JPH01268646A - Antitumor agent - Google Patents
Antitumor agentInfo
- Publication number
- JPH01268646A JPH01268646A JP63095724A JP9572488A JPH01268646A JP H01268646 A JPH01268646 A JP H01268646A JP 63095724 A JP63095724 A JP 63095724A JP 9572488 A JP9572488 A JP 9572488A JP H01268646 A JPH01268646 A JP H01268646A
- Authority
- JP
- Japan
- Prior art keywords
- cell
- monoclonal antibody
- mice
- antibody
- mouse
- 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
Links
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- CXQCLLQQYTUUKJ-ALWAHNIESA-N beta-D-GalpNAc-(1->4)-[alpha-Neup5Ac-(2->8)-alpha-Neup5Ac-(2->3)]-beta-D-Galp-(1->4)-beta-D-Glcp-(1<->1')-Cer(d18:1/18:0) Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@@H](CO)O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 CXQCLLQQYTUUKJ-ALWAHNIESA-N 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
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- FDJKUWYYUZCUJX-KVNVFURPSA-N N-glycolylneuraminic acid Chemical compound OC[C@H](O)[C@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-KVNVFURPSA-N 0.000 claims 1
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Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は新規なモノクローナル抗体Ａ１．２６７、ＡＩ
。Detailed Description of the Invention (Industrial Field of Application) The present invention provides novel monoclonal antibody A1.267, AI
.
４１０またはＡ１．４２５を有効成分とする抗腫瘍剤に
関するものである。The present invention relates to an antitumor agent containing A1.410 or A1.425 as an active ingredient.
更に詳しくは本発明は、Ｃ５７旧、／６マウス由来のＴ
細胞白血病細胞株［ＥＬ４でＡ系マウスを免疫すること
により得られる抗体産生細胞とＢａ１ｂ／ｃ系マウス骨
髄腫細胞ＮＳＩとを融合させることにより得られるハイ
ブリドーマが産生し、免疫グロブリンクラスが（ＩｇＧ
３、ｘ）であって、マウス及びヒトの腫瘍細胞の細胞表
面の種間共通の腫瘍関連抗原であるカングリオシトをＧ
Ｄ２＞ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）。More specifically, the present invention provides T
Hybridomas are produced by fusing antibody-producing cells obtained by immunizing A-lineage mice with cell leukemia cell line [EL4 and Ba1b/c-lineage mouse myeloma cells NSI, and the immunoglobulin class is (IgG).
3.
D2>GD3 (NeuAc, NeuAc).
ＧＤ３（ＮｅｕＧｃ、 ＮｅｕＡｃ）、 ＧＤｌｂ、
ＧＴｌａ、 ＧＱｌｂ）ＧＴＩの順に強く認識するモノ
クロナール抗体Ａ１．２６７を有効成分とする抗腫瘍剤
に関する。GD3 (NeuGc, NeuAc), GDlb,
The present invention relates to an antitumor agent containing a monoclonal antibody A1.267 that strongly recognizes GTI (GTla, GQlb) GTI as an active ingredient.
また、本発明は、 Ｃ５７ＢＬ／６マウス由来のＴ細
胞白血病細胞株ＥＬ４でＡ系マウスを免疫することによ
り得られる抗体産生細胞とＢａ１ｂ／ｃ系マウス由来の
骨髄腫細胞株ＮＳＩとを融合させることにより得られる
ハイブリドーマが産生し、免疫グロブリンクラスが（Ｉ
ｇＧ３、ｘ）であって、細胞表面ガングリオシドＧＤ２
を認識するモノクローナル抗体Ａｌ。Furthermore, the present invention involves fusing antibody-producing cells obtained by immunizing an A mouse with the T cell leukemia cell line EL4 derived from a C57BL/6 mouse and myeloma cell line NSI derived from a Ba1b/c mouse. A hybridoma obtained by is produced, and the immunoglobulin class is (I
gG3, x), which is a cell surface ganglioside GD2
Monoclonal antibody Al that recognizes.
４１０を有効成分とする抗腫瘍剤に関する。The present invention relates to an antitumor agent containing 410 as an active ingredient.
更に１本発明は、Ｃ５７ＢＬ／６マウス由来のＴ細胞白
血病細胞株ＥＬ４でＡ系マウスを免疫することにより得
られる抗体産生細胞とＢａ１ｂ／ｃ系マウス由来の骨髄
腫細胞株ＮＳＩとを融合させることにより得られるハイ
ブリドーマが産生し、免疫グロブリンクラスが（ＩｇＧ
３、ｘ）であって、細胞表面ガングリオシドＧＤ２を認
識するモノクローナル抗体ＡＩ。Furthermore, one aspect of the present invention is to fuse antibody-producing cells obtained by immunizing A lineage mice with the T cell leukemia cell line EL4 derived from C57BL/6 mice and myeloma cell line NSI derived from Ba1b/c lineage mice. The hybridoma obtained by
3, x) monoclonal antibody AI that recognizes cell surface ganglioside GD2.
４２５を有効威容とする抗腫瘍剤に関する。The present invention relates to an antitumor agent having an effective dose of 425.
なお、本発明においてガングリオシドの名前（ＧＤ２、
ＧＤ３など）は５ｖｅｎｎｅｒｈｏｌ Ｌｌｌの命名法
（Ｓｖｅｎｎｅｒｈｏｌｍ、 Ｌ、 Ｊ、 Ｌｉｐｉｄ
Ｒｅｓ、 ５．１４５−１５５（１９６４））による
二また、ＧＤ３のシアル酸誘導体は括弧の中に記した。In addition, in the present invention, the names of gangliosides (GD2,
GD3, etc.) are based on the nomenclature of 5vennerhol Lll (Svennerholm, L, J, Lipid
Res, 5.145-155 (1964)), and sialic acid derivatives of GD3 are listed in parentheses.
すなわち、ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）。That is, GD3 (NeuAc, NeuAc).
ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＧｃ）、ＧＤ３（Ｎｅｕ
Ｇｃ、 ＮｅｕＡｃ）、およびＧＤ３（ＮｅｕＧｃ、
Ｎｅｕ、Ｇｃ）はそれぞれ、■’ （ＮｅｕＡｃ）。GD3 (NeuAc, NeuGc), GD3 (Neu
Gc, NeuAc), and GD3 (NeuGc,
Neu, Gc) are respectively ■' (NeuAc).
