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｅｎｇ．），７４，２８８−２９４，（２００１）］、フコース［ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．），２７７，２６７３３−２６７４０（２００２）、ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．），２７８，３４６６−３４７３（２００３）］の含有量に影響を受けることが報告されている。中でも最も影響が大きいのはフコースであり、還元末端のＮ−アセチルグルコサミンの６位にフコースが付加した糖鎖の割合が少ないヒトＩｇＧ１型の抗体はＡＤＣＣ活性を顕著に増強する［ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．），２７７，２６７３３−２６７４０（２００２）、ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．），２７８，３４６６−３４７３（２００３）］。しかしこれらの報告はいずれも抗体単独でのＡＤＣＣ活性であり、他の薬剤によってフコースの量が少ないヒトＩｇＧ１型の抗体のＡＤＣＣ活性がさらに増強するかどうかは知られていない。
一方ＡＤＣＣ活性はエフェクター細胞を刺激するサイトカインによって上昇することが知られている。例えばインターロイキン（以下ＩＬ）−２［キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），４６，２１３（１９９８）、キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），５１，１７１（２００２）、ブラッド（Ｂｌｏｏｄ），９３，３９２２（１９９９）、ジャパニーズ・ジャーナル・オブ・キャンサー・リサーチ（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ．），８７，４９７（１９９６）］、ＩＬ−１２［ブラッド（Ｂｌｏｏｄ），９３，３９２２（１９９９）、ジャパニーズ・ジャーナル・オブ・キャンサー・リサーチ（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ．），８７，４９７（１９９６）］、ＩＬ−１５［ブラッド（Ｂｌｏｏｄ），９３，３９２２（１９９９）、ジャーナル・オブ・エクスペリメンタル・メディスン（Ｊ．Ｅｘｐ．Ｍｅｄ），１８０，１３９５（１９９４）］、インターフェロン（以下ＩＦＮ）−α［キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），４６，２１３（１９９８）、キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），５１，１７１（２００２）］、ＩＦＮ−γ［キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），４６，２１３（１９９８）、キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），５１，１７１（２００２）、ジャパニーズ・ジャーナル・オブ・キャンサー・リサーチ（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ．），８７，４９７（１９９６）］、マクロファージ−コロニー刺激因子（以下Ｍ−ＣＳＦ）［キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），４６，２１３（１９９８）、ブラッド（Ｂｌｏｏｄ），９３，３９２２（１９９９）、ジャパニーズ・ジャーナル・オブ・キャンサー・リサーチ（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ．），８７，４９７（１９９６）、ジャパニーズ・ジャーナル・オブ・キャンサー・リサーチ（Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ Ｒｅｓ．），８１，７９（１９９０）］等、様々なサイトカインをエフェクター細胞にｉｎ ｖｉｔｒｏで加えて刺激することにより、ＡＤＣＣ活性を上昇させることが知られている。
化学療法剤のような低分子の薬剤がｉｎ ｖｉｔｒｏのＡＤＣＣ活性を増強することは知られていないが、化学療法と抗体との併用により優れた治療効果が得られることが知られている。抗ＨＥＲ２／ｎｅｕヒト化抗体ｒｈｕＭＡｂ ＨＥＲ２（Ｈｅｒｃｅｐｔｉｎ、Ｒｏｃｈｅ社）はｔａｘａｎｅ系抗癌剤との併用療法により乳癌に対して顕著な効果を示すことが知られている［Ｃｌｉｎｉｃａｌ Ｔｈｅｒａｐｅｕｔｉｃｓ，２１，３０９（１９９９）］。また、抗ＣＤ２０ヒト型キメラ抗体ＩＤＥＣ−Ｃ２Ｂ８（Ｒｉｔｕｘａｎ、ＩＤＥＣ社）は多剤療法との併用療法によりＢ細胞リンパ腫に対して顕著な効果を示すことが知られている［Ｊ．Ｃｌｉｎ．Ｏｎｃｏｌ．，１７，２６８（１９９９）］。Antibody-dependent cytotoxicity (hereinafter abbreviated as ADCC activity) is that the Fc region of an antibody binds to Fcγ receptors expressed in cell populations called effector cells such as NK cells, monocytes / macrophages, and granulocytes. It happens. NK cells induce strong ADCC activity through FcγRIIIa, a type of Fcγ receptor on cells. The FcγRIIIa gene has a functional gene polymorphism at the 158th amino acid, and FcγRIIIa whose amino acid at 158 is Valine (hereinafter referred to as Val) induces stronger ADCC activity than that of Phenylalanine (hereinafter referred to as Phe). [Journal of Clinical Investigation (J. Clin. Invest.), 100, 1059-1070 (1997)]. Recently, it has been shown that patients with Val / Val genotypes have significantly higher clinical effects of Rituxan than patients with other genotypes [blood, 99, 754-758 (2002), Earth Lytis and Rheumatism (Arthritis Reum.), 48, 455-459 (2003)], it has been suggested that ADCC activity plays an important role in the clinical effects of antibody drugs of the human IgG1 subclass.
The sugar chains of glycoproteins such as antibodies are linked to asparagine (N-glycoside-linked sugar chains) and sugar chains (O-glycosyl-linked sugar chains) that bind to serine, threonine, etc., depending on the mode of binding to the protein moiety There are two main types. N-glycoside-linked sugar chains have a basic common core structure shown in the following structural formula (I) [Biochemical Experimental Method 23-Glycoprotein Sugar Chain Research Method (Academic Publishing Center) Etsuko Takahashi (1989) ]].

In the structural formula (I), GlcNAc represents N-acetylglucosamine, and Man 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.
An antibody molecule is configured as a tetramer in which two heavy chains and two light chains are associated. In the IgG1-type human antibody, a sugar chain having the above core structure is bound to the 297th asparagine from the N-terminus present in the Fc region of the heavy chain. In addition to this core structure, fucose may be added to the 6-position of N-acetylglucosamine, galactose, sialic acid, and N-acetylglucosamine at the reducing end on the non-reducing end side, but these components are usually not uniform. , Depending on the cell producing the antibody [Trends Biotechnol., 15, 26-32 (l997)]. The ADCC activity of human IgG1 is determined by galactose [Human Antibodies Hybridomas, 5, 143-151 (1994), Human Antibodies and Hybridomas ( Hum Antibodies Hybridomas, 6, 82-88 (1995)], N-acetylglucosamine [Nature. Biotechnol., 17, 176-180, (1999), Biotechnology and Bioengineering (Biotechnol). Bioeng.), 74, 288-294, (2001)], fucose [Journal of Biological Chemistry (J. Biol. Ch. em.), 277, 26733-26740 (2002), Journal of Biological Chemistry (J. Biol. Chem.), 278, 3466-3473 (2003)]. ing. Of these, fucose has the greatest effect, and a human IgG1-type antibody with a small proportion of sugar chain with fucose added to the 6-position of N-acetylglucosamine at the reducing end significantly enhances ADCC activity [Journal of Biological chemistry (J. Biol. Chem.), 277, 26733-26740 (2002), Journal of biological chemistry (J. Biol. Chem.), 278, 3466-3473 (2003)]. However, these reports are all ADCC activity of the antibody alone, and it is not known whether the ADCC activity of the human IgG1-type antibody with a small amount of fucose is further enhanced by other drugs.
On the other hand, ADCC activity is known to be increased by cytokines that stimulate effector cells. For example, interleukin (hereinafter IL) -2 [Cancer Immunol. Immunother., 46, 213 (1998), Cancer Immunol. Immunother., 51, 171 (2002). ), Blood, 93, 3922 (1999), Japanese Journal of Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996)], IL-12 [Blood, 93, 3922 (1999), Japanese Journal of Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996)], IL-15 [Blo od), 93, 3922 (1999), Journal of Experimental Medicine (J. Exp. Med), 180, 1395 (1994)], interferon (hereinafter, IFN) -α [Cancer Immunology Immunotherapy ( Cancer Immunol. (Immunother.), 46, 213 (1998), Cancer Immunol. Immunotherapy (Cancer Immunol. Immunother.), 51, 171 (2002)], IFN-γ [Cancer Immunol. (Cancer Immunol.). Immunother.), 46, 213 (1998), Cancer Immunol. Immunother., 51, 171 2002), Japanese Journal of Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996)], macrophage-colony stimulating factor (M-CSF) [Cancer Immunology Immunotherapy (Cancer). Immunol. Immunother.), 46, 213 (1998), Blood, 93, 3922 (1999), Japanese Journal of Cancer Research (Jpn. J. Cancer Res.), 87, 497 (1996). , Japanese journal of Cancer research (Jpn.J.Cancer Res.), 81,79 ( 1990)] , etc., to stimulate added in in vitro a variety of cytokines in effector cells More, it is known to increase the ADCC activity.
Although it is not known that a low molecular weight drug such as a chemotherapeutic agent enhances in vitro ADCC activity, it is known that an excellent therapeutic effect can be obtained by the combined use of chemotherapy and an antibody. The anti-HER2 / neu humanized antibody rhuMAb HER2 (Herceptin, Roche) is known to show a remarkable effect on breast cancer by combination therapy with a taxane anticancer agent [Clinical Therapeutics, 21, 309 (1999)]. . Further, it is known that the anti-CD20 human chimeric antibody IDEC-C2B8 (Rituxan, IDEC) has a remarkable effect on B cell lymphoma by combination therapy with multidrug therapy [J. Clin. Oncol. , 17, 268 (1999)].

