JPH0423997A - Monoclonal antibody - Google Patents
Monoclonal antibodyInfo
- Publication number
- JPH0423997A JPH0423997A JP2129680A JP12968090A JPH0423997A JP H0423997 A JPH0423997 A JP H0423997A JP 2129680 A JP2129680 A JP 2129680A JP 12968090 A JP12968090 A JP 12968090A JP H0423997 A JPH0423997 A JP H0423997A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- cells
- kato
- gom
- formula
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Landscapes
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Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明は、ＫＡＴＯ−ＩＩＩ細胞（ヒト印環胃癌細胞）
（以下ＫＡＴＯ−Ｉ１１と略称する）を認識するモノク
ローナル抗体、より詳しくはヒト胃癌細胞の免疫学的診
断、血中抗原測定、腫瘍特異的イメージング及びターゲ
ティング、癌治療等を可能にする新しいモノクローナル
抗体に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to KATO-III cells (human signet ring gastric cancer cells).
(hereinafter abbreviated as KATO-I11), more specifically, a new monoclonal antibody that enables immunological diagnosis of human gastric cancer cells, blood antigen measurement, tumor-specific imaging and targeting, cancer treatment, etc. .
日本人の癌患者の約１／４は胃癌であり、その早期発見
及び有効な治療法の開発が急務となっている。胃癌に特
異的に反応するモノクローナル抗体は癌の診断および治
療に大きな威力を発揮するものと考えられ、その開発が
望まれる。Approximately 1/4 of Japanese cancer patients have gastric cancer, and early detection and development of effective treatment methods are urgently needed. Monoclonal antibodies that specifically react with gastric cancer are thought to have great power in cancer diagnosis and treatment, and their development is desired.
近年、多くの腫瘍細胞、胎児細胞等を抗原として、哺乳
動物などに免疫することにより、正常細胞には反応せず
、癌細胞に特異的に反応するモノクローナル抗体の作成
が盛んに試みられている。In recent years, many attempts have been made to create monoclonal antibodies that specifically react with cancer cells but not with normal cells by immunizing mammals with many tumor cells, fetal cells, etc. as antigens. .
ＫＡＴＯ＝ｍは１９７８年に、胃癌患者より分離、純化
された胃印環細胞由来の株化癌細胞であり、今日代表的
な胃印環細胞癌として広く利用されている（Ｍ、 Ｓ
ｅｋｉｇｕｃｈｉ、 ｅｔ ａｌｌ、 Ｊａｐａｎ
、 Ｊ。KATO=m is a cancer cell line derived from gastric signet ring cells that was isolated and purified from a gastric cancer patient in 1978, and is widely used today as a typical gastric signet ring cell cancer (M, S
ekiguchi, et all, Japan
, J.
Ｅｘｐ、 Ｍｅｄ、、 ４８． ６１−６８ （１９
７８）） 。Exp, Med, 48. 61-68 (19
78)).
ＫＡＴＯ−ＩＩＩを免疫原吉したモノクローナル抗体の
作成は種々試みられているがＫＡＴ、Ｃ）−１１Ｉある
いは胃癌細胞に特異的な抗体は得られていない（Ｋ、
Ｉｍａｉ ｅｊ ａｌｌ、 Ｊ、 Ｉｍｍｕｎｏ
ｌ、、 １３２． ２９９２２９９７ （１９８４
）、 ； Ａ、 Ｙａｃｈｉ ｅｔ ａｌ、
、 Ｊ、 １ｍｍｕｎ。Various attempts have been made to create monoclonal antibodies that are immunogenic for KATO-III, but no antibodies specific to KAT, C)-11I or gastric cancer cells have been obtained (K,
Imai ej all, J, Immuno
l,, 132. 29922997 (1984
); A. Yachi et al.
, J, 1mmun.
１３２ ２９９７−３００４ （１９８４） ）。132 2997-3004 (1984)).
さらに、ヒト胃癌と反応する抗体としては、Ｃａｒｃｉ
ｎｏ Ｅｍｂｒｉｏｎｉｃ Ａｎｔｉｇｅｎ （ＣＥＡ
）を認識するモノクローナル抗体、Ｋｏｐｒｏｗｓｋｉ
らのＣＡ１９−９に反応するモノクローナル抗体、５
ｅｋｉｎｅらの開発した抗体２−７０等、多数作成され
ている（Ｂ、 ＦＡｆｋｉｎｓｏｎ ｅｔ ａｌ、
Ｃａｎｃｅｒ Ｒｅｓ、、 ４２． ４８２０−４
８２３（１９Ｂ２） ； Ｔ、 ５ｅｋｉｎｅ ｅ
ｌ ａｌ、、 Ｇａｎｎ、７５． １０６−１０８（１
９８３）等参照）。しかしながら、これまで胃癌に特異
的で抗原構造の解明されたモノクローナル抗体は知られ
ていない。Furthermore, as an antibody that reacts with human gastric cancer, Carci
no Embryonic Antigen (CEA
), a monoclonal antibody that recognizes
Monoclonal antibody that reacts with CA19-9 of et al., 5
Many antibodies have been created, such as antibody 2-70 developed by Ekinson et al. (B, FAfkinson et al.
Cancer Res, 42. 4820-4
823 (19B2); T, 5ekine e
l al,, Gann, 75. 106-108(1
983) etc.). However, to date, no monoclonal antibody specific for gastric cancer and whose antigenic structure has been elucidated has been known.
一方、下記式
％式％（１）
（式中ＧａｌはＤ−ガラクトース残基を、ＦｕｃはＬ−
フコース残基を、ＧｌｃＮＡｃはＮ−アセチル−Ｄ−グ
ルコサミン残基をそれぞれ示し、Ｒは糖脂質を示す。）
で表わされる構造を有する糖脂質は、ウサギの小腸に存
在することが知られているが、正常のヒト細胞には発現
されておらず、ＫＡＴＯ−Ｉ［［にのみ、その存在が認
められており、癌関連糖鎖抗原としての可能性が考えら
れている（Ｍ、 Ｅ、 Ｂｒｅｉｍｅｒ ｅｔａｌ、
、 Ｂｉｏｍｅｄ、 Ｍａｓｓ Ｓｐｅｃｊｒｏｍ、
、 ６． ２３１−２４１（１９７９） ； Ｍ、
Ｂｉａｓｚｃｚｙｋ ｅｔ、ａｌ、、Ｊ、 Ｂｉｏ
ｉ Ｃｈｅｍ２６２、 ３７２−３７９ （１９８７
））。On the other hand, the following formula % formula % (1) (in the formula, Gal represents a D-galactose residue, Fuc represents a L-
GlcNAc represents a fucose residue, GlcNAc represents an N-acetyl-D-glucosamine residue, and R represents a glycolipid. ) is known to exist in the small intestine of rabbits, but it is not expressed in normal human cells, and its presence has been observed only in KATO-I. and is considered to have the potential as a cancer-related carbohydrate antigen (M, E, Breimer et al.
, Biomed, Mass Specjrom,
, 6. 231-241 (1979); M.
Biaszczyk et al., J. Bio.
i Chem262, 372-379 (1987
)).
しかるに、上記式（１）で示される糖鎖構造を非還元末
端に有する化合物に特異的に反応するモノクローナル抗
体は、現在のところ得られていない。前記Ｋｏｐｒｏｗ
ｓｋｉ らが開発したモノクローナル抗体の中に、一部
上記式（１）で示される糖鎖構造と反応する抗体が含ま
れているが、該抗体はむしろＢ型糖鎖、あるいはＬｅＹ
型糖鎖により強く反応し、かならずしも上記糖鎖構造に
特異的に反応する抗体とは言えず、真に特異性の高い抗
体の開発が望まれている（Ｇ、 Ｃ，Ｈａｎｓｓｏｎ
ｅｔ ａｌ、 ＪＢｉｏｌ、 Ｃｈｅｍ ２
５８ ４０９１４１９８３） ： Ｍ、 Ｂｌ
ａｓｚｃｚｙｋｅｔ ａｌ Ｊ Ｂｉｏｌ Ｃｈ
ｅｍ、 ２６２３７２−３７９ （１９８７））。However, a monoclonal antibody that specifically reacts with a compound having a sugar chain structure represented by the above formula (1) at its non-reducing end has not yet been obtained. Said Koprow
Some of the monoclonal antibodies developed by Suki et al. contain antibodies that react with the sugar chain structure represented by the above formula (1), but these antibodies are more likely to react with B-type sugar chains or LeY.
The development of truly highly specific antibodies is desired, as they react more strongly with the type of sugar chains and cannot necessarily be said to specifically react with the above sugar chain structures (G, C, Hansson
et al, JBiol, Chem 2
58 409141983): M, Bl
aszczyket al J Biol Ch.
Em, 262372-379 (1987)).
一方式
％式％（２）
（Ｇａｌ、ＧｌｃＮＡｃ、Ｆｕｃ及びＲは前記式（１）
に同じ。）
で示される糖鎖構造を非還元末端に有する化合物を認識
するモノクローナル抗体は、数種市販されているが、こ
れらはいずれも１ｇＭ型の抗体である。しかして、抗体
はそのアイソタイプによって生理作用が異なり、ＩｇＧ
型の抗体はエフェクター細胞を介した細胞障害作用を発
現するが、１ｇＭ型にはその作用がない。また同じＩｇ
Ｇ型抗体でも１ｇＧ３型は補体と結合して標的細胞を破
壊する作用を有するが、１ｇＧ１と１ｇＧ４型にはその
作用がない。以上の点から上記式（２）の構造を認識す
る１ｇＧ３型のモノクローナル抗体の開発が望まれてい
る。One formula % Formula % (2) (Gal, GlcNAc, Fuc and R are from the above formula (1)
Same as . ) There are several types of monoclonal antibodies on the market that recognize compounds having the sugar chain structure shown at the non-reducing end, but all of these are 1gM type antibodies. However, antibodies have different physiological actions depending on their isotype, and IgG
This type of antibody exhibits a cytotoxic effect via effector cells, but the 1gM type has no such effect. Same Ig again
Among G-type antibodies, the 1gG3 type has the effect of binding to complement and destroying target cells, but the 1gG1 and 1gG4 types do not have this effect. From the above points, it is desired to develop a 1gG3 type monoclonal antibody that recognizes the structure of formula (2) above.