−ＬａｃＣｅｒ 、■’ （ＮｅｕＡｃ ａ ２→８Ｎ
ｅｕＧｃ）−ＬａｃＣｅｒ、 ＩＩ’（ＮａｕＧｃ ａ
２→８ＮｅｕＡｃ）−ＬａｃＣｅｒ、 Ｉ’Ｉ ３（
ＮａｕＧｃ）２−ＬａｃＣｅｒを意味する。そのほかの
物質の名称はＩＵＰＡＣ−ＩＵＢ会！ｉ （ＩＵＰＡＣ
−ＩＵＢ Ｃｏｍｍ１ｓｓｉｏｎ ｏｎ Ｂｉｏｃｈｅ
ｍｉｃａｌＮｏｍａｎｃｌａｔｕｒｅ、 Ｌｉｐｉｄｓ
１２．４５５−４６３（１９７７））による。-LacCer, ■' (NeuAc a 2→8N
euGc)-LacCer, II'(NauGc a
2→8NeuAc)-LacCer, I'I 3(
NauGc)2-LacCer. The names of other substances are from the IUPAC-IUB Association! i (IUPAC
-IUB Comm1ssion on Bioche
mical Nomanclature, Lipids
12.455-463 (1977)).
（従来の技術）
にｏｅｈｌｅｒとＭｉｌｓｔｅｉｎが抗体産生細胞と腫
瘍細胞との融合細胞（ハイブリドーマ）を用いてモノク
ローナル抗体の作成する方法（Ｎａｔｕｒｅ、μｉ６．
４９５（１９７５） ）を確立して以来、腫瘍細胞表面
に存在する腫瘍特異抗原に対するモノクロナール抗体を
獲得しようとする試みがなされてきたａ （Ｋｏｐｒｏ
ｗｓｋｉ、 ｔｌ、 ａｔ ａｌ、 Ｐｒｏｃ、Ｎａｔ
ｌ、 Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ。(Prior Art) Oehler and Milstein described a method for producing monoclonal antibodies using fused cells (hybridomas) of antibody-producing cells and tumor cells (Nature, μi6.
495 (1975)), attempts have been made to obtain monoclonal antibodies against tumor-specific antigens present on the surface of tumor cells.
wski, tl, at al, Proc, Nat
l, Acad, Sci, USA.
７５、３４０５（１９７ｇ）、Ｈｅｒｌｙｎ、 Ｍ、
ｅｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ。75, 3405 (197g), Herlyn, M.
et al., Proc., Natl.
Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ、 ７６、１４３８（１
９７９）） これはモノクローナル抗体の抗原に対す
る高い特異性・鋭敏性を利用して、基礎医学においては
腫瘍特異抗原の解析を飛躍的に発展させること、臨床分
野においては癌の的確な診断や癌を狙い打ちにする治療
への応用が期待されたからである。Acad, Sci, USA, 76, 1438 (1
979)) Utilizing the high specificity and sensitivity of monoclonal antibodies to antigens, this will dramatically advance the analysis of tumor-specific antigens in basic medicine, and in the clinical field, it will lead to accurate cancer diagnosis and cancer prevention. This is because it was expected to be applied to targeted treatments.
当初こうした研究は腫瘍抗原の抗原決定基として腫瘍細
胞蛋白質を対象として研究された。ところがこれらの抗
原に対するモノクローナル抗体は正常細胞とも反応し、
腫瘍細胞に対する特異性という観点からみると期待を裏
切るものであったのである。Initially, these studies focused on tumor cell proteins as antigenic determinants of tumor antigens. However, monoclonal antibodies against these antigens also react with normal cells;
From the viewpoint of specificity for tumor cells, the results were disappointing.
しかしながらこのようなモノクローナル抗体の中に数は
少ないながらも比較的腫瘍細胞に対する特異性の高いも
のがみられた。そこでこれら特異性の高いモノクローナ
ル抗体の認識している抗原を調べたところ、腫瘍細胞表
面の糖脂質、糖蛋白質の如き複合糖質の糖鎖部分を認識
していることが判明した。この中でもとりわけガングリ
オシド即ちシアル酸を有する糖脂質が特異性が高いこと
が判った。（ｌｌａｋｏｍｏｒｉ、 Ｓ、 Ｃａｎｃｅ
ｒ ｒｉｅｓ、 ４５．２４０５−２４１４（１９８５
））。キして、ガングリオシドを認識するモノクローナ
ル抗体の中には、既にモノシアロガングリオシドＣＡ］
、９−９を認識する１、１１６Ｎｓ１９−９（Ｋｏｐｒ
ｏｗｓｋｊ、、 ｔＬ ｅｔ ａｌ、 Ｓｏｍ
ａｔｉｃ Ｃｅ１ｌ Ｇｅｎｅｔｉｃｓ。However, among these monoclonal antibodies, although the number was small, some were found to have relatively high specificity for tumor cells. When we investigated the antigens recognized by these highly specific monoclonal antibodies, we found that they recognized sugar chain moieties of complex carbohydrates such as glycolipids and glycoproteins on the surface of tumor cells. Among these, gangliosides, that is, glycolipids containing sialic acid, were found to have particularly high specificity. (llakomori, S, Cance
r ries, 45.2405-2414 (1985
)). Among the monoclonal antibodies that recognize gangliosides, monosialoganglioside CA]
, 9-9 1, 116Ns19-9 (Kopr
owskj,, tL et al, Som
atic Ce1l Genetics.
５．９５７（１９７９）、 Ｍａｇｎａｎｉ、 Ｊ、
ｅｔ ａｌ、 ５ｃｉｅｎｃｅ、 ２１２．。5.957 (1979), Magnani, J.
et al., 5science, 212. .
５５（１９８１）））のように癌の診断に使用されてい
るものもある。55 (1981))) are used for cancer diagnosis.
ガングリオシドに対するモノクローナル抗体には以上の
ほか、ＧＤ３に対しては［’ｕｋｅｌ Ｃ，Ｓ、 ｅｔ
ａｌ。In addition to the above monoclonal antibodies against gangliosides, there are also monoclonal antibodies against GD3 ['ukel C, S, et al.
al.
Ｊ、 Ｅｘｐ、 Ｍａｄ、月５５−．１１３３−１１４
７（１９８２）やＮｕｄｅｌｍａｎ。J, Exp, Mad, May 55-. 1133-114
7 (1982) and Nudelman.
ε、 ｅｔ ａｌ、 Ｊ、 ［３ｉｏ１． Ｃｈｅｗ
、 ２５７．１２７５２−１２７５６（１９８２）、Ｇ
Ｄ２に対してはＣａｈａｎ、 Ｌ、 Ｄ、 ｅｔ ａｌ
。ε, et al, J, [3io1. Chew
, 257.12752-12756 (1982), G.
For D2, Cahan, L., D., et al.
.
Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａｄ、 Ｓｃｉ、 ＵＳ
Ａ ７９．７６２９−７６３３（１９８２）、Ｃｈｅｒ
ｅｓｈ、Ｄ、Ａ、ｅｔ ａｌ、 Ｐｒｏｃ、Ｎａｔｌ
。Proc, Natl, Acad, Sci, US
A 79.7629-7633 (1982), Cher.
esh, D, A, et al, Proc, Natl
.
Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ ８］、 ５７６７−５
７７１（１９８４）、Ｃｈａｕｎｇ ＋Ｎ、 Ｋ、 ｅ
ｔ ａｌ、 Ｃａｎｃｅｒ Ｒｅｓ、 ４５． ２６４
２−２６４９゜および５ａｉｔｏ Ｍ、 ｅｔ ａｌ、
Ｂｉｏｃｈｅｍ、 Ｂｉｏｐｈｙｓ、Ｒｅｓ。Acad, Sci, USA 8], 5767-5
771 (1984), Chaung +N, K, e
tal, Cancer Res, 45. 264
2-2649° and 5aito M, et al.
Biochem, Biophys, Res.
Ｃｏｍｍｕｎ、 ｕＺ、 １−７（１９８５）、 ０Ｍ
２に対してはＴａｉ、 Ｔ。Commun, uZ, 1-7 (1985), 0M
Tai, T for 2.
ｅｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａｄ
、 Ｓｃｉ、 ＵＳＡ ８０． ５３９２−５３９
６ （１９８３）やＮａＬｏｌｉ、 Ｅ、Ｊ、 ｅｔ
ａｌ、 Ｃａｎｃｅｒ Ｒｓｓ。et al, Proc, Natl, Acad
, Sci, USA 80. 5392-539
6 (1983) and NaLoli, E. J., et al.
al, Cancer Rss.
４万、 ４１６６−４１２０（＋９８６）、０Ｍ３に対
しては１１ｊｒａｂａｙａｓｈｊ、 Ｙ、 ｅｔ
ａｌ、 Ｊ、 ロｉｏ１． Ｃｈｅｗ、
２６０゜１３３２８−１３３３３ （１９８５）のよう
な研究がある。しかしながら現在までガングリシドに対
するモノクローナル抗体はそれほど数多く作られていな
いのが現状である。40,000, 4166-4120 (+986), 11jrabayashj, Y, et for 0M3
al, J, Roio1. Chew,
There are studies such as 260°13328-13333 (1985). However, to date, not many monoclonal antibodies against ganglyside have been produced.
これまで腫瘍細胞表面ガングリオシドに対するモノクロ
ーナル抗体が作られてこなかった大きな要因として、モ
ノクローナル抗体を産生ずるハイブリドーマを得るに際
し、ヒト腫瘍細胞で感作させたマウスの抗体産生細胞と
マウス骨髄腫細胞を融合する異種免疫の方法を用いてき
たことが挙げられる。即ち、抗〃ｘとして投与される腫
瘍細胞が異種であるヒト由来のものであるため、ヒトの
主要組織適応抗原系（Ｍａｊｏｒ Ｈｉｓｔｏｃｏｍｐ
ａｔｉｂｉｌｉｔｙＣｏｍｐｌｅｘ）である）ＩＬＡを
認識するモノクローナル抗体を産生ずるハイブリドーマ
ばかりが得られ、目的とするハイブリドーマを得る確率
が非常に小さかった。A major reason why monoclonal antibodies against tumor cell surface gangliosides have not been produced so far is that when obtaining hybridomas that produce monoclonal antibodies, mouse antibody-producing cells sensitized with human tumor cells are fused with mouse myeloma cells. One example is the use of heterologous immunization methods. That is, since the tumor cells administered as anti-
Only hybridomas that produced monoclonal antibodies that recognized ILA (Ability Complex) were obtained, and the probability of obtaining the desired hybridoma was extremely small.
このような状況下で本発明者らは上記の方法に代えて、
マウスリンパ系腫瘍細胞で感作されたマウスの抗体産生
細胞とマウス骨髄腫細胞とを融合させる同種免疫の方法
を採ることにより、 ＩＩＬＡを認識する七ツクロー
ナル抗体を産生ずる不要なハイブリドーマの生成が排除
され、マウスおよびヒトの腫瘍細胞の細胞表面の種間共
通の腫瘍関連抗原であるガングリオシド、特にＧＤ２を
特異的に認識するモノクローナル抗体を産生ずるハイブ
リドーマを極めて能率良く得ることができることを発見
した。そして本発明者らはこの発見に基づいて創製され
るモノクローナル抗体とそれを産生するハイブリドーマ
に関する発明について先に特許出願した（ＰＣＴ／ＪＰ
８７１００６９０） 。Under such circumstances, the present inventors replaced the above method,
By adopting a homologous immunization method in which mouse antibody-producing cells sensitized with mouse lymphoid tumor cells are fused with mouse myeloma cells, the generation of unnecessary hybridomas that produce seven clonal antibodies that recognize IILA is eliminated. They discovered that it is possible to very efficiently obtain hybridomas that produce monoclonal antibodies that specifically recognize gangliosides, particularly GD2, which are tumor-associated antigens common across species on the cell surface of mouse and human tumor cells. The present inventors previously filed a patent application for an invention related to a monoclonal antibody created based on this discovery and a hybridoma that produces it (PCT/JP
87100690).
一方、モノクローナル抗体の抗原に対する強い特異性を
利用して悪性腫瘍の治療に役立てようとする試みも精力
的になされてきた。その結果ガングリオシ ドを抗原す
るモノクローナル抗体が腫瘍の治療に有用であることが
知られ始めた（Ｉ（ａｋ。On the other hand, efforts have also been made to make use of the strong antigen specificity of monoclonal antibodies to help treat malignant tumors. As a result, it has become known that monoclonal antibodies that antigen gangliosides are useful for tumor treatment (I(ak)).
ｍｏｒｉ、 Ｓ、 Ｃａｎｃｅｒ Ｒｅｓ、 ４５．２
４０５−２４１４（１９８５））。mori, S, Cancer Res, 45.2
405-2414 (1985)).
そしてこのようなガングリオシドを抗原とするモノクロ
ーナル抗体の一部は既に臨床応用の段階にまで進んで来
ている。例えば、Ｄｉｐｐｏｌｄらがヒトメラノーマ細
胞株ＳＫ−ＭＥＬ２８をマウスに免疫することにより作
り出しくＤｉｐｐｏｌｄ Ｗ、 Ｇ、 ｅｔ ａｌ、
Ｐｒｏｃ。Some monoclonal antibodies that use gangliosides as antigens have already reached the stage of clinical application. For example, Dippold et al. created by immunizing mice with the human melanoma cell line SK-MEL28.
Proc.
Ｎａｔ、］、 Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ
７７、 ６１１４−６１１８（１９８０） ）、Ｐ
ｕｋｅｌらがＧＤ３を認識することを示した（Ｐｕｋｅ
ｌ、 Ｃ。Nat, ], Acad, Sci, USA
77, 6114-6118 (1980)), P.
showed that GD3 is recognized by Puke et al.
l, C.