上記構造式（Ｉ）において、アスパラギンと結合する糖鎖の末端を還元末端、反対側を非還元末端という。
Ｎ−グリコシド結合糖鎖としては、上記構造式（Ｉ）のコア構造を有するものであればいかなるものでもよいが、コア構造の非還元末端にマンノースのみが結合するハイマンノース型、コア構造の非還元末端側にガラクトース−Ｎ−アセチルグルコサミン（以下、Ｇａｌ−ＧｌｃＮＡｃと表記する）の枝を並行して１ないしは複数本有し、更にＧａｌ−ＧｌｃＮＡｃの非還元末端側にシアル酸、バイセクティングのＮ−アセチルグルコサミンなどの構造を有するコンプレックス型（複合型）、コア構造の非還元末端側にハイマンノース型とコンプレックス型の両方の枝を持つハイブリッド型などがあげられる。
抗体分子のＦｃ領域には、Ｎ−グリコシド結合糖鎖が１カ所ずつ結合する領域を有しているので、抗体１分子あたり２本の糖鎖が結合している。抗体分子に結合するＮ−グルコシド結合糖鎖としては、前記構造式（Ｉ）で示されるコア構造を含むいかなる糖鎖も包含されるので、抗体に結合する２本のＮ−グルコシド結合糖鎖には多数の糖鎖の組み合わせが存在することになる。
したがって、本発明における抗体組成物は、本発明の効果が得られる範囲であれば、単一の糖鎖構造を有する抗体分子から構成されていてもよいし、複数の異なる糖鎖構造を有する抗体分子から構成されていてもよい。
抗体分子としては、抗体のＦｃ領域を含む分子であればいかなる分子も包含される。具体的には、抗体、抗体の断片、Ｆｃ領域を含む融合蛋白質などがあげられる。
抗体としては、動物に抗原を免疫し、免疫動物の脾臓細胞より作製したハイブリドーマ細胞が分泌する抗体、遺伝子組換え技術により作製された抗体、すなわち、抗体遺伝子を挿入した抗体発現ベクターを、宿主細胞へ導入することにより取得された抗体などがあげられる。具体的には、ハイブリドーマが生産する抗体、ヒト化抗体、ヒト抗体などをあげることができる。
ハイブリドーマは、ヒト以外の哺乳動物に抗原を免疫して取得されたＢ細胞と、マウス、ラット等に由来するミエローマ細胞とを細胞融合させて得られる、所望の抗原特異性を有したモノクローナル抗体を生産する細胞をいう。
ヒト化抗体としては、ヒト型キメラ抗体、ヒト型ＣＤＲ移植抗体などがあげられる。
ヒト型キメラ抗体は、ヒト以外の動物の抗体Ｈ鎖Ｖ領域（以下、ＨＶまたはＶＨとも称す）および抗体Ｌ鎖Ｖ領域（以下、ＬＶまたはＶＬとも称す）とヒト抗体のＨ鎖Ｃ領域（以下、ＣＨとも称す）およびヒト抗体のＬ鎖Ｃ領域（以下、ＣＬとも称す）とからなる抗体をいう。ヒト以外の動物としては、マウス、ラット、ハムスター、ラビット等、ハイブリドーマを作製することが可能であれば、いかなるものも用いることができる。
ヒト型キメラ抗体は、モノクローナル抗体を生産するハイブリドーマより、ＶＨおよびＶＬをコードするｃＤＮＡを取得し、ヒト抗体ＣＨおよびヒト抗体ＣＬをコードする遺伝子を有する宿主細胞用発現ベクターにそれぞれ挿入してヒト型キメラ抗体発現ベクターを構築し、宿主細胞へ導入することにより発現させ、製造することができる。
ヒト型キメラ抗体のＣＨとしては、ヒトイムノグロブリン（以下、ｈＩｇと表記する）に属すればいかなるものでもよいが、ｈＩｇＧクラスのものが好適であり、更にｈＩｇＧクラスに属するｈＩｇＧ１、ｈＩｇＧ２、ｈＩｇＧ３、ｈＩｇＧ４といったサブクラスのいずれも用いることができる。また、ヒト型キメラ抗体のＣＬとしては、ｈＩｇに属すればいかなるものでもよく、κクラスあるいはλクラスのものを用いることができる。
ヒト型ＣＤＲ移植抗体は、ヒト以外の動物の抗体のＶＨおよびＶＬのＣＤＲのアミノ酸配列をヒト抗体のＶＨおよびＶＬの適切な位置に移植した抗体をいう。
ヒト型ＣＤＲ移植抗体は、ヒト以外の動物の抗体のＶＨおよびＶＬのＣＤＲ配列を任意のヒト抗体のＶＨおよびＶＬのＣＤＲ配列に移植したＶ領域をコードするｃＤＮＡを構築し、ヒト抗体のＣＨおよびヒト抗体のＣＬをコードする遺伝子を有する宿主細胞用発現ベクターにそれぞれ挿入してヒト型ＣＤＲ移植抗体発現ベクターを構築し、該発現ベクターを宿主細胞へ導入することによりヒト型ＣＤＲ移植抗体を発現させ、製造することができる。
ヒト型ＣＤＲ移植抗体のＣＨとしては、ｈＩｇに属すればいかなるものでもよいが、ｈＩｇＧクラスのものが好適であり、更にｈＩｇＧクラスに属するｈＩｇＧ１、ｈＩｇＧ２、ｈＩｇＧ３、ｈＩｇＧ４といったサブクラスのいずれも用いることができる。また、ヒト型ＣＤＲ移植抗体のＣＬとしては、ｈＩｇに属すればいかなるものでもよく、κクラスあるいはλクラスのものを用いることができる。
ヒト抗体は、元来、ヒト体内に天然に存在する抗体をいうが、最近の遺伝子工学的、細胞工学的、発生工学的な技術の進歩により作製されたヒト抗体ファージライブラリーならびにヒト抗体トランスジェニック非ヒト動物あるいはヒト抗体トランスジェニック植物から得られる抗体等も含まれる。
ヒト体内に存在する抗体は、例えば、ヒト末梢血リンパ球を単離し、ＥＢウイルス等を感染させ不死化、クローニングすることにより、該抗体を生産するリンパ球を培養でき、培養物中より該抗体を精製することができる。
ヒト抗体ファージライブラリーは、ヒトＢ細胞から調製した抗体遺伝子をファージ遺伝子に挿入することによりＦａｂ、一本鎖抗体等の抗体断片をファージ表面に発現させたライブラリーである。該ライブラリーより、抗原を固定化した基質に対する結合活性を指標として所望の抗原結合活性を有する抗体断片を発現しているファージを回収することができる。該抗体断片は、更に遺伝子工学的手法により、２本の完全なＨ鎖および２本の完全なＬ鎖からなるヒト抗体分子へも変換することができる。
ヒト抗体トランスジェニック非ヒト動物は、ヒト抗体遺伝子が細胞内に組込まれた動物をいう。具体的には、マウス胚性幹細胞へヒト抗体遺伝子を導入し、該胚性幹細胞を他のマウスの初期胚へ移植後、発生させることによりヒト抗体トランスジェニック非ヒト動物を作製することができる。また、動物の受精卵にヒト抗体遺伝子を導入し、該受精卵を発生させることにヒト抗体トランスジェニック非ヒト動物を作製することもできる。ヒト抗体トランスジェニック非ヒト動物からのヒト抗体の作製方法は、通常のヒト以外の哺乳動物で行われているハイブリドーマ作製方法によりヒト抗体ハイブリドーマを得、培養することで培養物中にヒト抗体を蓄積させることができる。
トランスジェニック非ヒト動物としては、ウシ、ヒツジ、ヤギ、ブタ、ウマ、マウス、ラット、ニワトリ、サルまたはウサギ等があげられる。
抗体の断片は、上記抗体の少なくともＦｃ領域の一部を含んだ断片をいう。Ｆｃ領域とは、抗体のＨ鎖のＣ末端側の領域、ＣＨ２領域およびＣＨ３領域をいう。少なくともＦｃ領域の一部とは、好ましくはＣＨ２領域を含む断片、より好ましくはＣＨ２領域内に存在する１番目のアスパラギン酸を含む領域をいう。ＩｇＧクラスのＦｃ領域は、カバット（Ｋａｂａｔ）らのＥＵ Ｉｎｄｅｘ［シーケンシズ・オブ・プロテインズ・オブ・イムノロジカル・インタレスト（Ｓｅｑｕｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ ｏｆ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ），５^ｔｈ Ｅｄ．Ｐｕｂｌｉｃ Ｈｅａｌｔｈ Ｓｅｒｖｉｃｅ，Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ，Ｂｅｔｈｅｓｄａ，ＭＤ．（１９９１）］のナンバリングで２２６番目のＣｙｓからＣ末端、あるいは２３０番目のＰｒｏからＣ末端までをいう。抗体の断片としては、Ｈ鎖の単量体、Ｈ鎖の２量体などがあげられる。本発明におけるＦｃ領域としては、天然型およびその変異型を包含する。
Ｆｃ領域の一部を有する融合蛋白質とは、抗体のＦｃ領域の一部を含んだ抗体あるいは抗体の断片と、酵素、サイトカインなどの蛋白質とを融合させた物質（以下、Ｆｃ融合蛋白質と称す）をいう。
本発明の抗体組成物中に含まれるＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％以上、好ましくは６０％以上、より好ましくは７０％以上、さらに好ましくは８０％以上、特に好ましくは９０％以上であり、抗体組成物中に含まれるＦｃ領域に結合する全てのＮ−グリコシド結合複合型糖鎖が、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖であることが最も好ましい。
以上のように抗体組成物中に含まれるＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が高ければ高いほど、抗体組成物は、高い抗体依存性細胞障害活性を有する。
Ｎ−グリコシド結合複合型糖鎖をＦｃ領域に有する抗体分子からなる組成物中に含まれる、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合は、抗体分子からヒドラジン分解や酵素消化などの公知の方法［生物化学実験法２３−糖タンパク質糖鎖研究法（学会出版センター）高橋禮子編（１９８９）］を用い、糖鎖を遊離させ、遊離させた糖鎖を蛍光標識又は同位元素標識し、標識した糖鎖をクロマトグラフィー法にて分離することによって決定することができる。また、遊離させた糖鎖をＨＰＡＥＤ−ＰＡＤ法［ジャーナル・オブ・リキッド・クロマトグラフィー（Ｊ．Ｌｉｑ．Ｃｈｒｏｍａｔｏｇｒ．），６，１５７７（１９８３）］によって分析することによっても決定することができる。
本発明において、高い治療効果としては、例えば細胞障害活性が高いこと等があげられる。
抗体組成物の有する細胞障害活性としては抗体依存性細胞障害活性（以下、ＡＤＣＣと略記する）、補体依存性細胞障害活性（モノクローナル・アンティボディズ）、抗原に結合することによる抗原発現細胞の増殖抑制活性などがあげられる。増殖抑制活性には標的細胞のアポトーシス誘導や分化誘導を促進するものも包含される［キャンサー・リサーチ（Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ）６０，７１７０（２０００）、ネイチャー・メディスン（Ｎａｔｕｒｅ Ｍｅｄｉｃｉｎｅ）１，６４４（１９９５）、セル・グロース・ディファレンシエーション（Ｃｅｌｌ Ｇｒｏｗｔｈ Ｄｉｆｆｅｒ．）３，４０１（１９９２）］。
ＡＤＣＣ活性とは、生体内で、腫瘍細胞等の細胞表面抗原などに結合した抗体が、抗体Ｆｃ領域とエフェクター細胞表面上に存在するＦｃ受容体との結合を介してエフェクター細胞を活性化し、腫瘍細胞等を障害する活性をいう［モノクローナル・アンティボディズ：プリンシプルズ・アンド・アプリケーションズ（Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｉｅｓ：Ｐｒｉｎｃｉｐｌｅｓ ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ），Ｗｉｌｅｙ−Ｌｉｓｓ，Ｉｎｃ．，Ｃａｐｔｅｒ２．１（１９９５）］。エフェクター細胞としては、ナチュラルキラー細胞、マクロファージ、単球、樹状細胞、顆粒球等の免疫細胞があげられる。またＦｃ受容体はＦｃα受容体Ｉ、Ｆｃε受容体Ｉ、Ｆｃε受容体ＩＩ、Ｆｃγ受容体Ｉ、Ｆｃγ受容体ＩＩａ、Ｆｃγ受容体ＩＩｂ、Ｆｃγ受容体ＩＩｃ、Ｆｃγ受容体ＩＩＩａ、Ｆｃγ受容体ＩＩＩｂ、Ｆｃ受容体ｎ等のタイプに分かれる。この内Ｆｃγ受容体ＩＩＩａは主にナチュラルキラー細胞上に発現し、ＡＤＣＣ活性に重要なＦｃ受容体の一つである［モノクローナル・アンティボディズ：プリンシプルズ・アンド・プラクティス（Ｍｏｎｏｃｌｏｎａｌ Ａｎｔｉｂｏｄｉｅｓ：ｐｒｉｎｃｉｐｌｅｓ ａｎｄ ｐｒａｃｔｉｃｅ），ＴｈｉｒｄＥｄｉｔｉｏｎ，Ａｃａｄ．Ｐｒｅｓｓ，１９９６（以下、モノクローナル・アンティボディズと略す）］。
したがって、本発明において、抗体分子のＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％以上である抗体組成物を単独で投与した場合よりも、高い治療効果を示すとは、抗体分子のＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％以上である抗体組成物と少なくとも１種類以上の薬剤とを組み合わせることにより、該抗体組成物単独で投与した場合に発揮される治療効果よりも高い治療効果を発揮する医薬をいう。
また、本発明の医薬は、抗体分子のＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％未満である抗体組成物と少なくとも１種類の薬剤とを組み合わせてなる医薬よりも、高い治療効果を示す。
本発明の医薬は、薬剤を単独で使用する場合よりも少ない薬剤の量にすることができ、薬剤を単独で高用量を患者に投与した場合に懸念された副作用を低減させることができる。
Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合した糖鎖構造を認識するレクチンに耐性を有する細胞（以下、α１，６−フコース／レクチン耐性細胞と称す）としては、酵母、動物細胞、昆虫細胞、植物細胞など抗体組成物を製造することができる細胞であって、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合した糖鎖構造を認識するレクチン耐性を有する細胞であればいかなる細胞でもよい。
具体的には、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合した糖鎖構造を認識するレクチンに耐性を有する、ハイブリドーマ細胞、ヒト抗体およびヒト化抗体を製造するための宿主細胞、ヒト抗体を生産するためのトランスジェニック非ヒト動物を製造するための胚性幹細胞および受精卵細胞、ヒト抗体を生産するためのトランスジェニック植物を製造するための植物カルス細胞、ミエローマ細胞、トランスジェニック非ヒト動物由来の細胞などがあげられる。ミエローマ細胞はハイブリドーマ細胞を製造する際に融合細胞として用いることができる。また、トランスジェニック非ヒト動物に抗原を免疫し、該動物の脾臓細胞を取り出し、ハイブリドーマ細胞を製造するために用いることができる。