また式
Ｇａｌβｌ→３ＧＩｃＮＡｃ→Ｒ（３）（式中Ｇａｌ、
ＧｌｃＮＡｃ及びＲは前記式（１）に同じ。）
で示される糖鎖構造を非還元末端に有する化合物を認識
する抗体も報告されているが、容易に入手できず、更に
高感度なモノクローナル抗体の開発が望まれている。Also, the formula Galβl→3GIcNAc→R(3) (in the formula, Gal,
GlcNAc and R are the same as in formula (1) above. ) Antibodies that recognize compounds having the sugar chain structure shown at the non-reducing end have also been reported, but they are not easily available, and there is a desire to develop monoclonal antibodies with even higher sensitivity.
本発明は上記所望の目的を達成するための新規なモノク
ローナル抗体を提供することをその目的とする。An object of the present invention is to provide a novel monoclonal antibody for achieving the above-mentioned desired objects.
課題を解決する為の手段
本発明者らは胃癌細胞により強く、特異的に反応するモ
ノクローナル抗体を得るために鋭意研究を重ねた結果、
株化胃癌細胞として代表的なＫＡＴＯ−ＩＩＩに反応性
を有するモノクローナル抗体を得、本発明を完成させた
。Means for Solving the Problems The present inventors have conducted intensive research to obtain monoclonal antibodies that react more strongly and specifically with gastric cancer cells.
The present invention was completed by obtaining a monoclonal antibody that is reactive with KATO-III, which is a typical gastric cancer cell line.
本発明によればＫＡＴＯ−ｍを認識するモノクローナル
抗体であって、下記（イ）、（ロ）及び（ハ）のいずれ
か少なくともひとつの化合物に特異反応性を有するモノ
クローナル抗体が提供される。According to the present invention, a monoclonal antibody that recognizes KATO-m and has specific reactivity with at least one of the following compounds (a), (b), and (c) is provided.
（イ）式
％式％
（Ｒユは糖、糖脂質又は糖蛋白質を示す。）で示される
糖鎖構造を非還元末端に有する化合物、（ロ）式
％式％（）
（Ｒ２は糖、糖脂質又は糖蛋白質を示す。）で示される
糖鎖構造を非還元末端に有する化合物及び（ハ）式
％式％（）
（Ｒ３は糖、糖脂質又は糖蛋白質を示す。）で示される
糖鎖構造を非還元末端に有する化合物。(a) A compound having a sugar chain structure represented by the formula % formula % (R represents a sugar, glycolipid, or glycoprotein) at the non-reducing end; Compounds having a sugar chain structure represented by the formula % (representing a glycolipid or glycoprotein) at the non-reducing end; A compound that has a chain structure at the non-reducing end.
（但し、各式中ＧａｌはＤ−ガラクトース残基を、Ｆｕ
ｃはＬ−フコース残基を、また
ＧｌｃＮＡｃはＮ−アセチル−Ｄ−グルコサミン残基を
それぞれ示す。）
本発明抗体は、ＫＡＴＯ−Ｈに反応性を有する。(However, in each formula, Gal represents a D-galactose residue, Fu
c represents an L-fucose residue, and GlcNAc represents an N-acetyl-D-glucosamine residue. ) The antibody of the present invention has reactivity with KATO-H.
即ち、ＫＡＴＯ−ＩＩＩの細胞膜に存在する糖鎖に特異
的に反応することをその最大の特徴としており、さらに
本発明抗体は正常血球細胞とは反応しない。That is, the greatest feature of the antibody is that it specifically reacts with sugar chains present in the cell membrane of KATO-III, and furthermore, the antibody of the present invention does not react with normal blood cells.
以下、本発明モノクローナル抗体の製造法につき詳述す
る。The method for producing the monoclonal antibody of the present invention will be described in detail below.
本発明抗体は、ＫＡＴＯ−ｍを免疫抗原として製造でき
る。上記ＫＡＴＯ−ｍを免疫抗原として利用した抗体の
製造は、常法に従うことができる（Ｒａｎ日ａｎｄ、
Ｐ、、 Ｃｈｅｍ、 Ｐｈｙｓ、 Ｌｉｐｉｄ
ｓ、 １５． １０５（１９７５） ； Ｈａｎｆ
ｌａｎｄ、 Ｐ、、 Ｃｂｅｍ、Ｐｈｙｓ、 Ｌｉｐ
ｉｄｓｌｏ、 ２０１ （１９７６） ； Ｋｏｓｃｉ
ｅｌａｋ、 Ｊ、、 Ｂｕｒ、 ＪＢｉｏｃｈｅｍ
、 ３７ ２１４ （１９７８） ）。The antibody of the present invention can be produced using KATO-m as an immunizing antigen. Antibodies can be produced using the above-mentioned KATO-m as an immunizing antigen according to conventional methods.
P, Chem, Phys, Lipid
s, 15. 105 (1975); Hanf
land, P,, Cbem, Phys, Lip
idslo, 201 (1976); Kosci
elak, J., Bur, J.Biochem
, 37 214 (1978)).
該方法は、より具体的には、例えば上記免疫抗原で免疫
した哺乳動物の形質細胞（免疫細胞）と哺乳動物の形質
細胞腫細胞との融合細胞（ｈ７ｂｒｉｄｏｍａ）を作成
し、これよりＫＡＴＯ−ｍを認識する所望抗体を産生ず
るクーロンを選択し、該クーロンの培養により製造でき
る。More specifically, in this method, for example, a fusion cell (h7bridoma) of a mammalian plasma cell (immune cell) immunized with the above-mentioned immunizing antigen and a mammalian plasmacytoma cell is created, and from this KATO-m The antibody can be produced by selecting a clone that produces the desired antibody that recognizes the antibody and culturing the clone.
また、本発明の抗体は抗体産生ハイブリドーマ培養上清
あるいはマウス腹水を抗体溶液としてそのままで使用で
きるものであり、さらには硫酸アンモニウム分画やイオ
ン交換クロマトグラフィーあるいはプロティンＡカラム
などによるアフイニティクロマトグラフイーにより精製
したものも抗体溶液として使用することも可能である。Furthermore, the antibody of the present invention can be used directly as an antibody solution using antibody-producing hybridoma culture supernatant or mouse ascites, and can also be used by affinity chromatography using ammonium sulfate fractionation, ion exchange chromatography, protein A column, etc. Purified products can also be used as antibody solutions.
上記方法において用いられる免疫抗原としては、ＫＡＴ
Ｏ−ｍ細胞を使用することが出来るほか、式（イ）、式
（ロ）及び式（ハ）に示す化合物を免疫抗原として、こ
れを適当なアジュバントと混合して使用することも出来
る。又、免疫される哺乳動物としては、特に制限はない
が、細胞融合に使用する形質細胞腫細胞との適合性を考
慮して選択するのが好ましく、一般にはマウス、ラット
等が有利に用いられる。The immunizing antigen used in the above method includes KAT
In addition to using O-m cells, compounds represented by formulas (a), (b), and (c) can also be used as immunizing antigens, mixed with appropriate adjuvants. There are no particular restrictions on the mammal to be immunized, but it is preferably selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and mice, rats, etc. are generally advantageously used. .
免疫は一般的方法により、例えば上記免疫抗原を哺乳動
物に静脈内、皮肉、皮下、腹腔内注射等により投与する
ことにより実施できる。Immunization can be carried out by a conventional method, for example, by administering the above-mentioned immunizing antigen to a mammal by intravenous, subcutaneous, subcutaneous, or intraperitoneal injection.
免疫は、例えばマウスの場合、免疫抗原をリン酸緩衝化
生理食塩水（Ｐ Ｂ Ｓ）や生理食塩水等で適当な濃度
に希釈し、所望により通常のアジュバントと併用して、
供試動物２〜１４日毎に数回投与し、総投与量が１００
〜５００μｇ／マウス程度になるようにして実施するの
が好ましい。最終免疫後、摘出した牌臓より所望の免疫
細胞を収得できる。尚、上記アジュバントとしては百日
咳ワクチン、完全又は不完全フロインドアジュバント、
あるいはアラムを用いるとよい。For example, in the case of mice, immunization is carried out by diluting the immunizing antigen to an appropriate concentration with phosphate buffered saline (PBS), physiological saline, etc., and using it in combination with a usual adjuvant if desired.
The test animals were administered several times every 2 to 14 days, with a total dose of 100
It is preferable to carry out the experiment at a concentration of about 500 μg/mouse. After the final immunization, desired immune cells can be obtained from the excised spleen. In addition, the above adjuvant includes pertussis vaccine, complete or incomplete Freund's adjuvant,
Alternatively, use arum.