Ｓ、 ｅｔ ａｌ、 Ｊ、 Ｅｘｐ、 Ｍｅｄ、 １５
５．１１３３−１１４７（１９８２））、抗ＧＤ３モノ
クローナル抗体Ｒ２４を、Ｈｏｕｇｈｔｏｎらが転移性
腫瘍を持つメラノーマ患者１２例に２週間にわたり約８
回全身投与したところ、３例に顕著な腫瘍の縮小を認め
た（ｌｌｏｕｇｈｔｏｎ、 Ａ、 Ｎ、 ａｔ ａｌ、
Ｐｒｏｃ。S. et al., J. Exp. Med, 15
5.1133-1147 (1982)), Houghton et al. administered the anti-GD3 monoclonal antibody R24 to 12 melanoma patients with metastatic tumors at approximately
After systemic administration, significant tumor shrinkage was observed in 3 cases (Loughton, A, N, at al.
Proc.
Ｎａｔｌ、 Ａｃａｄ、 Ｓｃｉ、 ＵＳＡ ８２．１
２４２−１２４６ （１９８５））。Natl, Acad, Sci, USA 82.1
242-1246 (1985)).
Ｒ２４については、これを使用してヒトの神経外胚葉性
悪性腫瘍と上皮性ガンの治療する方法に関する発明が、
特開昭６２−２８９５２４として出願されている。また
、Ｃｈｅｕｎｇがヒト神経芽細胞腫ＬＡＮ−１をマウス
に免疫して得られた抗ＧＤ２モノクローナル抗体３Ｆ８
を１２例の転移性腫瘍患者（メラノーマ６例、神経芽細
胞腫４例、骨肉腫２例）に全身投与して、３例に顕著な
効果が認められたと報告している（Ｃｈｅｕｎｇ、 Ｎ
、に、 ａｔ ａｌ、 Ｐｒｏｃ、 Ａｖａ、 Ａｓ５
ｏｃ。Regarding R24, the invention relates to a method for treating human neuroectodermal malignant tumors and epithelial cancer using R24.
It has been filed as Japanese Patent Application Laid-Open No. 62-289524. In addition, Cheung used anti-GD2 monoclonal antibody 3F8, which was obtained by immunizing mice with human neuroblastoma LAN-1.
reported that it was administered systemically to 12 patients with metastatic tumors (6 melanoma cases, 4 neuroblastoma cases, and 2 osteosarcoma cases), and remarkable effects were observed in 3 cases (Cheung, N.
, to, at al, Proc, Ava, As5
oc.
Ｃａｎｃｅｒ Ｒｅｓ、 ２７．３１８（１９８６））
、１更にはＩｒ１ｅらはメラノーマ患者の末梢血リンパ
球をＥＢウィルスで株化することにより得られた細胞が
生産する抗ＧＤ２モノクローナル抗体Ｌ７２を８人の患
者の合計２１箇所の転移性メラノーマに補体と共に投与
したところ、１６箇所で退縮が認められ、このうち１０
箇所では完全に消退したと報告している（Ｉｒｉｓ、
Ｒ，Ｆ。Cancer Res, 27.318 (1986))
1Furthermore, Ir1e et al. used the anti-GD2 monoclonal antibody L72, produced by cells obtained by cultivating peripheral blood lymphocytes of melanoma patients with EB virus, to metastatic melanoma in a total of 21 sites in 8 patients. When administered together with the body, regression was observed in 16 locations, of which 10
In some places, it has been reported that it has completely disappeared (Iris,
R,F.
ａｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａｄ、
Ｓｃｉ、 ＵＳＡ ８３．８６９４−８６０８（１９８
６））。at al, Proc, Natl, Acad,
Sci, USA 83.8694-8608 (198
6)).
そして本発明者らは上に記した特許出１１’［（ＰＣＴ
／Ｊｌ”８７１００６９０）の実施例に記載したモノク
ローナル抗体の中の一つにそれ自身で強い抗腫瘍性を持
つものを見い出し、用途発明（抗腫瘍剤）として特許出
願を行なった（特願昭６３−２１３１７）。The present inventors and the above-mentioned Patent No. 11' [(PCT
We discovered that one of the monoclonal antibodies described in the examples of ``/Jl'' 87100690) had strong antitumor properties by itself, and filed a patent application as a use invention (antitumor agent) (Patent application No. 63). -21317).
（発明が解決しようとする問題点）
本発明者らの先の出願（特願昭６３−２１３１７）は、
それまでのモノクローナル抗体を有効成分とする抗腫瘍
剤の殆どがメラノーマに対する効果であり、一部が神経
芽腫を退縮させるものであって、適用範囲はごく限られ
たものであったのに対し、白血病の如き悪性リンパ腫の
モノクローナル抗体による治療効果を見いだした点に大
きな意味がある。(Problems to be solved by the invention) The previous application by the present inventors (Japanese Patent Application No. 63-21317)
Until then, most of the antitumor drugs that had monoclonal antibodies as active ingredients were effective against melanoma, and some were effective for regression of neuroblastoma, so the range of application was extremely limited. The discovery of the therapeutic effect of monoclonal antibodies on malignant lymphomas such as leukemia is of great significance.
しかしながら同一の名称で呼ばれる腫瘍であっても腫瘍
の多様性は大きく、一つの抗腫瘍剤があらゆる症例に対
して著効を示すとは限らず、又腫瘍自体が抗腫瘍剤の投
与を継続するうちに薬剤に対して抵抗性を示すようにな
る場合が多い。そのために、癌の治療に際して治療開始
から完癒まで単一の抗腫瘍剤で事足りることは少なく、
複数の薬剤が求められている。However, even if tumors are called by the same name, there is a great diversity of tumors, and one anti-tumor drug may not be effective in all cases, and the tumor itself may require continued administration of anti-tumor drugs. In many cases, they develop resistance to drugs. For this reason, when treating cancer, a single antitumor agent is rarely sufficient from the start of treatment to complete recovery.
Multiple drugs are needed.
（問題点を解決するための手段）
そこで本発明者らは更に続けて先に出願した方法と同じ
でハイブリドーマを創製して、ハイブリドーマが産生ず
るモノクローナル抗体のうち抗腫瘍効果のあるものを探
索してきたところ、上述のモノクローナル抗体Ａ１．２
６７、Ａ１．４１０及びＡ１．４２５が強い抗腫瘍効果
を持つことを発見し１本発明を完成した。(Means for Solving the Problems) Therefore, the present inventors further created hybridomas using the same method as previously applied, and searched for monoclonal antibodies produced by the hybridomas that have antitumor effects. However, the above-mentioned monoclonal antibody A1.2
67, A1.410 and A1.425 have strong antitumor effects, and have completed the present invention.