レクチンに耐性を有する細胞とは、培養培地にレクチンを有効濃度与えて細胞培養を行ったときにも、生育が阻害されない細胞をいう。
本発明において、生育が阻害されないレクチン有効濃度は、細胞株に応じて適宜定めればよいが、通常１０μｇ／ｍｌ〜１０．０ｍｇ／ｍｌ、好ましくは０．５〜２．０ｍｇ／ｍｌである。親株細胞に変異を導入した場合のレクチンの有効濃度とは、該親株細胞が正常に生育できない濃度以上であり、好ましくは親株細胞が正常に生育できない濃度と同濃度、より好ましくは２〜５倍、さらに好ましくは１０倍、最も好ましくは２０倍以上の濃度をいう。
親株細胞とは、何らかの処理を施す前の細胞、すなわち本発明で用いるα１，６−フコース／レクチン耐性細胞を選択する工程を行う前の細胞、上述した酵素活性を低下または欠失するために遺伝子工学的な処理を行う前の細胞をいう。
親株細胞としては、特に限定はないが、種々の細胞株の親株細胞の具体例として、以下に示す細胞があげられる。
ＮＳ０細胞の親株細胞としては、バイオ／テクノロジー（ＢＩＯ／ＴＥＣＨＮＯＬＯＧＹ），１０，１６９（１９９２）、バイオテクノロジー・バイオエンジニアリング（Ｂｉｏｔｅｃｈｎｏｌ．Ｂｉｏｅｎｇ．），７３，２６１，（２００１）等の文献に記載されているＮＳ０細胞があげられる。また、理化学研究所細胞開発銀行に登録されているＮＳ０細胞株（ＲＣＢ０２１３）、あるいはこれら株を生育可能な培地に馴化させた亜株などもあげられる。
ＳＰ２／０−Ａｇ１４細胞の親株細胞としては、ジャーナル・オブ・イムノロジー（Ｊ．Ｉｍｍｕｎｏｌ．），１２６，３１７（１９８１）、ネイチャー（Ｎａｔｕｒｅ），２７６，２６９（１９７８）、ヒューマン・アンチィボディズ・アンド・ハイブリドーマズ（Ｈｕｍａｎ Ａｎｔｉｂｏｄｉｅｓ ａｎｄ Ｈｙｂｒｉｄｏｍａｓ），３，１２９（１９９２）等の文献に記載されているＳＰ２／０−Ａｇ１４細胞があげられる。また、ＡＴＣＣに登録されているＳＰ２／０−Ａｇ１４細胞（ＡＴＣＣ ＣＲＬ−１５８１）あるいはこれら株を生育可能な培地に馴化させた亜株（ＡＴＣＣ ＣＲＬ−１５８１．１）などもあげられる。
チャイニーズハムスター卵巣組織由来ＣＨＯ細胞の親株細胞としては、Ｊｏｕｒｎａｌ ｏｆ Ｅｘｐｅｒｉｍｅｎｔａｌ Ｍｅｄｉｃｉｎｅ，１０８，９４５（１９５８）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｅｉ．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，７７，４２１６（１９８０）、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．６０，１２７５（１９６８）、Ｃｅｌｌ，６，１２１（１９７５）、Ｍｏｌｅｃｕｌａｒ Ｃｅｌｌ Ｇｅｎｅｔｉｃｓ，Ａｐｐｅｎｄｉｘ Ｉ，ＩＩ（ｐ８８３−９００）等の文献に記載されているＣＨＯ細胞などがあげられる。また、ＡＴＣＣに登録されているＣＨＯ−Ｋ１株（ＡＴＣＣ ＣＣＬ−６１）、ＤＵＸＢ１１株（ＡＴＣＣＣＲＬ−９０９６）、Ｐｒｏ−５株（ＡＴＣＣ ＣＲＬ−１７８１）や、市販のＣＨＯ−Ｓ株（Ｌｉｆｅｔｅｃｈｎｏｌｏｇｉｅｓ社 Ｃａｔ＃１１６１９）、あるいはこれら株を生育可能な培地に馴化させた亜株などもあげられる。
ラットミエローマ細胞株ＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０細胞の親株細胞としては、Ｙ３／Ａｇ１．２．３細胞（ＡＴＣＣ ＣＲＬ−１６３１）から樹立された株化細胞が包含される。その具体的な例としては、Ｊ．Ｃｅｌｌ．Ｂｉｏｌ．，９３，５７６（１９８２）、Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ．，７３Ｂ，１（１９８１）等の文献に記載されているＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０細胞があげられる。また、ＡＴＣＣに登録されているＹＢ２／３ＨＬ．Ｐ２．Ｇ１１．１６Ａｇ．２０細胞（ＡＴＣＣ ＣＲＬ−１６６２）あるいはこれら株を生育可能な培地に馴化させた亜株などもあげられる。
Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合した糖鎖構造を認識するレクチンとしては、該糖鎖構造を認識できるレクチンであれば、いずれのレクチンも包含される。その具体的な例としては、レンズマメレクチンＬＣＡ（Ｌｅｎｓ Ｃｕｌｉｎａｒｉｓ由来のＬｅｎｔｉｌ Ａｇｇｌｕｔｉｎｉｎ）、エンドウマメレクチンＰＳＡ（Ｐｉｓｕｍ ｓａｔｉｖｕｍ由来のＰｅａ Ｌｅｃｔｉｎ）、ソラマメレクチンＶＦＡ（Ｖｉｃｉａ ｆａｂａ由来のＡｇｇｌｕｔｉｎｉｎ）、ヒイロチャワンタケレクチンＡＡＬ（Ａｌｅｕｒｉａ ａｕｒａｎｔｉａ由来のＬｅｃｔｉｎ）などがあげられる。
本発明において、α１，６−フコース／レクチン耐性細胞としては、一定の有効濃度のレクチン存在下で生育が阻害されない細胞であれば、いずれでもよいが、例えば、以下にあげる少なくとも１つの蛋白質の活性が親株細胞よりも低下または欠失した細胞などがあげられる。
（ａ）細胞内糖ヌクレオチドＧＤＰ−フコースの合成に関与する酵素蛋白質（以下、「ＧＤＰ−フコース合成酵素」と表記する）；
（ｂ）Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素蛋白質（以下、「α１，６−フコース修飾酵素」と表記する）；
（ｃ）細胞内糖ヌクレオチドＧＤＰ−フコースのゴルジ体への輸送に関与する蛋白質（以下、「ＧＤＰ−フコース輸送蛋白質」と表記する）。
ＧＤＰ−フコース合成酵素としては、細胞内で糖鎖へのフコースの供給源である糖ヌクレオチドＧＤＰ−フコースの合成に関与する酵素であればいかなる酵素も包含し、細胞内糖ヌクレオチドＧＤＰ−フコースの合成に影響を与える酵素などがあげられる。
細胞内の糖ヌクレオチドＧＤＰ−フコースは、ｄｅ ｎｏｖｏの合成経路あるいはＳａｌｖａｇｅ合成経路により供給されている。したがって、これら合成経路に関与する酵素はすべてＧＤＰ−フコース合成酵素に包含される。
ｄｅ ｎｏｖｏの合成経路に関与するＧＤＰ−フコース合成酵素としては、ＧＤＰ−ｍａｎｎｏｓｅ ４−ｄｅｈｙｄｒａｔａｓｅ（ＧＤＰ−マンノース ４−デヒドラターゼ；以下、ＧＭＤと表記する）、ＧＤＰ−ｋｅｔｏ−６−ｄｅｏｘｙｍａｎｎｏｓｅ ３，５−ｅｐｉｍｅｒａｓｅ，４−ｒｅｄｕｃｔａｓｅ（ＧＤＰ−ケト−デオキシマンノース ３，５−エピメラーゼ，４−リダクターゼ；以下、Ｆｘと表記する）などがあげられる。
Ｓａｌｖａｇｅ合成経路に関与するＧＤＰ−フコース合成酵素としては、ＧＤＰ−ｂｅｔａ−Ｌ−ｆｕｃｏｓｅ ｐｙｒｏｐｈｏｓｐｈｏｒｙｌａｓｅ（ＧＤＰ−ベータ−Ｌ−フコース−ピロホスフォリラーゼ；以下、ＧＦＰＰと表記する）、Ｆｕｃｏｋｉｎａｓｅ（フコキナーゼ）などがあげられる。ＧＭＤとしては、配列番号１、２または３で表されるアミノ酸配列を有するＧＭＤがあげられる。
細胞内糖ヌクレオチドＧＤＰ−フコースの合成に影響を与える酵素としては、上述の細胞内の糖ヌクレオチドＧＤＰ−フコースの合成経路に関与する酵素の活性に影響を与えたり、該酵素の基質となる物質の構造に影響を与える酵素も包含される。
α１，６−フコース修飾酵素としては、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合する反応に関与する酵素であればいかなる酵素も包含される。Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合する反応に関与する酵素としては、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合する反応に影響を与える酵素であればいかなる酵素も包含される。具体的には、α１，６−フコシルトランスフェラーゼやα−Ｌ−フコシダーゼなどがあげられる。α１，６−フコシルトランスフェラーゼとしては、配列番号４または５で表されるアミノ酸配列を有するα１，６−フコシルトランスフェラーゼがあげられる。
また、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合する反応に影響を与える酵素としては、上述のＮ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する反応に関与する酵素の活性に影響を与えたり、該酵素の基質となる物質の構造に影響を与える酵素も包含される。
ＧＤＰ−フコース輸送蛋白質としては、細胞内糖ヌクレオチドＧＤＰ−フコースのゴルジ体への輸送に関与する蛋白質であればいかなる蛋白質も包含され、具体的には、ＧＤＰ−フコーストランスポーターなどがあげられる。
また、細胞内糖ヌクレオチドＧＤＰ−フコースをゴルジ体内へ輸送する反応に影響を与える蛋白質もＧＤＰ−フコース輸送蛋白質に包含され、具体的には上述の細胞内糖ヌクレオチドＧＤＰ−フコースのゴルジ体への輸送に関与する蛋白質の活性に影響を与えたり、発現に影響を与える蛋白質などがあげられる。
本発明の製造方法に用いられる細胞を取得する方法としては、α１，６−フコース／レクチン耐性細胞を選択することができる手法であればいかなる手法でも用いることができる。具体的には、上述した蛋白質の活性を低下または欠失させる手法などがあげられる。上述の蛋白質の活性を低下または欠失させる手法としては、
（ａ）蛋白質の遺伝子を標的した遺伝子破壊の手法；
（ｂ）蛋白質の遺伝子のドミナントネガティブ体を導入する手法；
（ｃ）蛋白質についての突然変異を導入する手法；
（ｄ）蛋白質の遺伝子の転写または翻訳を抑制する手法；
などがあげられる（ＷＯ０２／３１１４０、ＷＯ０３／０８５１０７、ＷＯ０３／０８５１１８）。
本発明において、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素のゲノム遺伝子がノックアウトされた細胞としては、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素のゲノム遺伝子の発現または機能を完全に抑制された細胞などがあげられる。したがって、本発明において、ゲノム遺伝子がノックアウトされた細胞としては、以下に述べる手法を用いて、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素のゲノム遺伝子をノックアウトした細胞があげられる。
ゲノム遺伝子がノックアウトされた細胞の具体的な例としては、標的となる遺伝子のすべてまたは一部がゲノムから削除された細胞があげられる。このような細胞を取得する方法としては、目的とするゲノムの改変を行うことができれば、いずれの手法でも用いることができるが、遺伝子工学的な手法が望ましい。その具体的な手法としては、上述した酵素の活性を低下または欠失させる手法があげられる。
また、上述した、Ｎ−グリコシド結合糖鎖還元末端のＮ−アセチルグルコサミンの６位とフコースの１位がα結合した糖鎖構造を認識するレクチンに耐性な細胞株を選択する方法を用いることにより、Ｎ−グリコシド結合複合型糖鎖還元末端のＮ−アセチルグルコサミンの６位にフコースの１位がα結合する糖鎖修飾に関与する酵素のゲノム遺伝子がノックアウトされた細胞を選択することができる。
本発明における抗体組成物としては、疾患に関連する細胞に発現する抗原、もしくは疾患に関連する細胞の増殖や転移など、病態形成に関わる抗原などに対する抗体組成物などがあげられる。具体的にはＧＤ２、ＧＤ３、ＧＭ２、ＨＥＲ２、ＣＤ２０、ＣＤ２２、ＣＤ２５、ＣＤ３３、ＣＤ５２、ＭＡＧＥ、ＨＭ１．２４、ＣＣケモカイン受容体４（ＣＣＲ４）、副甲状腺ホルモン関連蛋白（ＰＴＨｒＰ）、塩基性線維芽細胞増殖因子、線維芽細胞増殖因子８、塩基性繊維芽細胞増殖因子受容体、線維芽細胞増殖因子８受容体、上皮細胞成長因子受容体（ＥＧＦＲ）、上皮性細胞接着分子（ＥｐＣａｍ）、インスリン様増殖因子、インスリン様増殖因子受容体、ＰＭＳＡ、血管内皮細胞増殖因子、血管内皮細胞増殖因子受容体、呼吸器合胞体ウイルス（ＲＳＶ）、ＩＬ−５受容体α鎖などに対する抗体組成物があげられる。