上記免疫細胞と融合される他方の親細胞としての哺乳動
物の形質細胞腫細胞としては、既に公知ノ種々ノモノ、
例エバＰ３／Ｘ６３−Ａｇ３ （Ｘ６３）（Ｎａｔｕｒ
ｅ、 ２５６． ４９５−４９７ （１９７５））
、Ｐ３／Ｘ６３−Ａｇ８Ｕｌ （Ｐ２Ｏ３） （Ｃ
ｕｒｒｅｎｔ Ｔｏｐｉｃｓ ｉｎ Ｍｉｃｒｏｂｉ
ｏｌｏｇｙａｎｄ ！ｍｍｕｎｏｌｏｇＬ ８１．
Ｉ−７（１９７８）） 、Ｐ３／ＮＳＩＩＡＮＳ
ｌｌＡｇ４−１（（Ｅｕｒ、 Ｊ、 １ｍｍｕｎｏ
１．、 ６． ５１１−５１９（１９７６）） 、Ｓｐ
２１０−Ａｇ１４（Ｓｐ２１０） （Ｎａｔｕｒｅ、
２７６２６９−２７０ （１９７ｇ）） 、Ｆ
Ｏ（Ｌ １ｍｍｕｎｏ、 Ｍｅｌｈ、 ３５１
−２１ （１９８０） ）等や、ラットにおける２１０
、 ＲＣＹ３． Ａｇ１．２．３． （Ｙ３） （Ｎ
ａｔｕｒｅ、 ２７７、 １３１−１３３． （１
９７９））等の骨髄腫細胞等を使用できる。As the mammalian plasmacytoma cells as the other parent cells to be fused with the above-mentioned immune cells, various known cells,
Example Eva P3/X63-Ag3 (X63) (Natur
e, 256. 495-497 (1975))
, P3/X63-Ag8Ul (P2O3) (C
Current Topics in Microbi
ologyand! mmunologL 81.
I-7 (1978)), P3/NSIIANS
llAg4-1((Eur, J, 1mmuno
1. , 6. 511-519 (1976)), Sp
210-Ag14(Sp210) (Nature,
276269-270 (197g)), F
O(L 1mmuno, Melh, 351
-21 (1980)) and 210 in rats.
, RCY3. Ag1.2.3. (Y3) (N
ture, 277, 131-133. (1
Myeloma cells such as 979)) can be used.
上記免疫細胞と形質細胞腫細胞との融合反応は、公知の
方法、例えばＭｉｌｓｔｅｉｎらの方法（Ｍｅｔｈｏｄ
ｉｎＥｎｚ７ｍθＩｏｇ７．７３．３ （１９１１１
））等に準じて行なうことができる。より具体的には上
記融合反応は、通常の融合促進剤、例えばポリエチレン
グリコール（ＰＥＧ）、センダイウィルス（ＨＶＪ）等
の存在下に、通常の培地中で実施され、培地には更に融
合効率を高めるためにジメチルスルホキシド等の補助剤
を必要に応じて添加することもできる。The above-mentioned fusion reaction between immune cells and plasmacytoma cells can be carried out using known methods, such as the method of Milstein et al.
inEnz7mθIog7.73.3 (19111
)) etc. More specifically, the above fusion reaction is carried out in a conventional medium in the presence of conventional fusion promoters, such as polyethylene glycol (PEG), Sendai virus (HVJ), etc., and the medium further enhances the fusion efficiency. For this purpose, an auxiliary agent such as dimethyl sulfoxide may be added as necessary.
また、電気処理（電気融合）による方法等を適宜採用す
ることもできる。免疫細胞と形質細胞腫細胞との使用比
は、通常の方法と変わりなく、例えば形質細胞腫細胞に
対して免疫細胞を約１〜１０程度度用いるのが普通であ
る。融合反応時の培地としては、形質細胞腫細胞の増殖
に通常使用される各種のもの、例えばＲＰＭＩ−１６４
０培地、ＭＥＭ培地、その他のこの種細胞培養に一般に
利用されるものを例示でき、通常これら培地は牛胎児血
清（Ｆ ＣＳ）等の血清補液を抜いておくのがよい。Further, a method using electrical processing (electrofusion) or the like may be appropriately employed. The ratio of immune cells to plasmacytoma cells used is the same as in conventional methods; for example, the ratio of immune cells to plasmacytoma cells is usually about 1 to 10 times. As a medium for the fusion reaction, various media commonly used for proliferation of plasmacytoma cells, such as RPMI-164, can be used.
Examples include 0 medium, MEM medium, and other media commonly used for this type of cell culture, and it is usually preferable to remove serum supplements such as fetal calf serum (FCS) from these media.
融合は上記免疫細胞と形質細胞腫細胞との所定量を、上
記培地内でよく混合し、予め３７℃程度に加温したＰＥ
Ｇ溶液、例えば平均分子量１０００〜６０００程度のも
のを、通常培地に約３０〜６０ｗ／ｖ％の濃度で加えて
混ぜ合わせることにより行なわれる。以後、適当な培地
を逐次添加して遠心し、上清を除去する操作を繰り返す
ことにより所望のハイブリドーマが形成される。For fusion, predetermined amounts of the above immune cells and plasmacytoma cells are mixed well in the above medium, and then heated to about 37°C in advance.
This is carried out by adding G solution, for example, one having an average molecular weight of about 1,000 to 6,000, to a normal medium at a concentration of about 30 to 60 w/v% and mixing. Thereafter, desired hybridomas are formed by repeating the steps of sequentially adding an appropriate medium, centrifuging, and removing the supernatant.
得られる所望のハイブリドーマの分離は、通常の選別培
地、例えばＨＡＴ培地（ヒポキサンチン、アミノプテリ
ンおよびチミジンを含む培地）で培養することにより行
なわれる。該ＨＡＴ培地での培養は、目的とするハイブ
リドーマ以外の細胞（未融合細胞等）が死滅するのに充
分な時間、通常数日〜数週間行なえばよい。かくして得
られるハイブリドーマは、通常の限界希釈法により目的
とする抗体の検索及び単一クーロン化に供される。The resulting desired hybridomas are isolated by culturing them in a conventional selection medium, such as HAT medium (a medium containing hypoxanthine, aminopterin, and thymidine). Culture in the HAT medium may be carried out for a sufficient period of time, usually from several days to several weeks, to kill cells other than the target hybridoma (unfused cells, etc.). The hybridoma thus obtained is subjected to the search for the desired antibody and single clonization using the conventional limiting dilution method.
目的抗体産生株の検索は、例えばＥＬＩＳＡ法（ＥＢｖ
＠ｌｌ、 Ｅ、、 Ｍｅｔｈ、 Ｅｎｘｙｍｏｌ、、
１θ、 ４１９−４３９（１９８０）） 、プラーク法
、スポット法、凝集反応法、オフタロニー（Ｏｕｃｈｔ
ｅｒｌ、ｏｎｙ）法、ラジオイムノアッセイ（ＲＩＡ）
法等の一般に抗体の検出に用いられている種々の方法（
「ハイブリドーマ法とモノクローナル抗体」、株式会社
Ｒ＆Ｄプラニング発行、第３０〜５３頁、昭和５７年３
月５日）に従い実施でき、この検索には前記免疫抗原が
利用できる。かくして得られるＮＡＴＯ−ｍを認識する
所望のモノクローナル抗体を産生ずるハイブリドーマは
、通常の培地で継代培養でき、また液体窒素中で長期保
存できる。Search for target antibody-producing strains can be performed, for example, by ELISA method (EBv
@ll, E,, Meth, Enxymol,,
1θ, 419-439 (1980)), plaque method, spot method, agglutination reaction method, Oucht.
erl, ony) method, radioimmunoassay (RIA)
Various methods commonly used for antibody detection, such as
"Hybridoma method and monoclonal antibodies", published by R&D Planning Co., Ltd., pp. 30-53, March 1982.
The above-mentioned immune antigen can be used for this search. The thus obtained hybridoma producing the desired monoclonal antibody that recognizes NATO-m can be subcultured in a conventional medium and can be stored for long periods in liquid nitrogen.
上記ハイブリドーマからの本発明モノクローナル抗体の
採取は、該ハイブリドーマを、常法に従って培養してそ
の培養上清として得る方法やハイブリドーマをこれと適
合性のある哺乳動物に投与して増殖させ、その腹水とし
て得る方法が採用される。前者の方法は、高純度の抗体
を得るのに適しており、後者の方法は、抗体の大量生産
に適している。また上記のごとくして得られる抗体は、
更に塩析、ゲル濾過法、アフィニティクロマトグラフィ
ー等の通常の手段により精製できる。The monoclonal antibody of the present invention can be collected from the above-mentioned hybridoma by culturing the hybridoma according to a conventional method and obtaining the culture supernatant, or by administering the hybridoma to a mammal compatible with the hybridoma and growing it, and then producing the ascites. The method of obtaining is adopted. The former method is suitable for obtaining highly purified antibodies, and the latter method is suitable for mass production of antibodies. In addition, the antibodies obtained as described above are
Further, it can be purified by conventional means such as salting out, gel filtration, and affinity chromatography.
かくして得られる本発明抗体は、これを利用して、例え
ば免疫沈降法、アフィニティクロマトグラフィー等の通
常の精製手段により式（イ）、式（ロ）及び式（ハ）で
示される糖鎖構造を有する化合物（抗原）を簡便且つ特
異的に精製することが可能である。The thus obtained antibody of the present invention can be used to obtain the sugar chain structures represented by formulas (a), (b), and (c) by ordinary purification methods such as immunoprecipitation and affinity chromatography. It is possible to easily and specifically purify a compound (antigen) containing the antigen.
更に本発明抗体の利用によれば放射免疫測定法（ＲＩＡ
）、酵素免疫測定法（ＥＬ Ｉ ＳＡ） 、凝集法等の
通常の免疫学的手段により、高感度、高精度に且つ高い
特異性をもって式（イ）、式（ロ）及び式（ハ）で示さ
れる抗原を簡易に測定することが出来る。Further, by using the antibodies of the present invention, radioimmunoassay (RIA) can be performed.
), enzyme-linked immunosorbent assay (ELISA), agglutination method, etc., with high sensitivity, high precision, and high specificity. The indicated antigen can be easily measured.