即ち本発明者らはマウスＴ細胞白血病細胞株を移植され
たマウスにモノクローナル抗体Ａ１．２６７、Ａ１．４
１０又はＡ１．４２５を投与したところ、これらモノク
ローナル抗体がこの担癌マウスの寿命を著量に延長し、
或は腫瘍を完全に拒絶させることを認めたのである。That is, the present inventors administered monoclonal antibodies A1.267 and A1.4 to mice transplanted with mouse T-cell leukemia cell lines.
10 or A1.425, these monoclonal antibodies significantly extended the lifespan of these tumor-bearing mice.
Or they allowed the tumor to be completely rejected.
本発明はモノクローナル抗体Ａ１．２６７、 ＡＩ、４
１０又はＡ１．４２５を有効成分とする新規な抗腫瘍剤
である。The present invention provides the monoclonal antibody A1.267, AI, 4
It is a novel antitumor agent containing A1.10 or A1.425 as an active ingredient.
そして、本発明に係るモノクローナル抗体ＡＩ。And monoclonal antibody AI according to the present invention.
２６７を産生ずるハイブリドーマＡ １ 、２６７は微
工研にＦＥＰＭ Ｐ−９９９２として寄託され、モノク
ローナル抗体Ａ１．４１０を産生ずるハイブリドーマ＾
１．４１０は微工研にＦＥＰＭ Ｐ−９９９３として寄
託され、そして、モノクローナル抗体Ａ１．４２５を産
生ずるハイブリドーマＡ１．４２５は微工研にＦＥＰＭ
Ｐ−９９９４として寄託されている。The hybridoma A 1 that produces 267, 267, has been deposited at FIKEN as FEPM P-9992, and is a hybridoma that produces monoclonal antibody A1.410.
1.410 has been deposited as FEPM P-9993 at FEPM, and the hybridoma A1.425 producing monoclonal antibody A1.425 has been deposited with FEPM P-9993 at FEPM.
It has been deposited as P-9994.
本発明の抗腫瘍剤を上記モノクローナル抗体を有効成分
として製剤化するには公知の方法を適用すればよい。投
与方法としては悪性リンパ腫の治療に用いる関係上、点
滴注射による投与が好ましい。Known methods may be applied to formulate the antitumor agent of the present invention using the above monoclonal antibody as an active ingredient. As for the administration method, administration by drip injection is preferable because it is used for the treatment of malignant lymphoma.
注射薬の製剤には、生理的食塩水、滅菌水リンゲル液等
の水溶性溶剤、非水性溶剤、等張化剤、烈痛化剤、溶解
補助剤、安定化剤、防腐剤、懸濁化剤、緩衝剤、乳化剤
等を任意に使用し得る。Injectable drug formulations include physiological saline, water-soluble solvents such as sterile water Ringer's solution, non-aqueous solvents, isotonic agents, sharpening agents, solubilizing agents, stabilizers, preservatives, and suspending agents. , buffers, emulsifiers, etc. may optionally be used.
−例として、生理的食塩水（塩濃度約０．９％）にヒト
血清アルブミンを５％にモノクローナル抗体を適量含む
ものを調製して注射剤とする。- As an example, an injection is prepared by preparing a physiological saline solution (salt concentration of approximately 0.9%) containing 5% human serum albumin and an appropriate amount of a monoclonal antibody.
モノクローナル抗体は点滴注射製剤中ｏ、ｉμｇ〜１０
０＠ｇ／＠Ｑ、好ましくは１μｇ〜１ ｍｇ／社含有さ
れる。The monoclonal antibody is in the infusion injection formulation, iμg ~ 10
The content is 0@g/@Q, preferably 1 μg to 1 mg/sha.
点滴投与量は症状、腫瘍の進行１年齢、性別等に応じて
５０〜１，０００ＷＩＱであり、毎日或は適当に日をお
いて与える。The intravenous dose is 50 to 1,000 WIQ depending on the symptoms, age of tumor progression, gender, etc., and is given daily or at appropriate intervals.
以下に本発明を実施例により具体的に説明する。The present invention will be specifically explained below using examples.
失施涜〔Ｌはヅブリドーでや作製）
Ｃ５７ＢＬマウス由来のＴ細胞腫瘍株ＥＬ４を１０’細
胞／マウス、１回／週、４週間に亙りＡ系マウスの腹腟
内に接種し、最終接種後３日日に肺臓を摘出してこれを
常法により組織培養液ＲＰＭ１１６４０（Ｇ、ＩＢＣＯ
社製）中の細胞浮遊液として調製した。Inoculation of T cell tumor line EL4 derived from C57BL mice into the abdomen and vagina of strain A mice at 10' cells/mouse once/week for 4 weeks, and after the final inoculation. On the 3rd day, the lungs were removed and cultured in tissue culture medium RPM11640 (G, IBCO) using a conventional method.
The cell suspension was prepared as a cell suspension in (manufactured by Sekisui Chemical Co., Ltd.).
一方融合の親細胞として１３ａｌｂ／ｃ系マウス由来の
骨髄腫細胞株ＮＳＩを培養して１０７細胞用意した。On the other hand, 107 cells were prepared by culturing the myeloma cell line NSI derived from 13alb/c mouse as the parent cells for fusion.
これらの細胞の融合はにｏｅｈ ｌｅｒとＭｉｌｓｔｅ
ｊｎの方法（Ｎａｔｕｒｅ、 ２５６．４９５（１９７
５））に従い、以下のように行なった。The fusion of these cells occurs in oehler and milste
jn method (Nature, 256.495 (197
5)), the following procedure was carried out.
前述のようにして調製した肺臓の浮遊細胞と骨髄腫細胞
を細胞数で５＝１となるように遠心チューブ内で混合し
、５００ｒ、ｐ、ｍ、で５分遠心した後上清を捨て、Ｒ
ＰＭＩ１６４０で濃度４０％（ｖ／ｖ）に調製したポリ
エチレングリコール（メルク社爬；平均分子２着／１，
０００）を０．２＋ｎＱ加えて２分間よく撹拌混合した
。The floating lung cells and myeloma cells prepared as described above were mixed in a centrifuge tube so that the number of cells was 5 = 1, and after centrifugation at 500 r, p, m for 5 minutes, the supernatant was discarded. R
Polyethylene glycol prepared with PMI 1640 to a concentration of 40% (v/v) (Merck Co., Ltd.; average molecular weight 2/1,
000) was added for 0.2+nQ and stirred and mixed well for 2 minutes.