上記の抗体組成物の具体例としては、抗ＧＤ２抗体［アンチ・キャンサー・リサーチ（Ａｎｔｉｃａｎｃｅｒ Ｒｅｓ．），１３，３３１（１９９３）］、抗ＧＤ３抗体［キャンサー・イムノロジー・イムノセラピー（Ｃａｎｃｅｒ Ｉｍｍｕｎｏｌ．Ｉｍｍｕｎｏｔｈｅｒ．），３６，２６０（１９９３）、ＷＯ０１／２３４３２、ＵＳ６４３７０９８］、抗ＧＭ２抗体［キャンサー・リサーチ（Ｃａｎｃｅｒ Ｒｅｓ．），５４，１５１１（１９９４）、ＵＳ６４２３５１１］、抗ＨＥＲ２抗体［プロシーディングス・オブ・ザ・ナショナル・アカデミー・オブ・サイエンス（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ），８９，４２８５（１９９２）］、抗ＣＤ２０抗体［ブラッド（Ｂｌｏｏｄ），８３，４３５（１９９４）、ＷＯ０３／５５９９３］、抗ＣＤ２２抗体［セミナーズ・イン・オンコロジー（Ｓｅｍｍｉｎ．Ｏｎｃｏｌ．），３０，２５３（２００３）］、抗ＣＤ２５抗体［ヒューマン・アンティボディズ（Ｈｕｍａｎ Ａｎｔｉｂｏｄｉｅｓ），１０，１２７−１４２（２００１）］、抗ＣＤ３３抗体［ジャーナル・オブ・クリニカル・オンコロジー（Ｊ．Ｃｌｉｎ．Ｏｎｃｏｌ．），１９，３２４４（２００１）］、抗ＣＤ５２抗体［プロシーディングス・オブ・ザ・ナショナル・アカデミー・オブ・サイエンス（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ），８９，４２８５（１９９２）］、抗ＭＡＧＥ抗体［ブリティッシュ・ジャーナル・オブ・キャンサー（Ｂｒｉｔｉｓｈ Ｊ．Ｃａｎｃｅｒ），８３，４９３（２０００）］、抗ＨＭ１．２４抗体［モレキュラー・イムノロジー（Ｍｏｌｅｃｕｌａｒ Ｉｍｍｕｎｏｌ．），３６，３８７（１９９９）］、抗ＣＣＲ４抗体［ＷＯ０１／６４７５４、ＷＯ０３／１８６３５、ＷＯ００／４２０７４、ＷＯ９９／１５６６６、ＵＳ６２４５３３２］、抗副甲状腺ホルモン関連蛋白（ＰＴＨｒＰ）抗体［キャンサー（Ｃａｎｃｅｒ），８８，２９０９（２０００）］、抗線維芽細胞増殖因子８抗体［プロシーディングス・オブ・ザ・ナショナル・アカデミー・オブ・サイエンス（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ），８６，９９１１（１９８９）、ＷＯ０３／００２６０８］、抗線維芽細胞増殖因子８受容体抗体［ジャーナル・オブ・バイオロジカル・ケミストリー（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．），２６５，１６４５５（１９９０）］、抗上皮細胞成長因子受容体抗体［キャンサー・リサーチ（Ｃａｎｃｅｒ Ｒｅｓ．），５９，１２３６（１９９９）］、抗上皮性細胞接着分子抗体［プロシーディングス・オブ・ザ・ナショナル・アカデミー・オブ・サイエンス（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ），７６，１４３８（１９７９）］、抗インスリン様増殖因子抗体［ジャーナル・オブ・ニューロサイエンス・リサーチ（Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．），４０，６４７（１９９５）、ＷＯ０３／９３３１７］、抗インスリン様増殖因子受容体抗体［ジャーナル・オブ・ニューロサイエンス・リサーチ（Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．），４０，６４７（１９９５）］、抗ＰＭＳＡ抗体［ジャーナル・オブ・ウロロジー（Ｊ．Ｕｒｏｌｏｇｙ），１６０，２３９６（１９９８）］、抗血管内皮細胞増殖因子抗体［キャンサー・リサーチ（Ｃａｎｃｅｒ Ｒｅｓ．），５７，４５９３（１９９７）］、抗血管内皮細胞増殖因子受容体抗体［オンコジーン（Ｏｎｃｏｇｅｎｅ），１９，２１３８（２０００）］、抗ＲＳＶ抗体［アンティバイラル・リサーチ（Ａｎｔｉｖｉｒａｌ Ｒｅｓ．），４７，５７−７７（２０００）］、抗ＩＬ−５受容体α鎖抗体（ＵＳ６０１８０３２、ＵＳ６５３８１１１）などがあげられる。具体的な抗体名としては、ハーセプチン（Ｈｅｒｃｅｐｔｉｎ）、リツキサン（Ｒｉｔｕｘａｎ）、キャンパス（Ｃａｍｐａｔｈ）、アバスチン（Ａｖａｓｔｉｎ）、ベクサー（Ｂｅｘｘａｒ）、リンフォサイド（ＬｙｍｐｈｏＣｉｄｅ）、マイロターグ（Ｍｙｌｏｔａｒｇ）、パノレックス（Ｐａｎｏｒｅｘ）、ゼバリン（Ｚｅｖａｌｉｎ）［ネイチャー・レビューズ・キャンサー（Ｎａｔ．Ｒｅｖ．Ｃａｎｃｅｒ），１，１１８（２００１）］、ゼナパックス（Ｚｅｎａｐａｘ）、シムレクト（Ｓｉｍｕｌｅｃｔ）、レミケード（Ｒｅｍｉｃａｄｅ）、シナジス（Ｓｙｎａｇｉｓ）、レオプロ（ＲｅｏＰｒｏ）［ヒューマン・アンティボディズ（Ｈｕｍａｎ Ａｎｔｉｂｏｄｉｅｓ），１０，１２７−１４２（２００１）］、エンブレル（Ｅｎｂｒｅｌ）［エキスパート・オピニオン・オン・ファーマコセラピー（Ｅｘｐｅｒｔ Ｏｐｉｎｉｏｎ ｏｎ Ｐｈａｒｍａｃｏｔｈｅｒａｐｙ），２，１１３７−１１４８（２００１）］、ゾレア（Ｚｏｌａｉｒ）［レスピラトリー・メディスン（Ｒｅｓｐｉｒａｔｏｒｙ Ｍｅｄｉｃｉｎｅ），９７，１２３−１２９（２００３）］などがあげられる。
本発明において、ＡＤＣＣ活性は、^５１Ｃｒ遊離法、ｌａｃｔａｔｅ ｄｅｈｙｄｒｏｇｅｎａｓｅ（ＬＤＨ）遊離法、フローサイトメトリー等によるｉｎ ｖｉｔｒｏの測定系、あるいは動物モデルを用いたｉｎ ｖｉｖｏの評価系等で測定することができる。
本発明において、抗体組成物中に含まれるＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％以上である抗体組成物とともに用いられる薬剤としては、サイトカイン、抗体などの蛋白質、低分子の薬剤、生物学的応答調節物質（以下ＢＲＭと称する）などがあげられる。
サイトカインとしては、インターロイキン（ＩＬ）類、コロニー刺激因子（ＣＳＦ）類、インターフェロン（ＩＦＮ）類、腫瘍壊死因子（ＴＮＦ）類、ケモカイン類、あるいはこれらの組み合わせがあげられる。具体的には、ＩＦＮ−α、ＩＦＮ−β、ＩＦＮ−γ、ＴＮＦ−α、ＴＮＦ−β、ＩＬ−１α、ＩＬ−１β、ＩＬ−２、ＩＬ−３、ＩＬ−４、ＩＬ−６、ＩＬ−７、ＩＬ−９、ＩＬ−１２、ＩＬ−１５、ＩＬ−１７、ＩＬ−１８、ＩＬ−２１、ｆｒａｃｔａｌｋｉｎｅ、ＧＭ−ＣＳＦ、Ｇ−ＣＳＦ、Ｍ−ＣＳＦ、ＳＣＦ、あるいはこれらの組み合わせがあげられる。好ましくはＩＬ−２、ＩＬ−１５、ＩＦＮ−α、ＩＦＮ−γがあげられる。
低分子の薬剤としては、アミフォスチン（エチオール）［ａｍｉｆｏｓｔｉｎｅ（ｅｔｈｙｏｌ）］、シスプラチン（ｃｉｓｐｌａｔｉｎ）、ダカルバジン（ＤＴＩＣ）［ｄａｃａｒｂａｚｉｎｅ（ＤＴＩＣ）］、ダクチノマイシン（ｄａｃｔｉｎｏｍｙｃｉｎ）、メクロレタミン（ナイトロジェンマスタード）［ｍｅｃｈｌｏｒｅｔｈａｍｉｎｅ（ｎｉｔｒｏｇｅｎｍｕｓｔａｒｄ）］、ストレプトゾシン（ｓｔｒｅｐｔｏｚｏｃｉｎ）、シクロフォスファミド（ｃｙｃｌｏｐｈｏｓｐｈａｍｉｄｅ）、カルムスチン（ＢＣＮＵ）［ｃａｒｍｕｓｔｉｎｅ（ＢＣＮＵ）］、ロムスチン（ＣＣＮＵ）［ｌｏｍｕｓｔｉｎｅ（ＣＣＮＵ）］、ドキソルビシン（アドリアマイシン）［ｄｏｘｏｒｕｂｉｃｉｎ（ａｄｒｉａｍｙｃｉｎ）］、ドキソルビシンリポ（ドキシル）［ｄｏｘｏｒｕｂｉｃｉｎｌｉｐｏ（ｄｏｘｉｌ）］、ゲムシタビン（ゲムザール）［ｇｅｍｃｉｔａｂｉｎｅ（ｇｅｍｚａｒ）］、ダウノルビシン（ｄａｕｎｏｒｕｂｉｃｉｎ）、ダウノルビシンリポ（ダウノゾーム）［ｄａｕｎｏｒｕｂｉｃｉｎｌｉｐｏ（ｄａｕｎｏｘｏｍｅ）］、プロカルバジン（ｐｒｏｃａｒｂａｚｉｎｅ）、マイトマイシン（ｍｉｔｏｍｙｃｉｎ）、シタラビン（ｃｙｔａｒａｂｉｎｅ）、エトポシド（ｅｔｏｐｏｓｉｄｅ）、メトトレキセート（ｍｅｔｈｏｔｒｅｘａｔｅ）、５−フルオロウラシル（５−ｆｌｕｏｒｏｕｒａｃｉｌ）、ビンブラスチン（ｖｉｎｂｌａｓｔｉｎｅ）、ビンクリスチン（ｖｉｎｃｒｉｓｔｉｎｅ）、ブレオマイシン（ｂｌｅｏｍｙｃｉｎ）、パクリタキセル（タキソール）［ｐａｃｌｉｔａｘｅｌ（ｔａｘｏｌ）］、ドセタキセル（タキソテア）［ｄｏｃｅｔａｘｅｌ（ｔａｘｏｔｅｒｅ）］、アルデスロイキン（ａｌｄｅｓｌｅｕｋｉｎ）、アスパラギナーゼ（ａｓｐａｒａｇｉｎａｓｅ）、ブスルファン（ｂｕｓｕｌｆａｎ）、カルボプラチン（ｃａｒｂｏｐｌａｔｉｎ）、クラドリビン（ｃｌａｄｒｉｂｉｎｅ）、カンプトテシン（ｃａｍｐｔｏｔｈｅｃｉｎ）、ＣＰＴ−１１、１０−ヒドロキシ−７−エチル−カンプトテシン（ＳＮ３８）［１０−ｈｙｄｒｏｘｙ−７−ｅｔｈｙｌ−ｃａｍｐｔｏｔｈｅｃｉｎ（ＳＮ３８）］、フロクスウリジン（ｆｌｏｘｕｒｉｄｉｎｅ）、フルダラビン（ｆｌｕｄａｒａｂｉｎｅ）、ヒドロキシウレア（ｈｙｄｒｏｘｙｕｒｅａ）、イホスファミド（ｉｆｏｓｆａｍｉｄｅ）、イダルビシン（ｉｄａｒｕｂｉｃｉｎ）、メスナ（ｍｅｓｎａ）、イリノテカン（ｉｒｉｎｏｔｅｃａｎ）、ミトキサントロン（ｍｉｔｏｘａｎｔｒｏｎｅ）、トポテカン（ｔｏｐｏｔｅｃａｎ）、ロイプロリド（ｌｅｕｐｒｏｌｉｄｅ）、メゲストロール（ｍｅｇｅｓｔｒｏｌ）、メルファラン（ｍｅｌｐｈａｒａｎ）、メルカプトプリン（ｍｅｒｃａｐｔｏｐｕｒｉｎｅ）、プリカマイシン（ｐｌｉｃａｍｙｃｉｎ）、ミトタン（ｍｉｔｏｔａｎｅ）、ペガスパラガーゼ（ｐｅｇａｓｐａｒｇａｓｅ）、ペントスタチン（ｐｅｎｔｏｓｔａｔｉｎ）、ピポブロマン（ｐｉｐｏｂｒｏｍａｎ）、ストレプトゾシン（ｓｔｒｅｐｔｏｚｏｃｉｎ）、タモキシフェン（ｔａｍｏｘｉｆｅｎ）、テニポシド（ｔｅｎｉｐｏｓｉｄｅ）、テストラクトン（ｔｅｓｔｏｌａｃｔｏｎｅ）、チオグアニン（ｔｈｉｏｇｕａｎｉｎｅ）、チオテパ（ｔｈｉｏｔｅｐａ）、ウラシルマスタード（ｕｒａｃｉｌ ｍｕｓｔａｒｄ）、ビノレルビン（ｖｉｎｏｒｅｌｂｉｎｅ）、クロラムブシル（ｃｈｌｏｒａｍｂｕｃｉｌ）、プレドニゾロン（ｐｒｅｄｎｉｓｏｌｏｎｅ）、ビンデシン（ｖｉｎｄｅｓｉｎｅ）、ニムスチン（ｎｉｍｓｔｉｎｅ）、セムスチン（ｓｅｍｕｓｔｉｎ）、カペシタビン（ｃａｐｅｃｉｔａｂｉｎｅ）、トムデックス（ｔｏｍｕｄｅｘ）、アザシチジン（ａｚａｃｙｔｉｄｉｎｅ）、ＵＦＴ、オキザロプラチン（ｏｘａｌｏｐｌａｔｉｎ）、ゲフィチニブ（イレッサ）［ｇｅｆｉｔｉｎｉｂ（Ｉｒｅｓｓａ）］、イマチニブ（ＳＴＩ５７１）［ｉｍａｔｉｎｉｂ（ＳＴＩ５７１）］、アムサクリン（ａｍｓａｃｒｉｎｅ）、オール−トランスレチノイン酸（ａｌｌ−ｔｒａｎｓ ｒｅｔｉｎｏｉｃ ａｃｉｄ）、サリドマイド（ｔｈａｌｉｄｏｍｉｄｅ）、ベキサロテン（ターグレチン）［ｂｅｘａｒｏｔｅｎｅ（ｔａｒｇｒｅｔｉｎ）］、デキサメタゾン（ｄｅｘａｍｅｔｈａｓｏｎｅ）、アナストロゾール（アリミデックス）［ａｎａｓｔｒｏｚｏｌｅ（Ａｌｉｍｉｄｅｘ）］、ロイプリン（ｌｅｕｐｌｉｎ）あるいはこれらの組み合わせがあげられる。好ましくは、ビンクリスチン、シクロフォスファミド、エトポシド、メトトレキセートあるいはこれらの組み合わせがあげられる。
抗体としては、上述した抗体があげられる。
本発明におけるＢＲＭとしては、ＢＣＧ、嫌気性コリネバクテリア、ムラミルジペプチド、トレハロースジミコール酸等の細菌製剤、ピラン共重合体、ＭＶＥ、ポリ Ｉ：Ｃ、ピリミジン、チモシン、チムリン、チモポエシン、ピシバニール、フコイダン、クレスチン等があげられる。
本発明の医薬の効果は、例えばｉｎ ｖｉｔｒｏの細胞障害活性測定系によって調べることができる。ｉｎ ｖｉｔｒｏの細胞障害活性測定系の例としては、ＡＤＣＣ活性の測定系があげられる。ＡＤＣＣ活性は、抗体の存在下で、抗原を発現する標的細胞と、ヒトあるいはその他の動物より採取した末梢血単核球、単球、マクロファージ、顆粒球等のエフェクター細胞を接触させ、障害された標的細胞の度合いを検出し、これを定量することにより測定することができる。障害された標的細胞の度合いは、^５１Ｃｒ遊離法、標的細胞の酵素活性を検出する方法、フローサイトメーターによる検出法などによって検出することができる。ＡＤＣＣ活性測定系における本発明の免疫担当細胞を活性化させる物質または抗腫瘍活性を有する物質の効果は、ＡＤＣＣ活性測定系中にこれらの物質を添加するか、あるいは標的細胞、またはエフェクター細胞、またはその両者にあらかじめこれらの物質を一定期間曝露してＡＤＣＣ活性に与える影響を観察することにより、測定することができる。
また本発明の医薬の効果は、動物モデルを用いたｉｎ ｖｉｖｏ抗腫瘍活性を測定することによっても調べることができる。
動物モデルとしては、ヌードマウス等の免疫不全マウスにヒト癌組織由来の培養細胞株を移植した異種移植モデル、培養マウス癌細胞株を正常な免疫系を有する野生型マウスへ移植した同系移植モデルなどがあげられる。
異種移植モデルはヌードマウス等の免疫不全マウスの皮下、皮内、腹腔内、静脈内等様々な部位にヒト癌細胞株を移植することにより作製することができる。
上記動物モデルを用いて抗体の単独投与、免疫担当細胞を活性化させる物質または抗腫瘍活性を有する物質の単独投与の効果と、本発明の医薬の効果とを比較することにより、本発明の医薬の抗腫瘍効果を評価することができる。
本発明の医薬は、単独で投与することも可能ではあるが、通常は薬理学的に許容される一つあるいはそれ以上の担体と一緒に混合し、製剤学の技術分野においてよく知られる任意の方法により製造した医薬製剤として提供するのが望ましい。
投与経路は、治療に際して最も効果的なものを使用するのが望ましく、経口投与、または口腔内、気道内、直腸内、皮下、筋肉内および静脈内等の非経口投与をあげることができ、蛋白質製剤の場合、望ましくは静脈内投与をあげることができる。
投与形態としては、噴霧剤、カプセル剤、錠剤、顆粒剤、シロップ剤、乳剤、座剤、注射剤、軟膏、テープ剤等があげられる。
経口投与に適当な製剤としては、乳剤、シロップ剤、カプセル剤、錠剤、散剤、顆粒剤等があげられる。
乳剤およびシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖等の糖類、ポリエチレングリコール、プロピレングリコール等のグリコール類、ごま油、オリーブ油、大豆油等の油類、ｐ−ヒドロキシ安息香酸エステル類等の防腐剤、ストロベリーフレーバー、ペパーミント等のフレーバー類等を添加剤として用いて製造できる。
カプセル剤、錠剤、散剤、顆粒剤等は、乳糖、ブドウ糖、ショ糖、マンニトール等の賦形剤、デンプン、アルギン酸ナトリウム等の崩壊剤、ステアリン酸マグネシウム、タルク等の滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、ゼラチン等の結合剤、脂肪酸エステル等の界面活性剤、グリセリン等の可塑剤等を添加剤として用いて製造できる。
非経口投与に適当な製剤としては、注射剤、座剤、噴霧剤等があげられる。
注射剤は、塩溶液、ブドウ糖溶液、あるいは両者の混合物からなる担体等を用いて調製される。
座剤はカカオ脂、水素化脂肪またはカルボン酸等の担体を用いて調製される。
また、噴霧剤は該医薬そのもの、ないしは受容者の口腔および気道粘膜を刺激せず、かつ該医薬を微細な粒子として分散させ吸収を容易にさせる担体等を用いて調製される。
担体として具体的には乳糖、グリセリン等が例示される。該医薬および用いる担体の性質により、エアロゾル、ドライパウダー等の製剤が可能である。また、これらの非経口剤においても経口剤で添加剤として例示した成分を添加することもできる。
投与量または投与回数は、目的とする治療効果、投与方法、治療期間、年齢、体重等により異なるが、通常成人１回当たり抗体量として０．１〜２０ｍｇ／ｋｇである。抗体と併用する薬剤は、単独で臨床に用いられる場合の投与量と同用量またはそれより少ない用量である。  An object of the present invention is to provide a medicament using an antibody composition having a high therapeutic effect and at least one kind of drug.
  The present invention relates to the following (1) to (14).