上記検定法において検体として用いられる体液としては
例えば血液、細胞組織液、リンパ液、胸水、腹水、羊水
、胃液、尿、膵液、髄液、唾液等を例示でき、これらの
うちでは血液、特に血清又は血漿が好ましい。Examples of body fluids used as specimens in the above assay include blood, tissue fluid, lymph, pleural fluid, ascites, amniotic fluid, gastric fluid, urine, pancreatic juice, cerebrospinal fluid, saliva, etc. Among these, blood, especially serum or plasma is preferred.
更に加えて、本発明抗体は液相系および固相系共に反応
性に優れ、液相系および固相系免疫検定法の適用に極め
て好ましい。更には本発明抗体は正常血球細胞に対し反
応しない為、癌の診断および／又はスクリーニングを正
確にすることが可能である。かかる本発明抗体を利用し
た精製系並びに測定系の設定、その改変ないし応用は当
業者にとり自明である。In addition, the antibodies of the present invention have excellent reactivity in both liquid-phase and solid-phase systems, and are extremely suitable for application to liquid-phase and solid-phase immunoassays. Furthermore, since the antibodies of the present invention do not react with normal blood cells, it is possible to accurately diagnose and/or screen for cancer. It will be obvious to those skilled in the art to set up a purification system and a measurement system using such an antibody of the present invention, and to modify or apply the same.
発明の効果
本発明によればＫＡＴＯ−ｍ細胞膜上の糖鎖を認識する
抗体が提供される。本発明抗体の利用によれば胃癌患者
等の血清診断、組織診断法、胃癌組織等のイメージング
法、胃癌部位等への薬物のターゲティング法、胃癌等の
抗体による治療法等が提供される。Effects of the Invention According to the present invention, an antibody that recognizes sugar chains on the KATO-m cell membrane is provided. Utilization of the antibodies of the present invention provides serological diagnosis of gastric cancer patients, tissue diagnosis methods, imaging methods of gastric cancer tissues, methods of targeting drugs to gastric cancer sites, methods of treating gastric cancer, etc. using antibodies, and the like.
実 施 例
本発明を更に詳しく説明するため、以下に実施例を挙げ
るが本発明はこれらに限定されるものではない。EXAMPLES In order to explain the present invention in more detail, Examples are given below, but the present invention is not limited thereto.
実施例 １
モノクローナル抗体の製造
■ ＫＡＴＯ−Ｉ１１細胞（ヒト印環胃癌細胞）はＭｉ
７ａｏｃｈｉ等の方法（Ｍｉ７ａｕｃｈｉ、 Ｔ、、
ｅｔ ａｔ。Example 1 Production of monoclonal antibody KATO-I11 cells (human signet ring gastric cancer cells) were Mi
The method of Mi7auchi et al. (Mi7auchi, T.,
et at.
Ｇａｎｎ、 ７３． ５８１−５８７ （１９ｇ２）
）に従って培養した。Gann, 73. 581-587 (19g2)
).
得られた培養癌細胞３×１０７個を８週齢のＢ Ａ Ｌ
Ｂ ／ ｃ雄性マウスの腹腔内に投与した。その後同
様に２回、３週問おきに追加投与して免疫した。最終免
疫の３日後に、牌臓を摘出した。摘出牌臓より上記免疫
したマウスの牌細胞を集め、３７℃に加温したＲＰＭＩ
培養液（ＲＰＭ１１６４０、日本製薬製、以下ＲＰＭＩ
培地と略称する。）で３回洗浄した。同様にマウス骨髄
腫瘍細胞（Ｐ３／ＮＳＩ−１ＮＳｌ−１−Ａ （Ｋｉｊ
ｈｌｅｔ、 Ｇ、 ＆ Ｍｉｌｓｌｅｉｎ。The obtained cultured cancer cells (3 x 107 cells) were transferred to 8-week-old BAL
B/c male mice were administered intraperitoneally. Thereafter, the animals were immunized twice in the same manner, with additional administration every 3 weeks. Three days after the final immunization, the spleens were removed. The tile cells of the immunized mice were collected from the removed spleen and heated to 37°C with RPMI.
Culture solution (RPM11640, manufactured by Nippon Pharmaceutical Co., Ltd., hereinafter referred to as RPMI
It is abbreviated as medium. ) was washed three times. Similarly, mouse bone marrow tumor cells (P3/NSI-1NSl-1-A (Kij
hlet, G., & Milslein.
Ｃ，、Ｅａｒ、 １． Ｉｍｍｕｎｏｌ、、 ６
： ５１１． １９７６） を洗浄した。これら、牌
細胞と骨髄腫瘍細胞を細胞数比５：１になるように５０
１１のチューブ内で混和し、２００ｇ、５分間遠心後、
上清をパスツールピペットで完全に除去した。これらの
操作は３７℃に保温した水槽内にて行なった。次に、ポ
リエチレングリコール１５００ （ベーリンガーマイン
ハイム山之内社製、以下ＰＥＧと略称する）２１１を加
え、ゆっくりと１〜２分間かき混ぜ１分間放置し、次い
で３゛７℃に保温した牛胎児血清（Ｆ ＣＳ）を含まな
いＲＰＭＩ培地１ ｘｉをゆっくりと１分間位かけて加
え、１分間放置し更に同波２ ｘｉを加えて２分間放置
し、更に同波４ ｘｉを加え４分間放置した。次いで、
３７℃に保温した１５％ＦＣ８゜０．０５℃力価／！の
硫酸ストレプトマイシン、６００００ｔＪ／ｌのペニシ
リンＧカリウム、５４■／！ゲンタマイシン及び１ｍＭ
ピルビン酸を含有するＲＰＭＩ−１，６４０培地（以下
、これを完全ＲＰＭＩ−１６４０培地という）８２１’
を２〜３分間かけて加えた後、２００ｇで５分間遠心分
離した。上清を吸引除去し、３７℃に保温した完全ＲＰ
ＭＩ培地に、稗細胞１×１０６個／ ｘｉとなるように
懸濁させた。次に、この懸濁液を９６穴プレート（コー
スタ−社製）に０．ｌｚｆずつ分注し、３７℃、５％Ｃ
０２，１００％湿度のインキュベータ内で培養した。２
４時間後、Ｑ、ｌｙＡ’ずつ１０％ＦＣ８添加ヒボキサ
ンチン１×１０−４Ｍ、アミノプテリン４ｘｌＯ−’Ｍ
及びチミジン１．６ｘｉｏ−５Ｍを含む完全ＲＰＭＩ培
地（以下、ＨＡＴ培地と略称する）を各ウェルに添加し
た。C,,Ear,1. Immunol, 6
: 511. 1976) was washed. These tile cells and bone marrow tumor cells were divided into 50 cells at a cell number ratio of 5:1.
After mixing in a tube of 11 and centrifuging at 200g for 5 minutes,
The supernatant was completely removed with a Pasteur pipette. These operations were performed in a water tank kept at 37°C. Next, polyethylene glycol 1500 (manufactured by Boehringer Meinheim Yamanouchi Co., Ltd., hereinafter abbreviated as PEG) 211 was added, stirred slowly for 1 to 2 minutes and left for 1 minute, then fetal bovine serum (FCS) kept at 3-7°C was added. ) was slowly added over a period of about 1 minute, and then allowed to stand for 1 minute, followed by adding 2 xi of same wave and leaving it for 2 minutes, and then adding 4 xi of same wave and leaving it for 4 minutes. Then,
15% FC8°0.05°C titer kept at 37°C/! of streptomycin sulfate, 60,000 tJ/l of penicillin G potassium, 54 ■/! Gentamicin and 1mM
RPMI-1,640 medium containing pyruvate (hereinafter referred to as complete RPMI-1640 medium) 821'
was added over 2-3 minutes, followed by centrifugation at 200g for 5 minutes. Aspirate the supernatant and keep the complete RP at 37°C.
Milia cells were suspended in MI medium at 1 x 10 6 cells/xi. Next, this suspension was placed in a 96-well plate (manufactured by Coaster) at 0.00%. Dispense 1zf at 37°C, 5%C
02, cultured in an incubator with 100% humidity. 2
After 4 hours, Q, lyA' each added 10% FC8-added hyboxanthin 1 x 10-4M, aminopterin 4xlO-'M
A complete RPMI medium (hereinafter abbreviated as HAT medium) containing 1.6 xio-5 M of thymidine was added to each well.
以後上清を２日目、３日目に０．１２７吸引し、０、ｌ
ｚｆの新しいＨＡＴ培地を加えて液替えを行なった。そ
の後、液替えは２〜３日おきに行なった。６日目に同様
に上清を吸引し、ｌＸｌ０’−４Ｍヒポキサンチン及び
１．６Ｘ１０−５Ｍチミジンを含む完全ＲＰＭＩ培地（
以下、ＨＴ培地と称する）に代えた。以後、完全ＲＰＭ
Ｉ培地で増殖維持した。融合後、１０〜１４日間でコロ
ニーが肉眼で観察されるようになり、細胞が９６ウエル
プレートの底面積の１／４を占めた時より、上清を免疫
抗原に用いたＫＡＴＯ−Ｉ［［を抗原とする酵素免疫測
定法（ＥＬＩＳＡ法）で試験し、陽性となったウェルか
ら直ちに限界希釈法によりクローニングを行なった。即
ち、Ｂ Ａ Ｌ Ｂ ／ ｃ系マウス胸腺細胞ｌＸＩＯ
３個を含むように調製した１０％ＦＣ８加ＲＰＭＩ−１
６４０培地の２０ｗ１を用いて、ハイブリドーマを３個
／ウェル、１個／ウェル及び０．３個／ウェルとなるよ
うに９６ウエルプレートに０．２ｘｌずつプレートに移
してクローニングし、目的とするハイブリドーマを樹立
した。Thereafter, the supernatant was aspirated at 0.127 on the second and third day, and 0,1
A new HAT medium of zf was added to perform a liquid change. Thereafter, the solution was changed every 2 to 3 days. On day 6, the supernatant was similarly aspirated and transferred to complete RPMI medium containing 1X10'-4M hypoxanthine and 1.6X10-5M thymidine (
(hereinafter referred to as HT medium). From then on, complete RPM
Growth was maintained in I medium. Colonies became visible to the naked eye in 10 to 14 days after fusion, and when the cells occupied 1/4 of the bottom area of the 96-well plate, the supernatant was used as the immunizing antigen for KATO-I [[ was tested by enzyme-linked immunosorbent assay (ELISA) using the antigen as an antigen, and cloning was immediately performed by limiting dilution from wells that were positive. That is, BALB/c mouse thymocytes IXIO
RPMI-1 with 10% FC8 prepared to contain 3
Using 20w1 of 640 medium, transfer 0.2xl of hybridomas to a 96-well plate at 3/well, 1/well, and 0.3/well for cloning, and clone the desired hybridoma. Established.