その後これに５ｍｆｌのｌ’ｌＰＭＩ１６４０を１滴づ
つ３分間かけて添加して希釈し、 ５００ｒ、ｐ、ｍ、
で５分遠心した後上清を捨てた。次いで細胞をｌ＋ＡＴ
培地（ＲＰＭ１１６４０培地に１０％（ν／ｖ）ウシ胎
児血清を加え、最終濃度でヒボキサンチン１０−４Ｍ、
アミノプテリン４×１０−７Ｍ、チミジン１．６ Ｘ
１０−’Ｍを加えたもの）に浮遊させて１００μＱ／穴
の割合で９６穴マイクロプレートに分注した。After that, 5 mfl of l'lPMI 1640 was added drop by drop over 3 minutes to dilute it, and 500 r, p, m,
After centrifugation for 5 minutes, the supernatant was discarded. The cells were then exposed to l+AT
Medium (RPM11640 medium with 10% (v/v) fetal bovine serum, final concentration of hyboxanthin 10-4M,
Aminopterin 4 x 10-7M, Thymidine 1.6X
10-'M) and dispensed into a 96-well microplate at a rate of 100 μQ/well.
温度３７°Ｃ，Ｃｏ２：空気＝５：９５の気相でこれを
培養し、培養３［］目および６日日にＩＩＡＴ培養で培
地交換を行なった。それ以後は３日毎にＩＩＴ培地（ｌ
ｌＡＴ椿地からアミノプテリンのみを除いた培地）で培
地交換を行なった。This was cultured at a temperature of 37°C in a gas phase of Co2:air = 5:95, and the medium was replaced with IIAT culture on the 3rd and 6th day of culture. Thereafter, every 3 days IIT medium (l
The medium was replaced with a medium obtained by removing only aminopterin from lAT Tsubakiji.
培養を続け、細胞のコロニーの形成が認められるように
なった時点（約１０−１４日１）で培養上清中の抗体活
性を測定してスクリーニングを行なった。The culture was continued, and at the time when cell colony formation was observed (approximately 10 to 14 days 1), screening was performed by measuring the antibody activity in the culture supernatant.
スクリーニングはウサギ血清補体を併用する細胞障害試
験法を用いた。具体的には、標的となるＣ５７１比マウ
ス由来のＴ細胞腫瘍株ＥＬ４の細胞浮遊液（、！Ｉ＋胞
濃度５ＸＩＯ’個／＋ｎｕ）２０μＱ、ウサギ血清補体
２０μＱおよび培養上清２０μＱを混合して時々撹拌し
つつ３７℃で４５分間反応させた。反応終了後水冷して
トリパンブルー水溶液５０μＱを加えた後、顕微鏡で観
察してトリパンブルーを取り込んだ細胞の有無により培
養上清の細胞障害活性を判断した。A cytotoxicity test method using rabbit serum complement was used for screening. Specifically, 20 μQ of a cell suspension of the target C571 mouse-derived T cell tumor line EL4 (!I + cell concentration of 5XIO' cells/+nu), 20 μQ of rabbit serum complement, and 20 μQ of culture supernatant were mixed. The reaction was carried out at 37° C. for 45 minutes with occasional stirring. After the reaction was completed, the culture supernatant was cooled with water and 50 μQ of trypan blue aqueous solution was added thereto, followed by observation under a microscope to determine the cytotoxic activity of the culture supernatant based on the presence or absence of cells that had taken up trypan blue.
スクリーニングを行なったハイブリドーマ４８６株のう
ち、細胞障害活性をもつ抗体を産生ずるものが１４個得
られ、そのうち１株のハイブリドーマについて、限界希
釈法によりクローン化した。更にクローニングを繰り返
して安定で単一の細胞由来のハイブリドーマＡ１．２６
７を得た。Of the 486 hybridoma strains screened, 14 were obtained that produced antibodies with cytotoxic activity, and one hybridoma among them was cloned by the limiting dilution method. Further cloning was repeated to create a stable, single cell-derived hybridoma A1.26.
I got a 7.
同様にして、ハイブリドーマＡ１．．４１０を得た。Similarly, hybridoma A1. ．． I got 410.
また、同様にして、ハイブリドーマＡ１．．４２５を得
た。Similarly, hybridoma A1. ．． I got 425.
なお、オフタロニー法により各ハイブリドーマが産生ず
る各モノクローナル抗体の免疫グロブリンのクラスを調
べたところ、いずれも（ＩｇＧ：＋、に）であった。In addition, when the immunoglobulin class of each monoclonal antibody produced by each hybridoma was investigated by the Ophthalony method, all of them were (IgG: +, 2).
失に性影はノ！−旦二土四五生旦、 ２６７Δ情ス）−
Ｂａ１ｂ／ｃ系のヌードマウスの腹腔にブリステン０．
５ｆｆｌｆｌ投与し、７日後に実施例１により得られた
ハイブリドーマＡ１゜２６７（ＦＥＲＭ Ｐ−９９９２
）約５〜１ＯＸＩＯ’細胞を腹腔的接種して腹水化を行
なった。What a shame! - 267Δjosu) -
Blisten 0.0.
After 7 days, hybridoma A1゜267 (FERM P-9992) obtained according to Example 1 was administered.
) Approximately 5-1 OXIO' cells were inoculated intraperitoneally to perform asciticization.
１〜２週間後に腹水を採取し、３．００Ｏｒ、ｐ、ｍ、
、１５分間の遠心により上清−ヒ層部分のブリステンと
沈澱物である細胞塊を取り除いたモノクローナル抗体が
含まれる中間層の部分を回収した。After 1 to 2 weeks, ascites was collected and 3.00 Or, p, m,
Then, by centrifugation for 15 minutes, the supernatant and the intermediate layer containing the monoclonal antibody were recovered from which the blisters and precipitated cell clusters in the supernatant layer were removed.
上記により回収された溶液１０ｍＱに硫酸アンモニウム
を赫終濃度５０％となるように加えて、塩析した。これ
を１０，０００ｒ、ｐ、ｍ、、３０分間の遠心にかけ、
沈澱した分画をＰＩ（ｓ（ｐＨ７，２の０，０］−Ｍ燐
酸緩衝液に０．１５Ｍ ＮａＣ１を加えた燐酸緩衝塩溶
液） １０ｍＱに溶かした。この溶液をＰｕｓ １，０
００ｍＱに対して３回透析を繰り返し、免疫グロブリン
分画とした。Ammonium sulfate was added to 10 mQ of the solution recovered above to a final concentration of 50% for salting out. This was centrifuged at 10,000 r, p, m, for 30 minutes,
The precipitated fraction was dissolved in 10 mQ of PI (a phosphate buffered salt solution prepared by adding 0.15 M NaCl to s (0,0 of pH 7,2)-M phosphate buffer). This solution was dissolved in Pus 1,0
Dialysis against 00mQ was repeated three times to obtain an immunoglobulin fraction.