  (1) Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% A pharmaceutical comprising a combination of the antibody composition as described above and at least one drug.
  (2) Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% The pharmaceutical for administering combining the antibody composition which is the above, and at least 1 sort (s) of medicine.
  (3) Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% A medicament for administering the antibody composition as described above and at least one drug simultaneously or sequentially.
  (4) Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% The medicament according to any one of (1) to (3) above, which exhibits a higher therapeutic effect than when the antibody composition as described above is administered alone.
  (5) The medicament according to (4) above, wherein the high therapeutic effect is high antibody-dependent cytotoxic activity.
  (6) An antibody composition is produced from a cell having resistance to a lectin that recognizes a sugar chain structure in which N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing terminal at position 6 and position 1 of fucose are α-linked. The medicament according to any one of (1) to (5) above, which is an antibody composition.
  (7) Among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the antibody composition is a sugar chain in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end. The medicament according to any one of (1) to (6) above, which is an antibody composition.
  (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-bonded to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing end ( The medicament according to any one of 1) to (7).
  (9) A cell in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is knocked out The medicament according to (7) or (8) above, which is an antibody composition produced from
  (10) The enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal is α1,6-fucosyltransferase (9) ) The pharmaceutical described.
  (11) The medicament according to any one of (1) to (10), wherein the drug is a substance selected from the group consisting of a protein, a low-molecular drug, and a biological response modifier.
  (12) The medicament according to (11) above, wherein the protein is a cytokine or an antibody.
  (13) The medicament according to (12), wherein the cytokine is a cytokine selected from IFN-γ, IL-2, and IL-15.
  (14) The medicament according to any one of (1) to (13) above, wherein the medicament is a therapeutic agent for a disease involving a tumor.
  As a pharmaceutical form of the present invention, among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, a sugar in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end A pharmaceutical comprising a combination of an antibody composition having a chain ratio of 50% or more and at least one drug, among all N-glycoside-linked complex sugar chains that bind to the Fc region contained in the antibody composition, Pharmaceutical composition and antibody composition for administration in combination of an antibody composition in which the ratio of sugar chain in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more and at least one kind of drug Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained therein, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more An antibody composition, a medicament for administration of at least one agent at the same time or sequentially and the like.
  Here, the combined medicine refers to the non-fucose bound to the N-acetylglucosamine at the reducing end of the sugar chain among all the N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition. The pharmaceutical composition may be prepared by separately preparing an antibody composition having a sugar chain ratio of 50% or more and at least one drug, and combining these drugs simultaneously or sequentially. It may be a mixture in which drug components are mixed. In the mixture in which each drug component is mixed, fucose binds to N-acetylglucosamine at the reducing end of the sugar chain among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition. Also included are fusion antibodies in which at least one drug is bound to an antibody composition in which the proportion of untreated sugar chains is 50% or more.
  In the present invention, among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the ratio of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end The number of sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain with respect to the total number of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the composition Refers to the proportion of The proportion of sugar chains is preferably the proportion of sugar chains in which the 1-position of fucose is not α-bonded to the 6-position of N-acetylglucosamine at the sugar chain reducing end.
  In the present invention, a sugar chain in which fucose is not bound to N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing end means that the fucose is N-acetyl at the N-glycoside-linked complex type sugar chain reducing end. This refers to a sugar chain that is not α-bonded to glucosamine, specifically, a sugar chain in which the 1-position of the fucose is not α-bonded to the 6-position of N-acetylglucosamine of the N-glycoside-bonded complex sugar chain. .
  The antibody composition in the present invention includes any composition as long as it contains an antibody molecule having an N-glycoside-linked complex sugar chain in the Fc region.
  The antibody molecule is a tetramer in which two molecules of two kinds of polypeptide chains, a heavy chain and a light chain (hereinafter referred to as H chain and L chain, respectively) are associated with each other. About one-quarter of the N-terminal side of the H chain and about one-half (each 100 amino acids) of the N-terminal side of the L chain are called V regions and are rich in diversity and directly involved in antigen binding To do. Most of the parts other than the V region are called C regions. Antibody molecules are classified into IgG, IgM, IgA, IgD, and IgE classes based on C region homology.
  The IgG class is further classified into IgG1 to IgG4 subclasses based on the homology of the C region.