クローニングはＫＡＴＯ−ＩＩＩをコートした９６ウエ
ルプレートを用い２回以上行なった。かくして、所望の
反応特異性を有する本発明のモノクローナル抗体を産生
ずるハイブリドーマ（クーロンＮＯＧＯＭＨＢ−２、Ｇ
ＯＭＨＢ−３およびＧＯＭＨＢ−５）を得た。Cloning was performed two or more times using a 96-well plate coated with KATO-III. Thus, a hybridoma (Coulomb NOGOMHB-2, G
OMHB-3 and GOMHB-5) were obtained.
上記モノクローナル抗体産生細胞ＧＯＭＨＢ２は、工業
技術院微生物工業技術研究所にｒＧＯＭＨＢ−２Ｊなる
表示で寄託されており、その寄託番号は微工研菌寄第１
１１６１号（ＦＥＲＭＰ−１１１６１）である。また、
ＧＯＭＨＢ−３はｒＧＯＭＨＢ−３Ｊの名称で微工研菌
寄第１１４４０号（ＦＥＲＭ Ｐ−１１４４０）として
、更にＧＯＭＨＢ−５はｒＧＯＭＨＢ−５Ｊの名称で微
工研菌寄第１１３３２号（ＦＥ、ＲＭ Ｐ−１１３３２
）としてそれぞれ寄託されている。The monoclonal antibody-producing cell GOMHB2 has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under the designation rGOMHB-2J, and its deposit number is
No. 1161 (FERMP-11161). Also,
GOMHB-3 is named rGOMHB-3J and is published as FERM P-11440 (FERM P-11440), and GOMHB-5 is named rGOMHB-5J and is published as FERM P-11332 (FE, RM P). -11332
) have been deposited respectively.
■ 上記■で得られたクローンＮｏ、ＧＯＭＨＢ−２、
ＧＯＭＨＢ−３およびＧＯＭＨＢ−５を、それぞれ完全
ＲＰＭＩ培地にて５％ＣＯ２条件下で、３７℃にて９６
時間培養した。培養液を３００Ｏｒｐｍ 、 Ｉ Ｑ分
間遠心分離して、目的モノクローナル抗体（以下これら
をそれぞれＧＯＭ−２、ＧＯＭ−３およびＧＯＭ−５と
いう）を含む培養上清を得た。■ Clone No. obtained in ■ above, GOMHB-2,
GOMHB-3 and GOMHB-5 were grown at 96°C at 37°C under 5% CO2 in complete RPMI medium, respectively.
Cultured for hours. The culture solution was centrifuged at 300 rpm for IQ minutes to obtain culture supernatants containing the target monoclonal antibodies (hereinafter referred to as GOM-2, GOM-3, and GOM-5, respectively).
■ 前記■で得たクローンＮｏ、ＧＯＭＨＢ−２、ＧＯ
ＭＨＢ−３およびＧＯＭＨＢ−５のそれぞれ１×１０６
個を、予めプリスタン（アルドリッチ社製）を接種して
おいたＢ Ａ Ｌ Ｂ ／ ｃ系マウスに腹腔内投与し
た。１０〜１４日後、蓄積した腹水を採取し、本発明抗
体を含む腹水を得た。■ Clone No., GOMHB-2, GO obtained in above ■
1 x 106 each of MHB-3 and GOMHB-5
The sample was intraperitoneally administered to BALB/c mice that had been previously inoculated with pristane (manufactured by Aldrich). After 10 to 14 days, the accumulated ascites was collected to obtain ascites containing the antibody of the present invention.
実施例 ２
本発明抗体の性状
■ 抗体のサブクラス
マウスモノクローナル抗体サブクラス同定用キット（ザ
イメッド社製）を用いて決定した上記抗体のサブクラス
は、ＧＯＭ−２が１ｇＧ３に、ＧＯＭ−３が１ｇＧ３に
、及びＧＯＭ−５がＩｇＭにであった。Example 2 Properties of the antibody of the present invention ■ Antibody subclass The subclasses of the above antibody determined using a mouse monoclonal antibody subclass identification kit (manufactured by Zymed) are GOM-2 to 1gG3, GOM-3 to 1gG3, and GOM-5 was IgM.
■ 抗体産生レベル
実施例１−■で得られたハイブリドーマ（クローンＮｏ
、ＧＯＭＨＢ−２、ＧＯＭＨＢ−３およびＧＯＭＨＢ−
５）を１０％ＦＣ８添加ＲＰＭＩ培地にて３７℃、５％
ＣＯ２の条件で培養した。■ Antibody production level Example 1-Hybridoma obtained in ■ (Clone No.
, GOMHB-2, GOMHB-3 and GOMHB-
5) at 37°C in RPMI medium supplemented with 10% FC8.
Culture was performed under CO2 conditions.
ハイブリドーマが最大細胞密度になったときの培養上清
中のＧＯＭ−２のＩｇＧ量は約５０ｇｇ／ｚｌであった
。また、ＧＯＭ−３のＩｇＧ量は約３０ａｇ／ｙＩｌで
あった。ＧＯＭ−５のＩｇＭ量は約４０１Ｉｇ／ｚ／で
あった。The amount of GOM-2 IgG in the culture supernatant when the hybridomas reached the maximum cell density was about 50 gg/zl. Moreover, the IgG amount of GOM-3 was about 30 ag/yIl. The IgM amount of GOM-5 was approximately 401 Ig/z/.
■力価
力価の測定は１０％ホルマリン−ＰＢＳで固定したＮＡ
ＴＯ−１１１細胞５Ｘ１０’と希釈した培養上清とを４
℃で一夜反応させた。ＰＢＳで洗浄後、抗マウスＩ ｇ
Ｇ−Ｆ ＩＴＣ又は抗マウスＩｇＭＦ Ｉ Ｔ Ｃ（ｆ
ｌｕｏｒｅｓｃｅｉｎ １ｓｏｔｈｉｏｃｙａｎａｔｅ
２００倍希釈、カッペル社製）を加えて４℃で４時間
反応させた。ＰＢＳで洗浄してフローサイトメーター（
オーツ社製）にて蛍光強度を測定した。■Titer Measurement of titer is performed using NA fixed in 10% formalin-PBS.
TO-111 cells 5X10' and the diluted culture supernatant were
The reaction was allowed to occur overnight at °C. After washing with PBS, anti-mouse Ig
G-F ITC or anti-mouse IgMF ITC (f
fluorescein 1sothiocyanate
(200 times diluted, manufactured by Kappel) was added and reacted at 4°C for 4 hours. Wash with PBS and use a flow cytometer (
(manufactured by Oats) to measure the fluorescence intensity.
このときの平均蛍光強度が２０を示す抗体活性を×１と
し、その時の培養上清を培養力価とした。The antibody activity with an average fluorescence intensity of 20 at this time was defined as ×1, and the culture supernatant at that time was defined as the culture titer.
上記方法にて測定した培養上清中の細胞結合性の力価は
ＧＯＭ−２がＸ１００Ｏであった。The cell binding titer of GOM-2 in the culture supernatant measured by the above method was X100O.
またＧＯＭ−３は×５００で、ＧＯＭ−５はＸ１００Ｏ
であった。Also, GOM-3 is ×500, and GOM-5 is ×100O.
Met.
■ 分子量
ＧＯＭ−２及びＧＯＭ−３は、前記実施例１■で得た腹
水をＩｇＧ精製キッ）（ＭＡＰＳキット、バイオラッド
社）を用いてＴｇＧに精製した。(2) Molecular weight GOM-2 and GOM-3 were purified into TgG from the ascites obtained in Example 1 (2) using an IgG purification kit (MAPS kit, Bio-Rad).
ＧＯＭ−５は、ＧＯＭＨＢ−５細胞を完全無血清培地Ｈ
Ｌ−１（ベントレックス社）にて培養し、その培養上清
を限外濾過（ＸＭ３００、アミコン社）にて濃縮し、１
／２飽和硫安にて塩析し、その沈殿をＰＢＳに再溶解後
、ゲル濾過
（Ｓｅｐｂａｒｏｓｅ ＣＬ−６Ｂ、ファルマシア社）
にてＩｇＭに精製した。GOM-5 was prepared by growing GOMHB-5 cells in complete serum-free medium H.
L-1 (Ventrex), the culture supernatant was concentrated by ultrafiltration (XM300, Amicon), and 1
/2 Salting out with saturated ammonium sulfate, redissolving the precipitate in PBS, and gel filtration (Sepbarose CL-6B, Pharmacia)
It was purified to IgM.
精製したＧＯＭ−２、ＧＯＭ−３及びＧＯＭ５を５ＤＳ
−ＰＡＧＥにて分子量を求めた。尚、ＩｇＧは重鎖と軽
鎖の和とし、ＩｇＭは重鎮と軽鎖の和の５量体として分
子量を計算した。Purified GOM-2, GOM-3 and GOM5 in 5DS
-Molecular weight was determined by PAGE. The molecular weight of IgG was calculated as the sum of heavy chains and light chains, and the molecular weight of IgM was calculated as a pentamer of the sum of heavy chains and light chains.