次いで該免疫グロブリン分画を、０．０３Ｍ ＮａＣＱ
を含むρ１１８ 、０の燐酸緩衝液に対して十分に透析
した。The immunoglobulin fraction was then treated with 0.03M NaCQ.
The sample was extensively dialyzed against a phosphate buffer containing ρ118,0.
別にこれと同じ緩衝液で平衡化しておいたイオン交換樹
脂ＤＥ５２カラム（ワットマン社製）に該透析液をかけ
、ＤＥ５２カラムに吸着されなかった両分を回収した。Separately, the dialysate was applied to an ion exchange resin DE52 column (manufactured by Whatman) that had been equilibrated with the same buffer, and both fractions that were not adsorbed to the DE52 column were collected.
この回収された両分をＰＢＳに対して透析を行なったも
のを精製ＩｇＧ抗体とし、モノクローナル抗体Ａ１．２
６７を得た。Both recovered fractions were dialyzed against PBS to obtain purified IgG antibody, and monoclonal antibody A1.2
I got 67.
実施例良」孟工仁久ｐ−ナル抗体Ａ１．２６７ｑ狩濃」
ひモノクローナル抗体Ａ １ 、２６７が認識している
ガングリオシドを決定するために、薄層クロマトグラフ
ィー（以下ＴＬＣと称す）上での酸素免疫染色法を行な
った。Good example "Meng Ko Jinkyu p-nal antibody A1.267q Karino"
In order to determine the ganglioside recognized by monoclonal antibody A 1,267, oxygen immunostaining on thin layer chromatography (hereinafter referred to as TLC) was performed.
（１）ヒトメラノーマ細胞株Ｍ１４のガングリオシド分
画、ガングリオシド純品および中性糖脂質純品の調製
ヒトメラノーマ細胞株ＵＣＬＡＳＯ−Ｍ １４ （以下
Ｍ１４という）はカルホルニア大学のＲ，Ｔｒｉｅ博士
より入手したもので、Ｍ’１４のガングリオシド分画の
調製はｒａｔ。(1) Preparation of ganglioside fraction, pure ganglioside product, and pure neutral glycolipid product of human melanoma cell line M14 Human melanoma cell line UCLASO-M 14 (hereinafter referred to as M14) was obtained from Dr. R. Trie of the University of California. The ganglioside fraction of M'14 was prepared using rat.
Ｔ、 ａｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａ
ｄ、 Ｓｃｉ、 ＵＳＡ ８０．５３９２−５３９６（
１９８３）に従った。T, at al, Proc, Natl, Aca
d, Sci, USA 80.5392-5396 (
(1983).
ガングリオシドＧＭ１． ＧＤｌａ、 ＧＤｌｂ、 Ｇ
Ｔｌｂはウシの脳から調製し、Ｋａｎｆｅｒ、 Ｊ、
Ｎ、 Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ。Ganglioside GM1. GDla, GDlb, G
Tlb was prepared from bovine brain and was described by Kanfer, J.
N. Methods Enzymol.
１．４．６６０−６６４（１，９６９）に従って精製し
た。ＧＱｌｂはヤトロン社から購入した。ヒト脳の０Ｍ
３はスウエーデンのＧｏｔｅｂｏｒｇ大学のり、 Ｓｖ
ｅｎｎｅｒｈｏ１ｍ博士から入手した。０Ｍ２とＣＤ２
はそれぞれ０Ｍ２とＧＤｌｂから、ウシ來丸β−ガラク
トシダーゼ（アメリカのミシガン大学のＪ、 ＩＪ、
Ｊｏｕｒｄｉａｎ博士から入手）を用いて調製した。ヒ
トミニリンの０Ｍ４とヒト脳のＧＴｌａは東京都臨床医
学総合研究所の有賀博士から入手した。クマ赤血球のＧ
Ｄ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）、ＧＤ３ （Ｎｅｕ
Ａｃ 。Purified according to 1.4.660-664 (1,969). GQlb was purchased from Yatron. 0M of human brain
3 is from Goteborg University in Sweden, Sv
Obtained from Dr. Ennerho1m. 0M2 and CD2
were obtained from 0M2 and GDlb, respectively, using Ushiraimaru β-galactosidase (J, IJ, University of Michigan, USA;
(obtained from Dr. Jourdian). Human minirin 0M4 and human brain GTla were obtained from Dr. Ariga of the Tokyo Metropolitan Institute of Clinical Medicine. bear red blood cell G
D3 (NeuAc, NeuAc), GD3 (Neu
Ac.
ＮｅｕＧｃ）、ＣＤ３（ＮｅｕＧｃ、 ＮｅｕＡｃ）、
およびＧＤ３（ＮａｕＧｃ。NeuGc), CD3 (NeuGc, NeuAc),
and GD3 (NauGc.
ＮｅｕＧｃ）は東京都臨床医学総合研究所の橋本博士と
鈴木明身傅士から入手した。ＬａｃＣａｒ、 ＧｇＯｓ
、Ｃａｒ。NeuGc) was obtained from Dr. Hashimoto and Fuji Suzuki of the Tokyo Metropolitan Institute of Clinical Medicine. LacCar, GgOs
, Car.
およびＧｇ０ｓ４Ｃｅｒはそれぞれ０Ｍ３．０Ｍ２．
ＧＭＩから弱酸で処理し、イアトロビーズカラムクロマ
トグラフィーで精製して調製した。ＧｌｃＣｅｒはＴａ
ｉ、 Ｔ。and Gg0s4Cer are 0M3.0M2.
It was prepared from GMI by treatment with a weak acid and purification by Iatrobead column chromatography. GlcCer is Ta
i, T.
ａｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａｄ、
Ｓｃｉ、 ＵＳＡ ８０．５３９２−５３９６（＋９８
３）に従って調製した。ＧｂＯ８３ＣｅｒとＧｂＯ８４
Ｃｓｒは５ｕｐｅｌｃｏ社（アメリカ、ペンシルバニア
州、ベラフオンテ）から購入した。at al, Proc, Natl, Acad,
Sci, USA 80.5392-5396 (+98
3). GbO83Cer and GbO84
Csr was purchased from 5upelco (Bellajuonte, Pennsylvania, USA).
（２） ＴＬＣの方法
メルク社製のシリカゲルを塗布済みのＴＬＣプレート「
シリカゲル６０」（厚さ２００μＩ）と同じくメルク社
製のシリカゲルを塗布済みのＴＬＣプラスチックシート
を使用した。クロマトグラムの展開溶媒はクロロフォル
ム／メタノール１０．２２％ＣａＣ１□水溶液を容積比
５５／４５／１０としたものを用いた。なお。(2) TLC method TLC plate coated with Merck's silica gel
A TLC plastic sheet coated with silica gel manufactured by Merck & Co., Ltd., as well as "Silica Gel 60" (thickness 200 μI), was used. The developing solvent for the chromatogram was a chloroform/methanol 10.22% CaCl□ aqueous solution with a volume ratio of 55/45/10. In addition.