  The H chain is divided into four immunoglobulin domains, VH, CH1, CH2, and CH3, from the N-terminal side. Between CH1 and CH2, there is a highly flexible peptide region called a hinge region, and CH1 and CH2 are separated. . A structural unit consisting of CH2 and CH3 after the hinge region is called an Fc region, and an N-glycoside-linked sugar chain is bound thereto. In addition, this region is a region to which Fc receptor, complement, and the like are bound (Immunology Illustrated Original 5th Edition, issued February 10, 2000, Nanedo Edition, Introduction to Antibody Engineering, January 25, 1994) First edition, Jinjinshokan).
  The sugar chains of glycoproteins such as antibodies are linked to asparagine (N-glycoside-linked sugar chains) and to sugar chains (O-glycosyl-linked sugar chains) that bind to serine, threonine, etc. There are two main types. N-glycoside-linked sugar chains have a basic common core structure shown in the following structural formula (I) [Biochemical Experimental Method 23-Glycoprotein Sugar Chain Research Method (Academic Publishing Center) Etsuko Takahashi (1989) ]].

  In the structural formula (I), the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
  Any N-glycoside-linked sugar chain may be used as long as it has the core structure of the above structural formula (I), but a high mannose type in which only mannose binds to the non-reducing end of the core structure, non-core structure. One or more branches of galactose-N-acetylglucosamine (hereinafter referred to as Gal-GlcNAc) are provided in parallel on the reducing end side, and sialic acid and bisecting N are provided on the non-reducing end side of Gal-GlcNAc. -Complex type (complex type) having a structure such as acetylglucosamine, and a hybrid type having both a high mannose type and a complex type branch on the non-reducing terminal side of the core structure.
  The Fc region of an antibody molecule has a region where N-glycoside-linked sugar chains are bonded one by one, so that two sugar chains are bound per antibody molecule. The N-glucoside-linked sugar chain that binds to the antibody molecule includes any sugar chain that includes the core structure represented by the structural formula (I). Therefore, the two N-glucoside-linked sugar chains that bind to the antibody There will be many combinations of sugar chains.
  Therefore, the antibody composition in 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 are obtained. It may be composed of molecules.
  The antibody molecule includes any molecule that contains the Fc region of an antibody. Specific examples include antibodies, antibody fragments, and fusion proteins containing an Fc region.
  As an antibody, an antibody is immunized with an antigen and secreted by a hybridoma cell prepared from a spleen cell of the immunized animal, an antibody prepared by gene recombination technology, that is, an antibody expression vector into which an antibody gene is inserted, And antibodies obtained by introduction into. Specific examples include antibodies produced by hybridomas, humanized antibodies, human antibodies, and the like.
  A hybridoma is a monoclonal antibody having a desired antigen specificity obtained by fusing a B cell obtained by immunizing a mammal other than a human with a myeloma cell derived from a mouse, a rat or the like. A cell that produces.
  Examples of humanized antibodies include human chimeric antibodies and human CDR-grafted antibodies.
  Human-type chimeric antibodies include non-human animal antibody H chain V regions (hereinafter also referred to as HV or VH) and antibody L chain V regions (hereinafter also referred to as LV or VL) and human antibody H chain C regions (hereinafter referred to as LV or VH). , Also referred to as CH) and an L chain C region of a human antibody (hereinafter also referred to as CL). Any animal other than humans can be used as long as it can produce hybridomas such as mice, rats, hamsters, rabbits and the like.
  Human chimeric antibodies are obtained by obtaining cDNAs encoding VH and VL from hybridomas producing monoclonal antibodies and inserting them into expression vectors for host cells having genes encoding human antibody CH and human antibody CL, respectively. A chimeric antibody expression vector can be constructed and expressed by being introduced into a host cell.
  The CH of the human chimeric antibody may be any as long as it belongs to human immunoglobulin (hereinafter referred to as hIg), but the hIgG class is preferred, and hIgG1, hIgG2, hIgG3, Any of the subclasses such as hIgG4 can be used. The CL of the human chimeric antibody may be any as long as it belongs to hIg, and those of κ class or λ class can be used.
  The human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of non-human animal antibodies to appropriate positions of the human antibody VH and VL.
  The human CDR-grafted antibody constructs a cDNA encoding a V region obtained by grafting the VH and VL CDR sequences of a non-human animal antibody into 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 CL of a human antibody, and the human CDR-grafted antibody is expressed by introducing the expression vector into the host cell. Can be manufactured.
  The CH of the human CDR-grafted antibody may be any as long as it belongs to hIg, but the hIgG class is preferable, and any of the subclasses such as hIgG1, hIgG2, hIgG3, and hIgG4 belonging to the hIgG class may be used. it can. The CL of the human CDR-grafted antibody may be any CL as long as it belongs to hIg, and those of κ class or λ class can be used.
  Human antibodies originally refer to antibodies that exist naturally in the human body, but human antibody phage libraries and human antibody transgenics created by recent advances in genetic engineering, cell engineering, and developmental engineering techniques. Antibodies obtained from non-human animals or human antibody transgenic plants are also included.
  The antibody present in the human body can be cultured, for example, by isolating human peripheral blood lymphocytes, infecting and immortalizing and cloning EB virus and the like, and culturing lymphocytes that produce the antibody. Can be purified.
  The human antibody phage library is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by inserting antibody genes prepared from human B cells into the phage genes. From the library, phages expressing antibody fragments having a desired antigen-binding activity can be collected using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted into a human antibody molecule comprising two complete heavy chains and two complete light chains by genetic engineering techniques.
  A human antibody transgenic non-human animal refers to an animal in which a human antibody gene is incorporated into cells. Specifically, a human antibody transgenic non-human animal can be produced by introducing a human antibody gene into a mouse embryonic stem cell, transplanting the embryonic stem cell to an early embryo of another mouse, and generating it. A human antibody transgenic non-human animal can also be produced by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg. A human antibody is produced from a non-human animal transgenic non-human animal by using a conventional method for producing a hybridoma in a mammal other than a human to obtain a human antibody hybridoma and cultivate it to accumulate the human antibody in the culture. Can be made.
  Examples of transgenic non-human animals include cattle, sheep, goats, pigs, horses, mice, rats, chickens, monkeys, and rabbits.
  An 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 H chain of an antibody, 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. The Fc region of the IgG class is described in Kabat et al. EU Index [Sequences of Proteins of Immunological Interest, 5^th  Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)], from the 226th Cys to the C-terminal, or from the 230th Pro to the C-terminal. Examples of antibody fragments include H chain monomers and H chain dimers. The Fc region in the present invention includes natural types and mutants thereof.
  A fusion protein having a part of an Fc region is a substance obtained by fusing an antibody or antibody fragment containing a part of an 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 all the N-glycoside-bonded complex 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 bound to N-acetylglucosamine at the sugar chain reducing end is 50%. Or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more, and all N-glycosidic conjugates that bind to the Fc region contained in the antibody composition Most preferably, the type sugar chain is a sugar chain in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end.
  As described above, among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is high. The higher the antibody composition, the higher the antibody-dependent cytotoxic activity.
  The ratio of the sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain contained in the composition consisting of the antibody molecule having an N-glycoside-linked complex type sugar chain in the Fc region is determined from the antibody molecule to the hydrazine Using known methods such as degradation and enzymatic digestion [Biochemical Experimental Method 23-Glycoprotein Glycan Research Method (Academic Publishing Center) Takahashi Etsuko (1989)], sugar chains are released and the released sugar chains are fluorescent. It can be determined by labeling or isotope labeling and separating the labeled sugar chain by a chromatographic method. It can also be determined by analyzing the released sugar chain by the HPAED-PAD method [J. Liq. Chromatogr., 6, 1577 (1983)].
  In the present invention, examples of the high therapeutic effect include high cytotoxic activity.
  The cytotoxic activity of the antibody composition includes antibody-dependent cytotoxic activity (hereinafter abbreviated as ADCC), complement-dependent cytotoxic activity (monoclonal antibodies), and antigen-expressing cells that bind to the antigen. Examples thereof include growth inhibitory activity. Antiproliferative activity includes those that promote apoptosis and differentiation induction of target cells [Cancer Research]60, 7170 (2000), Nature Medicine.1644 (1995), Cell Growth Differ.3, 401 (1992)].
  ADCC activity means that an antibody bound to a cell surface antigen such as a tumor cell in a living body activates an effector cell through binding of an antibody Fc region to an Fc receptor present on the effector cell surface. It refers to the activity of damaging cells and the like [Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc. , Capter 2.1 (1995)]. Examples of effector cells include immune cells such as natural killer cells, macrophages, monocytes, dendritic cells, and granulocytes. Fc receptor is Fcα receptor I, Fcε receptor I, Fcε receptor II, Fcγ receptor I, Fcγ receptor IIa, Fcγ receptor IIb, Fcγ receptor IIc, Fcγ receptor IIIa, Fcγ receptor IIIb, It is divided into types such as Fc receptor n. Among them, Fcγ receptor IIIa is mainly expressed on natural killer cells and is one of Fc receptors important for ADCC activity [Monoclonal Antibodies: principals and practices. ), Third Edition, Acad. Press, 1996 (hereinafter abbreviated as Monoclonal Antibodies)].
  Therefore, in the present invention, among all N-glycoside-bonded complex sugar chains that bind to the Fc region of an antibody molecule, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. N-acetyl at the reducing end of the sugar chain of all N-glycoside-bonded complex sugar chains that bind to the Fc region of the antibody molecule means higher therapeutic effect than when the antibody composition is administered alone. By combining an antibody composition in which the proportion of the sugar chain in which fucose is not bound to glucosamine is 50% or more and at least one kind of drug, the therapeutic effect exhibited when the antibody composition alone is administered. Refers to a drug that exhibits a high therapeutic effect.
  In addition, the pharmaceutical of the present invention has a ratio of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end of all N-glycoside-bonded complex sugar chains that bind to the Fc region of the antibody molecule. % Higher than that of a pharmaceutical comprising a combination of an antibody composition of less than% and at least one drug.
  The medicament of the present invention can reduce the amount of the drug as compared with the case where the drug is used alone, and can reduce the side effects which are a concern when a high dose of the drug alone is administered to a patient.
  A cell having resistance to a lectin recognizing a sugar chain structure in which the 6-position of N-acetylglucosamine at the N-glycoside-binding complex type sugar chain reducing end and the 1-position of fucose are α-linked (hereinafter referred to as α1,6-fucose / lectin resistance) Cell)) is a cell capable of producing an antibody composition such as yeast, animal cell, insect cell, plant cell, etc., which is the 6-position of N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing end. Any cell may be used as long as it has lectin resistance and recognizes a sugar chain structure in which the 1-position of fucose is α-linked.
  Specifically, a hybridoma cell, a human antibody, which has resistance to a lectin that recognizes a sugar chain structure in which the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal and the 1-position of fucose are α-linked Host cells for producing humanized antibodies, embryonic stem cells and fertilized egg cells for producing transgenic non-human animals for producing human antibodies, and for producing transgenic plants for producing human antibodies Plant callus cells, myeloma cells, cells derived from transgenic non-human animals and the like can be mentioned. Myeloma cells can be used as fusion cells when producing hybridoma cells. Moreover, it can be used for immunizing a transgenic non-human animal with an antigen, taking out the spleen cells of the animal, and producing hybridoma cells.
  A cell having resistance to lectin refers to a cell whose growth is not inhibited even when cell culture is performed with an effective concentration of lectin applied to a culture medium.
  In the present invention, the effective lectin concentration at which growth is not inhibited may be appropriately determined according to the cell line, but is usually 10 μg / ml to 10.0 mg / ml, preferably 0.5 to 2.0 mg / ml. When the mutation is introduced into the parent cell, the effective concentration of the lectin is not less than the concentration at which the parent cell cannot grow normally, preferably the same concentration as the concentration at which the parent cell cannot grow normally, more preferably 2 to 5 times. More preferably, the concentration is 10 times, most preferably 20 times or more.
  The parent cell is a cell before any treatment, that is, a cell before the step of selecting α1,6-fucose / lectin resistant cells used in the present invention, a gene for reducing or eliminating the above-mentioned enzyme activity. A cell before engineering treatment.
  The parent cell is not particularly limited, but specific examples of the parent cell of various cell lines include the cells shown below.
  NS0 cell parent cell lines include Bio / Technology (BIO / TECHNOLOGY),10169 (1992), Biotechnol. Bioeng.,73, 261, (2001), NS0 cells described in the literature. In addition, NS0 cell line (RCB0213) registered with RIKEN Cell Development Bank, or sub-strains in which these strains are conditioned to a medium capable of growth.