その結果、ＧＯＭ−２の分子量は１．６×１０２Ｋｄ、
ＧＯＭ−３の分子量は１．６×１０２Ｋｄ１またＧＯＭ
−５の分子量は１．０×１０３Ｋｄであった。As a result, the molecular weight of GOM-2 was 1.6×102Kd,
The molecular weight of GOM-3 is 1.6×102Kd1 and GOM
The molecular weight of -5 was 1.0 x 103 Kd.
■ 培養癌細胞との反応性
ＨＬ−６０（ヒト前骨髄性白血病細胞）、ＡＣＨＮ （
ヒト賢腺癌）、ＷｉＤｒ（大腸腺癌）及びＵ−９３７（
組織球性白血病）とＫＡＴＯ−■をインビトロにて培養
した。培養細胞を回収しＰＢＳで洗浄した後、０．５■
／ ｘｉのポリリジンをプリコートした９６ウエルプレ
ートにウェル当り５Ｘ１０’個の細胞を分注し、細胞が
ウェルの底面に接着したところで、グルタルアルデヒド
（終濃度０．１％）を加えて固定化した。これを２％ウ
シ血清アルブミン（ＢＳＡ）／リン酸緩衝生理食塩水（
Ｐ Ｂ Ｓ）で３７℃、１時間ブロッキングし、ＰＢＳ
で洗浄後、培養上清に５倍希釈した各モノクローナル抗
体溶液０．０５ｚ／を加え、４℃で一夜反応させた。こ
れをＰＢＳで洗浄後、更に二次抗体（抗マウスＩｇＧ−
パーオキシダーゼ複合体（ａｎｔｉ ＩｇＧ−Ｐｏｘ）
又は抗マウスＩ ｇＭ−パーオキシターゼ複合体（ａｎ
ｔｉ ＩｇＭ−Ｐｏｘ） （カッペル社製）を４℃で
一夜反応させた。最後に洗浄液で洗浄し発色液（オルト
フェニレンジアミン（ＯＰ Ｄ）溶液）をウェル当り５
０μｍ加え、室温で３０分反応させた後、５０ｆｉｌの
２規定硫酸を加えて反応を停止し、４９２ｎｍの吸光度
を測定した。■ Reactivity with cultured cancer cells HL-60 (human promyelocytic leukemia cells), ACHN (
human adenocarcinoma), WiDr (colon adenocarcinoma) and U-937 (
histiocytic leukemia) and KATO-■ were cultured in vitro. After collecting the cultured cells and washing with PBS, 0.5
5 x 10' cells per well were dispensed into a 96-well plate pre-coated with polylysine of /xi, and when the cells adhered to the bottom of the well, glutaraldehyde (final concentration 0.1%) was added to fix them. This was mixed with 2% bovine serum albumin (BSA)/phosphate buffered saline (
Block for 1 hour at 37°C with PBS)
After washing with water, 0.05z/of each monoclonal antibody solution diluted 5 times was added to the culture supernatant, and the mixture was allowed to react at 4°C overnight. After washing this with PBS, the secondary antibody (anti-mouse IgG-
Peroxidase complex (anti IgG-Pox)
or anti-mouse I gM-peroxidase complex (an
tiIgM-Pox) (manufactured by Kappel) was reacted at 4°C overnight. Finally, wash with washing solution and add coloring solution (ortho-phenylenediamine (OPD) solution) to 5 wells per well.
After adding 0 μm and reacting at room temperature for 30 minutes, 50 fil of 2N sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured.
その結果、ＧＯＭ−２モノクロ一ナル抗体は、ＫＡＴＯ
’−１［との反応性を１００とすると、ＨＬ−６０が０
．２、ＡＣＨＮが１．７、Ｕ−９３７が１．７であり、
Ｗ ｉ Ｄ ｒとはほとんど反応せず、このことがらＨ
Ｌ−６０、ＡＣＨＮ。As a result, GOM-2 monoclonal antibody
'-1 [If the reactivity with 100, HL-60 is 0
．． 2, ACHN is 1.7, U-937 is 1.7,
It hardly reacts with W i Dr, and this shows that H
L-60, ACHN.
Ｕ−９３７及びＷ ｉ Ｄ ｒ細胞に対する反応性は弱
いことが判った。It was found that the reactivity towards U-937 and WiDr cells was weak.
すなわち、本発明ＧＯＭ−２モノクローナル抗体は、特
異的にＫＡＴＯ−ｍ細胞を認識することが認識された。That is, it was recognized that the GOM-2 monoclonal antibody of the present invention specifically recognizes KATO-m cells.
また、同様にＧＯＭ−３モノクロ一ナル抗体はＫＡＴＯ
−ＩＩＩとの反応性を１００とするとＡＣＨＮには１．
０、Ｗ ｉ Ｄ ｒには３３．９であり、ＨＬ−６０と
Ｕ−９３７にはほとんど反応しなかった。Similarly, GOM-3 monoclonal antibody is KATO
If the reactivity with -III is 100, then ACHN has 1.
0, W i Dr was 33.9, and there was almost no reaction with HL-60 and U-937.
更に、ＧＯＭ−５モノクロ一ナル抗体においてはＫＡＴ
Ｏ−Ｉｌｌとの反応性を１００とするとＨＬ−６０が４
．８で、ＡＣＨＮが４３．６で、Ｗ ｉ Ｄ ｒが４５
．４で、Ｕ−９３７が５６．１であった。Furthermore, in the GOM-5 monoclonal antibody, KAT
If the reactivity with O-Ill is 100, HL-60 is 4.
．． 8, ACHN is 43.6, W i Dr is 45
．． 4, and U-937 was 56.1.
この結果、ＫＡＴＯ−Ｈに比較して反応性は弱いが、Ｇ
ＯＭ−３はＷ ｉ Ｄ ｒに、ＧＯＭ−５はＡＣＨＮ及
びＷ ｉ Ｄ ｒ及びＵ−９３７細胞に対しても反応性
を有することが判明した。As a result, although the reactivity is weaker than that of KATO-H, G
OM-3 was found to be reactive with Wi Dr, and GOM-5 was also found to be reactive with ACHN, Wi Dr, and U-937 cells.
又、この点よりＧＯＭ−２及びＧＯＭ−３及びＧＯＭ−
５はそれぞれ特異性を異にするモノクローナル抗体であ
ることが判明した。Also, from this point, GOM-2, GOM-3, and GOM-
5 were found to be monoclonal antibodies with different specificities.
■ 正常ヒト血球細胞との反応性
正常血球細胞に対する本発明各種モノクローナル抗体、
ＧＯＭ−２とＧＯＭ−３及びＧＯＭ−５の陽性率をフロ
ーサイトメトリーにより分析した。■ Reactivity with normal human blood cells Various monoclonal antibodies of the present invention against normal blood cells,
The positive rate of GOM-2, GOM-3, and GOM-5 was analyzed by flow cytometry.
人血法は、ヘパリン採血した人血液を溶血操作後ＰＢＳ
で洗浄したものを培養上清と４℃、１時間反応させて、
洗浄後二次抗体（抗マウスＩｇＧ−ＦＩＴＣまたは抗マ
ウスＩｇＭ−ＦＩＴＣ（カッペル社製））を４℃、１時
間反応させフローサイトメタ−（オーツ社製）で測定し
た（太田和雄、「フローサイトメトリー手技と実際」、
蟹書房、１９８４年、細胞工学、４．９５７ （１９８
５）参照）。In the human blood method, human blood collected with heparin is hemolyzed and then PBS is used.
The washed product was reacted with the culture supernatant at 4°C for 1 hour,
After washing, a secondary antibody (anti-mouse IgG-FITC or anti-mouse IgM-FITC (manufactured by Kappel)) was reacted at 4°C for 1 hour and measured using a Flow Cytometer (manufactured by Oats) (Kazuo Ota, "Flow Cyto ``Metric techniques and practice''
Kani Shobo, 1984, Cell Engineering, 4.957 (198
5)).
本発明抗体ＧＯＭ−２とＧＯＭ−３及びＧＯＭ−５が正
常リンパ球と正常単球、正常顆粒球に対して反応性を有
するか否を検討した。It was examined whether the antibodies GOM-2, GOM-3, and GOM-5 of the present invention have reactivity with normal lymphocytes, normal monocytes, and normal granulocytes.
その結果、本発明モノクローナル抗体ＧＯＭ−２に陽性
を示した細胞の割合は、リンパ球０． １％、単球と顆
粒球は反応せず、正常ヒト血球細胞との反応性は認めら
れなかった。As a result, the percentage of cells that were positive for the monoclonal antibody GOM-2 of the present invention was 0.00% for lymphocytes. 1%, monocytes and granulocytes did not react, and no reactivity with normal human blood cells was observed.
同様に本発明モノクローナル抗体ＧＯＭ−３もリンパ球
０．１％、単球と顆粒球は反応せず、ＧＯＭ−５もリン
パ球０．４％、単球には反応せず、顆粒球とは０． ２
％反応した。すなわち、ＧＯＭ−３及びＧＯＭ−５も正
常ヒト血球細胞と反応しないことが判明した。Similarly, the monoclonal antibody GOM-3 of the present invention has 0.1% of lymphocytes and does not react with monocytes and granulocytes, and GOM-5 has 0.4% of lymphocytes, does not react with monocytes and does not react with granulocytes. 0. 2
% reacted. That is, it was found that GOM-3 and GOM-5 also did not react with normal human blood cells.