  As the parent cell line of SP2 / 0-Ag14 cells, Journal of Immunology (J. Immunol.),126, 317 (1981), Nature,276269 (1978), Human Antibodies and Hybridomas,3, 129 (1992), and the like, SP2 / 0-Ag14 cells. In addition, SP2 / 0-Ag14 cells (ATCC CRL-1581) registered in the ATCC or sub-strains (ATCC CRL-1581.1) in which these strains are conditioned in a viable medium are also included.
  As a parent cell of CHO cells derived from Chinese hamster ovary tissue, Journal of Experimental Medicine,108945 (1958), Proc. Natl. Acad. Sei. USA,60, 1275 (1968), Genetics,55, 513 (1968), Chromosoma,41129 (1973), Methods in Cell Science,18, 115 (1996), Radiation Research,148, 260 (1997), Proc. Natl. Acad. Sci. USA,774216 (1980), Proc. Natl. Acad. Sci.60, 1275 (1968), Cell,6121 (1975), Molecular Cell Genetics, Appendix I, II (p883-900), and the like. In addition, CHO-K1 strain (ATCC CCL-61), DUXB11 strain (ATCC CRL-9096), Pro-5 strain (ATCC CRL-1781) registered in ATCC, commercially available CHO-S strain (Lifetechnologies Cat #) 11619), or substrains obtained by acclimating these strains to a medium capable of growth.
  Rat myeloma cell line YB2 / 3HL. P2. G11.16 Ag. The parent cell of 20 cells includes a cell line established from Y3 / Ag1.2.3 cells (ATCC CRL-1631). Specific examples thereof include J. Org. Cell. Biol. ,93576 (1982), Methods Enzymol. ,73B, 1 (1981) and the like described in the literature such as YB2 / 3HL. P2. G11.16 Ag. There are 20 cells. In addition, YB2 / 3HL. P2. G11.16 Ag. Examples thereof include 20 cells (ATCC CRL-1662) or sub-strains obtained by acclimating these strains to a medium capable of growth.
  As a lectin that recognizes a sugar chain structure in which the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal and the 1-position of fucose are α-bonded, any lectin that can recognize the sugar chain structure can be used. Lectins are also included. Specific examples thereof include lentil lectin LCA (Lens  CulinarisLentil Agglutinin), pea lectin PSA (Pisum  sativumPea Lectin), broad bean lectin VFA (Vicia  fabaDerived from Agglutinin), Hilochawantake lectin AAL (Aleuria  aurantia(Lectin of origin) and the like.
  In the present invention, the α1,6-fucose / lectin-resistant cell may be any cell as long as growth is not inhibited in the presence of a certain effective concentration of lectin. For example, the activity of at least one protein listed below Are cells that are reduced or deleted from the parent cell line.
  (A) an enzyme protein involved in the synthesis of intracellular sugar nucleotide GDP-fucose (hereinafter referred to as “GDP-fucose synthase”);
  (B) Enzyme protein involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing terminal (hereinafter referred to as “α1,6-fucose modifying enzyme”) );
  (C) Protein involved in transport of intracellular sugar nucleotide GDP-fucose to the Golgi apparatus (hereinafter referred to as “GDP-fucose transport protein”).
  The GDP-fucose synthase includes any enzyme that is involved in the synthesis of the sugar nucleotide GDP-fucose, which is the source of fucose to the sugar chain in the cell, and the synthesis of the intracellular sugar nucleotide GDP-fucose. Enzymes that affect
  The intracellular sugar nucleotide GDP-fucose is supplied by a de novo synthesis route or a salvage synthesis route. Therefore, all enzymes involved in these synthetic pathways are included in the GDP-fucose synthase.
  The 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).
  Examples of the GDP-fucose synthase involved in the Salvage synthesis pathway include GDP-beta-L-fucose pyrophosphorylase (GDP-beta-L-fucose-pyrophosphorylase; hereinafter referred to as GFPP), Fucokinase (fucokinase) and the like. 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 intracellular sugar nucleotide GDP-fucose include the substances that affect the activity of the enzyme involved in the above-described intracellular sugar nucleotide GDP-fucose synthesis pathway, and substances that serve as substrates for the enzyme. Enzymes that affect structure are also included.
  The α1,6-fucose modifying enzyme includes any enzyme as long as it is an enzyme involved in a reaction in which the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end and the 1-position of fucose are α-bonded. The As an enzyme involved in the reaction of α-bonding the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end and the 1-position of fucose, N-acetylglucosamine at the N-glycosidically linked sugar chain reducing end Any enzyme can be used as long as it affects the reaction of α-bonding the 6th position of 1 and the 1st position of fucose. Specific examples include α1,6-fucosyltransferase and α-L-fucosidase. Examples of α1,6-fucosyltransferase include α1,6-fucosyltransferase having the amino acid sequence represented by SEQ ID NO: 4 or 5.
  In addition, as an enzyme that affects the reaction in which the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end and the 1-position of fucose are α-bonded, the above-mentioned N-glycoside-linked complex sugar chain reducing end Also included are enzymes that affect the activity of the enzyme involved in the α-bonding of the 1-position of fucose to the 6-position of N-acetylglucosamine and the structure of the substance that is the substrate of the enzyme.
  The GDP-fucose transport protein includes any protein as long as it is a protein involved in the transport of intracellular sugar nucleotide GDP-fucose to the Golgi body, and specifically includes a GDP-fucose transporter.
  In addition, proteins that affect the reaction of transporting intracellular sugar nucleotide GDP-fucose into the Golgi are also included in the GDP-fucose transport protein. Specifically, transport of the above-mentioned intracellular sugar nucleotide GDP-fucose to the Golgi body. Examples include proteins that affect the activity of proteins involved in or 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 it can select α1,6-fucose / lectin resistant cells. Specific examples include a technique for reducing or deleting the activity of the above-described protein. As a technique for reducing or deleting the activity of the above-mentioned protein,
  (A) a gene disruption technique targeting protein genes;
  (B) a technique for introducing a dominant negative form of a protein gene;
  (C) a technique for introducing a mutation in a protein;
  (D) a technique for suppressing transcription or translation of a protein gene;
  (WO02 / 31140, WO03 / 085107, WO03 / 085118).
  In the present invention, N-glycoside-linked complex type sugar chain reducing terminal N-acetylglucosamine 6-position N-acetylglucosamine, the cell where the genomic gene of the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked is knocked out -Cells in which the expression or function of the genome gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of the glycoside-linked complex is completely suppressed. It is done. Therefore, in the present invention, the cell in which the genomic gene is knocked out can be obtained by using the method described below, wherein the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end. Examples include cells in which the genomic gene of an enzyme involved in sugar chain modification is knocked out.
  A specific example of a cell in which a genomic gene has been knocked out is a cell in which all or part of the target gene has been deleted from the genome. As a method for obtaining such cells, any method can be used as long as the target genome can be modified, but a genetic engineering method is desirable. Specific examples of the technique include a technique for reducing or deleting the activity of the enzyme described above.
  Moreover, by using the above-mentioned method of selecting a cell line resistant to a lectin that recognizes a sugar chain structure in which the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain and the 1-position of fucose are α-linked. A cell in which the genomic gene of the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-bonded to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out can be selected.
  Examples of the antibody composition in the present invention include an antibody composition against an antigen expressed in a cell associated with a disease or an antigen involved in pathogenesis such as proliferation and metastasis of a cell associated with a disease. Specifically, GD2, GD3, GM2, HER2, CD20, CD22, CD25, CD33, CD52, MAGE, HM1.24, 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, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCam), insulin Antibody composition against insulin-like growth factor, insulin-like growth factor receptor, PMSA, vascular endothelial growth factor, vascular endothelial growth factor receptor, respiratory syncytial virus (RSV), IL-5 receptor α chain, etc. It is done.
  Specific examples of the antibody composition include an anti-GD2 antibody [Anticancer Res.,13331 (1993)], anti-GD3 antibody [Cancer Immunol. Immunother.],36, 260 (1993), WO01 / 23432, US6437098], anti-GM2 antibody [Cancer Res.,54, 1511 (1994), US6423511], anti-HER2 antibody [Procedure of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),894285 (1992)], anti-CD20 antibody [Blood,83, 435 (1994), WO 03/55993], anti-CD22 antibody [Semimin. Oncol.,30, 253 (2003)], anti-CD25 antibody [Human Antibodies],10127-142 (2001)], anti-CD33 antibody [J. Clin. Oncol.],193244 (2001)], anti-CD52 antibody [Procedure of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),894285 (1992)], anti-MAGE antibody [British J. Cancer,83493 (2000)], anti-HM1.24 antibody [Molecular Immunol.,36, 387 (1999)], anti-CCR4 antibody [WO01 / 64754, WO03 / 18635, WO00 / 42074, WO99 / 15666, US62453332], anti-parathyroid hormone-related protein (PTHrP) antibody [Cancer,88, 2909 (2000)], anti-fibroblast growth factor 8 antibody [Procedure of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),86, 9911 (1989), WO 03/002608], anti-fibroblast growth factor 8 receptor antibody [J. Biol. Chem.],265, 16455 (1990)], anti-epidermal growth factor receptor antibody [Cancer Res.,59, 1236 (1999)], anti-epithelial cell adhesion molecule antibody [Procedure of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA),761438 (1979)], anti-insulin-like growth factor antibody [J. Neurosci. Res.,40, 647 (1995), WO 03/93317], anti-insulin-like growth factor receptor antibody [J. Neurosci. Res.],40, 647 (1995)], anti-PMSA antibody [J. Urology, 160, 2396 (1998)], anti-vascular endothelial growth factor antibody [Cancer Research.574593 (1997)], anti-vascular endothelial cell growth factor receptor antibody [Oncogene,19, 2138 (2000)], anti-RSV antibody [Antiviral Research.47, 57-77 (2000)], anti-IL-5 receptor α chain antibody (US6018032, US65538111) and the like. Specific antibody names include Herceptin, Rituxan, Campath, Avastin, Bexar, Lymphoside, Mylotarg, Panorex, Zevalin [Nature. Rev. Cancer,1, 118 (2001)], Zenapax, Simulect, Remicade, Synagis, ReoPro [Human Antibodies,10, 127-142 (2001)], Enbrel [Expert Opinion on Pharmacotherapy,21137-1148 (2001)], Zolair [Respiratory Medicine,97, 123-129 (2003)].
  In the present invention, ADCC activity is⁵¹By Cr release method, lactate dehydrogenase (LDH) release method, flow cytometry, etc.in vitroUsing a measurement system or animal modelin vivoIt is possible to measure with an evaluation system.
  In the present invention, of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50 Examples of the drug used together with the antibody composition of at least% include cytokines, proteins such as antibodies, low molecular drugs, biological response modifiers (hereinafter referred to as BRM), and the like.
  Cytokines include interleukins (IL), colony stimulating factor (CSF), interferon (IFN), tumor necrosis factor (TNF), chemokines, or combinations thereof. Specifically, IFN-α, IFN-β, IFN-γ, TNF-α, TNF-β, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL -7, IL-9, IL-12, IL-15, IL-17, IL-18, IL-21, fractalkine, GM-CSF, G-CSF, M-CSF, SCF, or combinations thereof . IL-2, IL-15, IFN-α, and IFN-γ are preferable.
  Examples of low molecular weight drugs include amifostine (ethiol) [amifostine (ethyl)], cisplatin (daplatin) (DTIC) [dacarbazine (DTIC)], dactinomycin (dactinomycin), mechloretamine (nitrogen mustard) nitrogenmustard)], streptozocin, cyclophosphamide, carmustine (BCNU) [admuthine (BCNU)], romustine (CCNU) , Doxo Bicine lipo (doxyl) [doxorubicinlipo (doxil)], gemcitabine (gemzar) [gemcitabine (gemzar)], daunorubicin (daunorubicin), daunorubicinpine (daunorubicinpine) (Cytarabine), etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, bleomycin (Paclitaxel (taxol)), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, carboplatin, carboplatin, carboplatin camptothecin), CPT-11, 10-hydroxy-7-ethyl-camptothecin (SN38) [10-hydroxy-7-ethyl-camptothecin (SN38)], floxuridine, fludarabine, hydroxyurea (urix) ), Ifosfamide (Ifosfamide), idarubicin, mesna, irinotecan, mitoxantrone, topotecan, leuprolide, megestol, megestol Purine (mercaptopurine), prikamycin, mitotane, pegasparagase, pentostatin, pipobroman, streptozocide (instreptozect) oside), test lactone (testolactone), thioguanine, thiotepa, uracil mustard (uracil mustard), vinorelbine (n), chlorambucil (n) Semestin, capecitabine, tomudex, azacitidine, UFT, oxaloplatin, gefitinib (Iressa) [gefitinib (ST) matinib (STI571)], amsacrine, all-trans retinoic acid, thalidomide, bexarotene (targretin) (ex), dexamethasole (ex) ) [Anatrozole (Alimidex)], leuplin, or a combination thereof. Preferably, vincristine, cyclophosphamide, etoposide, methotrexate, or a combination thereof is used.
  Examples of the antibody include the above-described antibodies.
  BRM in the present invention includes BCG, anaerobic corynebacteria, muramyl dipeptide, trehalose dimycolic acid and other bacterial preparations, pyran copolymer, MVE, poly I: C, pyrimidine, thymosin, thymrin, thymopoesin, pisibanil, fucoidan And krestin.
  The effect of the medicament of the present invention is, for example,in vitroIt can be examined by a cytotoxic activity measuring system.in vitroExamples of the cytotoxic activity measurement system include an ADCC activity measurement system. ADCC activity was impaired by contacting target cells expressing the antigen with effector cells such as peripheral blood mononuclear cells, monocytes, macrophages and granulocytes collected 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 target cells damaged is⁵¹It can be detected by a Cr release method, a method for detecting enzyme activity of a target cell, a detection method using a flow cytometer, and the like. The effect of the substance that activates the immunocompetent cell of the present invention or the substance having antitumor activity in the ADCC activity measurement system is determined by adding these substances in the ADCC activity measurement system, or by targeting the target cell, effector cell, or It can be measured by exposing these substances to both of them in advance for a certain period and observing the effect on ADCC activity.
  The effect of the medicament of the present invention was obtained using an animal model.in vivoIt can also be examined by measuring antitumor activity.
  Animal models include xenotransplantation models in which cultured cell lines derived from human cancer tissue are transplanted into immunodeficient mice such as nude mice, syngeneic transplantation models in which cultured mouse cancer cell lines are transplanted into wild-type mice having a normal immune system, etc. Can be given.
  A xenograft model can be prepared by transplanting human cancer cell lines to various sites such as subcutaneous, intradermal, intraperitoneal, intravenous, etc. of immunodeficient mice such as nude mice.
  By comparing the effects of single administration of an antibody, a substance that activates immunocompetent cells or a substance having antitumor activity, and the effect of the medicament of the present invention using the above animal model, the medicament of the present invention The antitumor effect of can be evaluated.
  The medicament of the present invention can be administered alone, but usually mixed together with one or more pharmacologically acceptable carriers, and any well known in the technical field of pharmaceutics 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, and oral administration or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous can be used. In the case of a preparation, intravenous administration can be preferably mentioned.
  Examples of the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  Liquid preparations such as emulsions and syrups include saccharides such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxybenzoic acid Preservatives such as esters, and flavors such as strawberry flavor and peppermint can be used as additives.
  Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy A binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, a plasticizer such as glycerin and the like can be used as additives.
  Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
  The injection is prepared using a carrier made of a salt solution, a glucose solution, or a mixture of both.
  Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  The spray is prepared using a carrier that does not irritate the drug itself or the recipient's oral cavity and airway mucosa, and disperses the drug as fine particles to facilitate absorption.
  Specific examples of the carrier include lactose and glycerin. Depending on the properties of the medicine and the carrier used, preparations such as aerosols and dry powders are possible. In these parenteral preparations, the components exemplified as additives for oral preparations can also be added.
  The dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 0.1-20 mg / kg as the amount of antibody per adult. The drug used in combination with the antibody is the same dose or less than the dose when used clinically alone.

FIG. 1 shows the enhancement effect of various cytokines on the ADCC activities of KM2760-1 and KM3060. The vertical axis represents cytotoxic activity (%). (2) indicates no cytotoxic activity, □ indicates IL-2 addition, and hatched lines indicate cytotoxic activity when IL-15 is added.
FIG. 2 shows the enhancing effect of various cytokines on ADCC activity of KM 3065 and Rituxan. The vertical axis represents cytotoxic activity (%). In the figure, ■ represents the absence of cytokine, □ represents the addition of IL-2, and the diagonal line represents the cytotoxic activity upon addition of IL-15.
FIG. 3 shows the enhancement effect of various cytokines on ADCC activity of KM 3065 and Rituxan. The vertical axis represents cytotoxic activity (%). In the figure, ■ represents the absence of cytokine, □ represents the addition of IFN-α, and the diagonal line represents the cytotoxic activity when IFN-γ was added.
Hereinafter, the present invention will be described in detail. This application claims the priority of the Japan patent application 2003-406589 for which it applied on December 4, 2003, and includes the content described in the specification and / or drawing of the said patent application.

第１図に結果を図示した。サイトカイン非添加時のＫＭ２７６０−１のＡＤＣＣ活性はＫＭ３０６０のＡＤＣＣ活性よりも高いが、サイトカインの添加によりさらに増強された。この結果は、抗体組成物中に含まれるＦｃ領域に結合する全Ｎ−グリコシド結合複合型糖鎖のうち、糖鎖還元末端のＮ−アセチルグルコサミンにフコースが結合していない糖鎖の割合が５０％以上である抗体組成物の高いＡＤＣＣ活性は、サイトカインによってさらに上昇することを示す。Combined effect of anti-CCR4 antibody and cytokine on cytotoxicity in vitro
The combined effect of anti-CCR4 human chimeric antibodies KM2760-1 and KM3060 (WO02 / 31140) and cytokines in in vitro cytotoxic activity 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 expression strain into the rat myeloma cell line YB2 / 0 cells. The KM2760-1 # 58-35-16 strain has lectin resistance, and the sugar chain content to which fucose in all the sugar chains of KM2760-1 does not bind is 87%. On the other hand, KM3060 is an anti-CCR4 chimeric antibody produced from the cell line 5-03 obtained by introducing an anti-CCR4 chimeric antibody expression strain into the CHO / DG44 cell line. KM3060 does not have lectin resistance, and the glycan content in which fucose in all glycan chains of KM3060 does not bind is 8%. KM2760-1 and KM3060 have the same amino acid sequence and antigen binding activity is also equivalent.
(A) Preparation of effector cell solution 50 mL of healthy human venous blood was collected and 0.5 mL of heparin sodium (manufactured by Shimizu Pharmaceutical Co., Ltd.) was added and gently mixed. This was centrifuged (400 g, 20 minutes) using a MONO-POLY separation solution (manufactured by Dainippon Pharmaceutical Co., Ltd.) to separate the mononuclear cell layer. After washing by centrifugation 3 times with RPMI1640-FCS (5) medium, the suspension was resuspended at a concentration of 3 × 10 ⁶ cells / mL using the same medium to obtain an effector cell solution.
(B) Stimulation of effector cells by cytokines 50 μL of the effector cell solution obtained in (a) above was dispensed into 96-well U-bottom plates (Falcon). Further, 50 μL of the following solutions diluted with RPMI 1640-FCS (5) were added, and allowed to stand in a 5% CO ₂ incubator for 3 days.
(1) RPMI 1640-FCS (5) only (control without cytokine addition)
(2) IL-2 (manufactured by Peprotech) 2 ng / mL
(3) IL-15 (manufactured by Peprotech) 2 ng / mL
(C) Preparation of target cell solution CCR4 / cultured in RPMI 1640-FCS (10) medium (RPMI 1640 medium (GIBCO BRL) containing 10% FCS) containing G418 (Nacalai Tesque) at 0.5 mg / mL EL4 cells (mouse thymoma cells introduced with CCR4 gene, WO01 / 64754) were washed with RPMI1640-FCS (5) medium (RPMI1640 medium (GIBCO BRL) containing 5% FCS) by centrifugation and suspension. Then, it adjusted to 2 * 10 < ⁵ > cell / mL with RPMI1640-FCS (5) culture medium, and it was set as the target cell solution.
(D) Measurement of cytotoxic activity 50 μL (1 × 10 ⁴ cells / well) of the target cell solution prepared in (c) was dispensed into each well of the 96-well U-bottom plate prepared in (b). At this time, the ratio of effector cells to target cells is 15: 1. Further, KM2760-1 or KM3060 was added so as to have a final concentration of 1 ng / mL or 100 ng / mL to make a total volume of 200 μL, and the mixture was reacted at 37 ° C. for 4 hours. After the reaction, the plate was centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant was measured using CytoTox96 Non-Radioactive Cytotoxicity Assay (manufactured by Promega) by acquiring absorbance data according to the attached instructions. did. Absorbance data of spontaneous release of target cells uses only medium instead of effector cell solution and antibody solution. Absorbance data of spontaneous release of effector cells uses only medium instead of target cell solution and antibody solution. Obtained by performing the same operation as above. Absorbance data for target cell total release was obtained by using medium instead of antibody solution and effector cell solution, adding 15 μL of 9% Triton X-100 solution 45 minutes before the end of the reaction, and performing the same operation as above. It was determined by measuring the LDH activity of Qing. ADCC activity was determined by the following formula.

The results are shown in FIG. The ADCC activity of KM2760-1 when no cytokine was added was higher than the ADCC activity of KM3060, but was further enhanced by the addition of cytokine. As a result, the ratio of sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain out of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition is 50. The high ADCC activity of an antibody composition that is greater than or equal to% indicates that it is further increased by cytokines.

Effect of combined use of anti-CD20 antibody and cytokine on in vitro cytotoxic activity
The combined effect of the anti-CD20 human chimeric antibody KM 3065 (WO03 / 55993) and the commercially available anti-CD20 human chimeric antibody Rituxan (Genentech) and cytokine in the in vitro cytotoxic activity was measured by the method described in Example 1.
Cytokines are IL-2, IL-15 (all manufactured by Peprotech, final concentration 1 ng / mL), IFN-α (manufactured by Peprotech, final concentration 100 ng / mL), IFN-γ (manufactured by R & D Systems, final concentration 100 ng). / ML) was used. KM 3065 is an anti-CD20 chimeric antibody produced from the cell line KM 3065 obtained by introducing an anti-CD20 chimeric antibody expression strain into the rat myeloma cell line YB2 / 0 cells. The KM 3065 strain has lectin resistance, and the sugar chain content to which fucose in all the sugar chains of KM 3065 does not bind is 96%. On the other hand, the sugar chain content in which fucose is not bound in all sugar chains of Rituxan is 6%. KM 3065 and Rituxan have the same amino acid sequence and antigen binding activity is also equivalent.
The results are shown in FIG. 2 and FIG. The ADCC activity of KM 3065 when no cytokine was added was higher than that of Rituxan, but was further enhanced by the addition of cytokine. As a result, the ratio of sugar chains in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain out of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition is 50. The high ADCC activity of an antibody composition that is greater than or equal to% indicates that it is further increased by cytokines.

  Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. A medicine using an antibody composition and at least one drug is expected to have a high therapeutic effect.

Claims (14)

Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. A medicament comprising a combination of an antibody composition and at least one drug. Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. A medicament for administering an antibody composition in combination with at least one drug. Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. A medicament for administering an antibody composition and at least one drug simultaneously or sequentially. Of all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition, the proportion of sugar chains in which fucose is not bound to N-acetylglucosamine at the sugar chain reducing end is 50% or more. The medicine according to any one of claims 1 to 3, which exhibits a higher therapeutic effect than when the antibody composition is administered alone. The medicament according to claim 4, wherein the high therapeutic effect is a high antibody-dependent cytotoxic activity. Antibody composition produced from a cell having resistance to a lectin that recognizes a sugar chain structure in which N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing terminal at position 6 of N-acetylglucosamine and position 1 of fucose are α-linked The medicine according to any one of claims 1 to 5, which is a product. An antibody in which the antibody composition is a sugar chain in which fucose is not bound to N-acetylglucosamine at the reducing end of the sugar chain among all N-glycoside-bonded complex sugar chains that bind to the Fc region contained in the antibody composition The pharmaceutical according to any one of claims 1 to 6, which is a composition. The sugar chain to which fucose is not bonded is a sugar chain in which the 1-position of the fucose is not α-bonded to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside bond complex type sugar chain. The medicament according to any one of the above. An antibody composition is produced from a cell in which the genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is knocked out. The medicament according to claim 7 or 8, which is an antibody composition. 10. The pharmaceutical according to claim 9, wherein the enzyme involved in the sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the N-glycoside-linked complex sugar chain reducing end is α1,6-fucosyltransferase. . 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 low-molecular drug, and a biological response modifier. The medicament according to claim 11, wherein the protein is a cytokine or an antibody. The medicament according to claim 12, wherein the cytokine is a cytokine selected from IFN-γ, IL-2 and IL-15. The medicament according to any one of claims 1 to 13, wherein the medicament is a therapeutic agent for a disease involving a tumor.

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