■ 本発明抗体の認識する抗原構造の決定■−１モノク
ローナル抗体ＧＯＭ−２の認識する抗原構造の決定
ＫＡＴＯ−ＩＩＩ （ヒト印環胃癌細胞）はＭｉｙａｕ
ｃｈｉ等の方法（Ｍｉｙａｕｃｈｉ、Ｔ、、 ｅｔ
ａｌ、、 Ｇａｎｎ、 ７３５８１−５８７． （
１９８２） ）に従って培養した。培養細胞１００ｇに
イソプロパツール：ヘキサン：水（ＩＨＷ）＝５５ ：
２０ ： ２５の混合溶液を５００２／加えてワーリ
ングブレンダーにより、細胞を破砕した。４℃で４分間
抽出し、ガラスフィルターで濾過した。この操作を５回
行なった後、抽出液をロータリーエバポレーターで濃縮
乾固した後、クロロホルム：メタノール（ＣＭ）＝２
：１約１００ｚ／に溶解し不溶物を遠心分離（３０００
ｔｐｍＸ２０分）で除去し、抽出液の１１５容の水を添
加してゆるやかに攪拌し静置した。上層を分離して残っ
た下層に再度水を添加し、同様の操作を３回繰り返した
。３回の操作で得られた上清をロータリーエバポレータ
ー（東京理化社製）で濃縮し、水に対して３日間分画サ
イズ３５００の透析膜スペクトラボアＮｏ、３（スペク
トラム メディカルインダストリーズ社製）を使用し透
析した。得られた透析内液を粗製ＫＡＴＯ−■糖脂質と
した。■ Determination of the antigenic structure recognized by the antibody of the present invention ■-1 Determination of the antigenic structure recognized by the monoclonal antibody GOM-2 KATO-III (human signet ring gastric cancer cells)
The method of Chi et al. (Miyauchi, T., et al.
al., Gann, 73581-587. (
(1982)). Isopropanol: hexane: water (IHW) = 55 for 100 g of cultured cells:
A mixed solution of 20:25 was added at a ratio of 5002/2, and the cells were crushed using a Waring blender. Extraction was performed at 4°C for 4 minutes and filtered through a glass filter. After performing this operation 5 times, the extract was concentrated to dryness using a rotary evaporator, and then chloroform:methanol (CM) = 2
:1 Dissolve at about 100z/centrifuge to remove insoluble matter (3000
tpm x 20 minutes), 115 volumes of water from the extract was added, and the mixture was gently stirred and allowed to stand still. The upper layer was separated, water was added again to the remaining lower layer, and the same operation was repeated three times. The supernatant obtained from the three operations was concentrated using a rotary evaporator (manufactured by Tokyo Rika Co., Ltd.), and dialysis membrane Spectrabore No. 3 (manufactured by Spectrum Medical Industries, Inc.) with a fraction size of 3500 was used against water for 3 days. I underwent dialysis. The obtained dialysis fluid was used as crude KATO-■ glycolipid.
この粗製ＫＡＴＯ−１［糖脂質を乾燥固化し、クロロホ
ルム：メタノール：水（ＣＭＷ）＝３０ ：６０：８に
溶解し、遠心分離により不溶物を除去した。次にこれを
、予めＣＭＷ液で平衡化したＤＥＡＥ−セファデックス
Ａ−２５カラム（１６Ｘ２００ｍｍ）（ファルマシア社
製）に添加し、５倍量のＣＭＷ液で洗浄後、クロロホル
ム：メタノール： ０．８Ｍ ＮＨ４Ａｃ＝３０ ：
６０ ： ８で溶出した。カラムを素通りした液と洗
浄液をＫＡＴＯ−ＩＩＩ糖脂質中性画分とし、吸着画分
をＫＡＴＯ−ＩＩＩ糖脂質酸性画分とした。濃縮乾固し
た中性画分をイソプロパノールニヘキサン：水（ＩＨＷ
）＝５５ ： ２０ ： ２５に溶解し、イアトロビー
ズカラム６Ｒ８８０１０（８Ｘ６００薗）（ダイアヤト
ロン社製）を用いて高速液体クロマトグラフィー（ＨＰ
Ｌｃ）（ファルマシア社製）により分画した。分画のた
めの溶媒はＡ溶液［イソプロパツール：ヘキサン：水＝
５５ ： ４０ ： ５］とＢ溶液［イソプロパツール
：ヘキサン：水；５５ ： ２０ ： ２５］を用い、
流速０．５ｍｌ／分で溶出した。溶出液は各々分取して
ＧＯＭ−２抗体と反応する画分を集めた。This crude KATO-1 [glycolipid was dried and solidified, dissolved in chloroform:methanol:water (CMW) = 30:60:8, and insoluble materials were removed by centrifugation. Next, this was added to a DEAE-Sephadex A-25 column (16 x 200 mm) (manufactured by Pharmacia) that had been equilibrated with a CMW solution in advance, and after washing with 5 times the amount of CMW solution, chloroform: methanol: 0.8M NH4Ac =30:
It eluted at a ratio of 60:8. The liquid that passed through the column and the washing liquid were designated as a KATO-III glycolipid neutral fraction, and the adsorbed fraction was designated as a KATO-III glycolipid acidic fraction. The neutral fraction concentrated to dryness was mixed with isopropanol nihexane:water (IHW
)=55:20:25 and subjected to high performance liquid chromatography (HP
Lc) (manufactured by Pharmacia). The solvent for fractionation is A solution [isopropanol:hexane:water=
55: 40: 5] and B solution [isopropanol: hexane: water; 55: 20: 25],
Elution was performed at a flow rate of 0.5 ml/min. The eluate was separated and the fractions that reacted with the GOM-2 antibody were collected.
このようにして得られたＧＯＭ−２反応性糖脂質画分を
エンドグリコセラミダーゼ（生化学工業社製）処理後、
ＮａＢ ［’Ｈ］ ４で還元標識した。After treating the GOM-2-reactive glycolipid fraction thus obtained with endoglycoceramidase (manufactured by Seikagaku Corporation),
Reduction labeling was performed with NaB['H]4.
本発明モノクローナル抗体ＧＯＭ−２をプロティンＡ−
セファロースカラム（ファルマシア社製）に結合させて
作製したＧＯＭ−２抗体カラムを予め０．１５Ｍ Ｎ
ａＣ１を含む１０ｍＭ トリスＨＣ／緩衝液ｐＨ７，８
にて平衡化した。次いで上記［３１（コー標識オリゴ糖
（２〜４×１１０３ｄｐ／１００μｍ）を本抗体カラム
にのせ１時間吸着させた後、流速５１／ ／ ｈ ｒで
溶出した。The monoclonal antibody GOM-2 of the present invention was combined with protein A-
A GOM-2 antibody column prepared by binding to a Sepharose column (manufactured by Pharmacia) was prepared in advance at 0.15M N.
10mM Tris HC/buffer pH 7,8 containing aC1
Equilibrated at . Next, the above [31 (co-labeled oligosaccharide (2-4 x 1103 dp/100 μm)) was placed on the antibody column and adsorbed for 1 hour, and then eluted at a flow rate of 51//hr.
本抗体カラムに親和性を示した［３Ｈ］−標識オリゴ糖
を分離し、その糖鎖構造を解析した結果、後記する式（
４）の構造が得られた。この結果から該式（４）の構造
が、本発明モノクローナル抗体ＧＯＭ−２の認識する抗
原糖鎖構造であることが判明した。As a result of separating the [3H]-labeled oligosaccharide that showed affinity to this antibody column and analyzing its sugar chain structure, we found the following formula (
The structure 4) was obtained. These results revealed that the structure of formula (4) is the antigen sugar chain structure recognized by the monoclonal antibody GOM-2 of the present invention.
次にモノクローナル抗体ＧＯＭ−２の認識する抗原構造
の特異性を明らかにすることを目的として、後記式（５
）及び式（６）で示される各［３Ｈ］−標識オリゴ糖の
ＧＯＭ−２抗体カラムへの結合性を、上記と同様の方法
で調べた。Next, in order to clarify the specificity of the antigen structure recognized by monoclonal antibody GOM-2, we used the formula (5
) and the binding of each [3H]-labeled oligosaccharide represented by formula (6) to the GOM-2 antibody column was examined in the same manner as above.
式 （４） ：Ｇａ１ａｌ−＋３Ｇａｌ （２←１ａ
Ｆｕｃ） β１→４ＧｌｃＮＡｃ （３←ｌαＦｕｃ）
βｌ”３Ｇａｌβ１→４ＧＩＣＯＴ式（５） ：Ｇａｌ
（２＝ｌａＦｕｃ）β１−４ＧｌｃＮＡｃ （３←Ｉ
ａＦｕｃ）βｌ→３．Ｇａｌβｌ→４ＧｌｃＯＴ式（６
） ：Ｇａ１ａｌ−３Ｇａｌ （２←１ａＦｕｃ）β１
−４ＧｌｃＮＡｃβ１→３Ｇａｌβ１→４ＧＩｃＯＴ［
ＯＴは還元末端がトリチウムで還元されていることを示
す。］
その結果を下記第１表に示す。Formula (4): Ga1al-+3Gal (2←1a
Fuc) β1→4GlcNAc (3←lαFuc)
βl"3Galβ1→4GICOT formula (5): Gal
(2=laFuc)β1-4GlcNAc (3←I
aFuc)βl→3. Galβl → 4GlcOT formula (6
) :Gal-3Gal (2←1aFuc)β1
-4GlcNAcβ1→3Galβ1→4GIcOT[
OT indicates that the reducing end has been reduced with tritium. ] The results are shown in Table 1 below.
第 １ 表 ＋＋＋；強い親和性を有する ；極めて弱い親和性を有する ；親和性が認められない。Chapter 1 Table +++; has strong affinity ; has extremely weak affinity ; No affinity observed.
第１表より、本発明ＧＯＭ−２モノクローナル抗体は、
糖鎖式（４）に強い親和性を示したにもかかわらず、糖
鎖式（５）とは全（親和性が認められず、また糖鎖式（
６）とは極めて弱い親和性しか示さなかった。尚、ＧＯ
Ｈ−２抗体カラムからの各糖鎖の溶出位置から判断する
と、本発明ＧＯＭ−２モノクローナル抗体の糖鎖式（６
）への親和性は糖鎖式（４）への親和性の約１／４０と
極めて弱いものであった。From Table 1, the GOM-2 monoclonal antibody of the present invention is
Although it showed strong affinity for glycan formula (4), no affinity was observed for glycan formula (5), and glycan formula (
6) showed only extremely weak affinity. In addition, GO
Judging from the elution position of each sugar chain from the H-2 antibody column, the sugar chain formula (6
) was extremely weak, about 1/40 of the affinity for sugar chain formula (4).
一方、Ｋｏｐｒｏｖｓｋｉ らの報告している抗体（Ｂ
ｒ５５−２）は各・種部脂質を組み込んだリポソームに
よるインヒビジョンアッセイの結果から、式（５）の末
端構造を持つ糖脂質に対する結合性が式（４）の末端構
造を持つ糖脂質に対するそれに比べて約５００倍強いこ
とが示されている（ＭＢＩａｓｘｃｘ７に、 ｅｔ
ａｌ、、 Ｊ、Ｂｉｏｌ、 Ｃｈｅｍ、、 ２６
２． ３７２３７９ （１９８７））。On the other hand, the antibody (B
r55-2) has binding properties to glycolipids with the terminal structure of formula (5) that are similar to those of glycolipids with the terminal structure of formula (4), based on the results of inhibition assay using liposomes incorporating various lipids. It has been shown that it is about 500 times stronger than MBIasxcx7, et
al,, J,Biol,Chem,, 26
2. 372379 (1987)).
又、同様にＫｏｐｒｏｗｓｋｉ らの報告している抗体
Ｅ２８３−５２は、式（４）の末端構造を持つ糖脂質と
式（６）の末端構造を持つ糖脂質の混合物にも反応性を
示しているが、式（４）よりもむしろ式（６）の末端構
造を持つ糖脂質に強く反応している（Ｇ、Ｃ，Ｍａｎｓ
ｓｏｎ ｅｔ ａｌ、、 Ｊ、 Ｂｉｏｌ、 Ｃｈ
ｅｍ、。Similarly, antibody E283-52 reported by Koprowski et al. also shows reactivity to a mixture of a glycolipid having the terminal structure of formula (4) and a glycolipid having the terminal structure of formula (6). reacts strongly with the glycolipid having the terminal structure of formula (6) rather than that of formula (4) (G, C, Mans
son et al., J., Biol, Ch.
Em,.
２５８、４０９１−４０９７ （１９８３） ）。258, 4091-4097 (1983)).
以上の結果から本発明ＧＯＭ−２モノクローナル抗体は
、従来知られている抗体よりも式（イ）の糖鎖構造を非
還元末端に有する糖鎖と強（結合する極めて特異性の高
い抗体であることが明らかである。From the above results, the GOM-2 monoclonal antibody of the present invention is an extremely highly specific antibody that binds more strongly to sugar chains having the sugar chain structure of formula (A) at the non-reducing end than conventionally known antibodies. That is clear.
■−２モノクローナル抗体ＧＯＭ−３及びＧＯＭ−５の
認識する抗原構造の決定
後記第２表に示した各種合成糖脂質（バイオカーボ社製
）及び合成糖鎖のＢＳＡ複合体（ＴＨＩ社製）を各々９
６ウエルマイクロプレートにウェル当り１０１ｇずつ分
注し、吸着させ固相化した。これに２％Ｂ Ｓ Ａ／Ｐ
Ｂ Ｓで３７℃、１時間ブロッキングしＰＢＳで洗浄
後、培養上清を５倍希釈したモノクローナル抗体ＧＯＭ
−３及びＧＯＭ−５溶液０．０５ｘｌを加え４℃で一夜
反応させた。これをＰＢＳで洗浄後、更に二次抗体（抗
マウス（ＩｇＧ＋Ｉｇ＾＋ＩｇＭ）−Ｐｏｘ （カッ
ペル社製）を４℃で４時間反応させた。最後にＰＢＳで
洗浄後発色液（ＯＰＤ溶液）をウェル当り５０μｍ加え
、室温で３０分反応させた後、５０ｐ１の２規定硫酸を
加えて反応を停止し、４９２ｎｍの吸光度を測定し、反
応性の有無を判定した。■-2 Determination of antigen structures recognized by monoclonal antibodies GOM-3 and GOM-5 Various synthetic glycolipids (manufactured by Biocarbo) and BSA complexes of synthetic sugar chains (manufactured by THI) shown in Table 2 below were used. 9 each
101 g per well was dispensed into a 6-well microplate and adsorbed to form a solid phase. 2% BSA/P for this
After blocking with BS at 37°C for 1 hour and washing with PBS, monoclonal antibody GOM was prepared by diluting the culture supernatant 5 times.
-3 and 0.05xl of GOM-5 solutions were added and reacted overnight at 4°C. After washing this with PBS, it was further reacted with a secondary antibody (anti-mouse (IgG+Ig^+IgM)-Pox (manufactured by Kappel) at 4°C for 4 hours.Finally, after washing with PBS, a coloring solution (OPD solution) was added to the well. After adding 50 μm per sample and reacting for 30 minutes at room temperature, the reaction was stopped by adding 50 pl of 2N sulfuric acid, and the absorbance at 492 nm was measured to determine the presence or absence of reactivity.
結果を第２表に示す。The results are shown in Table 2.
−；反応しない １１１１強く反応する但し表中の糖
鎖構造におけるＣＥＴＥは２（２−カルボメトキシエチ
ルチオ）エチル基を、ＢＳＡは牛血清アルブミンを、Ｐ
Ａ１４は炭素数１４のフェニルアルキル基を、ＰＡ８は
炭素数８のフェニルアルキル基をそれぞれ示す。-: Does not react 1111 Strongly reacts However, in the sugar chain structure in the table, CETE represents 2(2-carbomethoxyethylthio)ethyl group, BSA represents bovine serum albumin, P
A14 represents a phenylalkyl group having 14 carbon atoms, and PA8 represents a phenylalkyl group having 8 carbon atoms.
第２表の結果から、モノクローナル抗体ＧＯＭ−３は式
（ロ）に示す糖鎖構造を、またモノクローナル抗体ＧＯ
Ｍ−５は式（ハ）に示す糖鎖構造を、それぞれ最小抗原
構造として認識することが明らかとなった。From the results in Table 2, monoclonal antibody GOM-3 has the sugar chain structure shown in formula (b), and monoclonal antibody GO
It was revealed that M-5 recognizes each of the sugar chain structures shown in formula (c) as the minimum antigen structure.
（以 上）(that's all)
Claims (1)
するモノクローナル抗体であって、下記（イ）、（ロ）
及び（ハ）のいずれか少なくともひとつの化合物に特異
反応性を有することを特徴とするモノクローナル抗体。 （イ）式 Ｇａｌａ１→３Ｇａｌ（２←１ａＦｕｃ）β１→４Ｇｌ
ｃＮＡｃ（３←１ａＦｕｃ）→Ｒ＿１（Ｒ＿１は糖、糖
脂質又は糖蛋白質を示す。）で示される糖鎖構造を非還
元末端に有する化合物 （ロ）式 Ｇａｌβ１→３ＧｌｃＮＡｃ（４←１ａＦｕｃ）→Ｒ＿
２（Ｒ＿２は糖、糖脂質又は糖蛋白質を示す。）で示さ
れる糖鎖構造を非還元末端に有する化合物 （ハ）式 Ｇａｌβ１→３ＧｌｃＮＡｃ→Ｒ＿３ （Ｒ＿３は糖、糖脂質又は糖蛋白質を示す。）で示され
る糖鎖構造を非還元末端に有する化合物 （但し、各式中ＧａｌはＤ−ガラクトース残基を、Ｆｕ
ｃはＬ−フコース残基を、またＧｌｃＮＡｃはＮ−アセ
チル−Ｄ−グルコサミン残基をそれぞれ示す。）(1) A monoclonal antibody that recognizes KATO-III cells (human signet ring gastric cancer cells), comprising the following (a) and (b):
A monoclonal antibody characterized by having specific reactivity with at least one compound of (c). (B) Formula Gala1→3Gal(2←1aFuc)β1→4Gl
cNAc(3←1aFuc)→R_1 (R_1 represents sugar, glycolipid, or glycoprotein) A compound having the sugar chain structure at the non-reducing end (b) Formula Galβ1→3GlcNAc(4←1aFuc)→R_
2 (R_2 represents a sugar, glycolipid, or glycoprotein) at the non-reducing end of the compound (c) Formula Galβ1→3GlcNAc→R_3 (R_3 represents a sugar, glycolipid, or glycoprotein). ) has a sugar chain structure at the non-reducing end (however, in each formula, Gal represents a D-galactose residue, Fu
c represents an L-fucose residue, and GlcNAc represents an N-acetyl-D-glucosamine residue. )
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2129680A JPH0423997A (en) | 1990-05-18 | 1990-05-18 | Monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2129680A JPH0423997A (en) | 1990-05-18 | 1990-05-18 | Monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0423997A true JPH0423997A (en) | 1992-01-28 |
Family
ID=15015519
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2129680A Pending JPH0423997A (en) | 1990-05-18 | 1990-05-18 | Monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0423997A (en) |
-
1990
- 1990-05-18 JP JP2129680A patent/JPH0423997A/en active Pending
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