ＴＬＣプレート上に展開されたガングリオシドを染色す
る場合にはレゾルシノール（ｒｅｓｏｒｃｉｎｏｌ ）
染色を、中性糖脂質の染色にはオルシノール（ｏｒｃｉ
ｎｏｌ）染色を用いた。When staining gangliosides developed on TLC plates, resorcinol
For neutral glycolipid staining, orcinol (orci
nol) staining was used.
（３） ＴＬＣプレート上でのモノクローナル抗体 Ａ
Ｉ、２６７による酵素免疫染色法
ガングリオシドのＴＬＣ終了後、ＴＬＣプレートをウシ
血清アルブミン１％とポリビニールピロリドン（ｐｏｌ
ｙｖｉｎｙｌｐｙｒｏｌｉｄｏｎｅ） １％とを含有す
る燐酸緩衝塩溶液に浸した。空気中で乾燥させた後、プ
レートをモノクローナル抗体Ａ１．２６７溶液（１０μ
ｇ１社）と２時間２５℃で反応させた６次に燐酸緩衝塩
溶液（以下ＰＢＳと称す）を５回交換しつつその中でク
ロマトグラムを洗浄した。そして西洋ワサビペルオキシ
ダーゼを結合させたヤギ抗マウスＩｇＧおよび工ｇＭ抗
体をこのクロマトグラムと２５℃で２時間反応させた。(3) Monoclonal antibody A on TLC plate
Enzyme immunostaining method according to I, 267 After TLC of gangliosides, the TLC plate was treated with 1% bovine serum albumin and polyvinyl pyrrolidone (pol
yvinylpyrolidone) in a phosphate buffered saline solution containing 1%. After drying in air, the plate was coated with monoclonal antibody A1.267 solution (10μ
Next, the chromatogram was washed in a phosphate buffered saline solution (hereinafter referred to as PBS) which was exchanged five times. Then, goat anti-mouse IgG and engineered gM antibodies conjugated with horseradish peroxidase were reacted with this chromatogram at 25° C. for 2 hours.
再びＰＢＳを５回交換しつつその中でクロマトグラムを
洗浄した。染色に当たっては４００μｙ；／ｍＱの０−
フェニレンジアミンと０．１２％の過酸化水素を含むク
エン酸−燐酸緩衝液（ｐＨ５，０，８０ａ＋Ｍ）を用い
、 １５分間反応させて、発色させた。その抜水に浸し
て反応を停止させた。The chromatogram was washed again in 5 changes of PBS. For staining, 400μy;/mQ of 0-
Using a citric acid-phosphate buffer (pH 5, 0, 80a+M) containing phenylene diamine and 0.12% hydrogen peroxide, the mixture was reacted for 15 minutes to develop color. The reaction was stopped by immersing it in the drained water.
（４）ガングリオシド純品混合物及びＭ１４のガングリ
オシドのモノクローナル抗体Ａ１．２６７と反応させた
ＴＬＣプレート上での酵素免疫染色法の結果第１図は左
のレーンから、ガングリオシド純品のｍ合物（ＧＭ３＋
ＧＭ２＋ＧＭＩ＋ＧＤ３＋ＧＤ２）を几ｃにかけテレゾ
ルシノールで染色したもの、ヒトメラノーマ細胞株旧４
のガングリオシド分画をＴＣＬにがけてレゾルシノール
で染色したもの、Ｍ１４のガングリオシド分画をＴＣＬ
にかけた後Ａ１．２６７で酵素免疫染色を行なったもの
である。（レーン番号３）この図のレーン番号３で示さ
れるように、モノクロナール抗体Ａ１．２６７はガング
リオシドＧＤ２とＧＤ３とに反応している。(4) Results of enzyme immunostaining on a TLC plate reacted with a pure ganglioside mixture and M14 ganglioside monoclonal antibody A1.267.
GM2+GMI+GD3+GD2) stained with telesorcinol, human melanoma cell line Old 4
The ganglioside fraction of M14 was applied to TCL and stained with resorcinol, and the ganglioside fraction of M14 was applied to TCL and stained with resorcinol.
After exposure to A1.267, enzyme immunostaining was performed. (Lane No. 3) As shown in lane No. 3 of this figure, monoclonal antibody A1.267 reacts with gangliosides GD2 and GD3.
その他のガングリオシド純品および中性糖脂質純品につ
いても各々ＴＣＬにかけた後モノクローナル抗体Ａ１．
２６７で酵素免疫染色を行なった。これらの結果につい
ては図には示さず、第１図の内容と合わせて、Ａ１．２
６７との反応性を表１に示す。Other pure ganglioside products and pure neutral glycolipids were also subjected to TCL, followed by monoclonal antibody A1.
Enzyme immunostaining was performed with 267. These results are not shown in the figure, but together with the contents of Figure 1, A1.2
Table 1 shows the reactivity with 67.
表 １
ＧＤ３ ＋＋ ０Ｍ３ −
ＧＤ２ ＋＋＋ ０Ｍ２ −
ＧＤｌａ −ＧＭＩ −
ＧＤｌｂ ＋＋ ＧｌｃＣｅｒ −ＧＴｌａ
＋＋ ＬａｃＣｅｒ −ＧＴｌｂ ＋ Ｇｇ０
ｓ３Ｃｅｒ −ＧＱｌｂ ＋＋ ＧｇＯｓ、Ｃｅｒ
−−ＧＴ３ ＧｂＯｓ、Ｃｅｒ−
ＧＴ２ ＧｂＯｓ、Ｃｅｒ −
０Ｍ４ −
表１から分かるように本発明のモノクローナル抗体は七
ノーシアロシルーガングリオシド（０Ｍ４．０Ｍ３．０
Ｍ２およびＧＭＩ）、および中性糖脂質（ＧｌｃＣｅｒ
、ＬａｃＣｅｒ、ＧｇＯｓ、Ｃｅｒ、 ＧｇＯｓ、Ｃｅ
ｒ、 Ｇｇ０ｓ３ＣｅｒおよびＧｂＯｓ４Ｃａｒ）とは
反応しない。Table 1 GD3 ++ 0M3 - GD2 +++ 0M2 - GDla -GMI - GDlb ++ GlcCer -GTla
++ LacCer −GTlb + Gg0
s3Cer −GQlb ++ GgOs, Cer
--GT3 GbOs, Cer- GT2 GbOs, Cer-0M4- As can be seen from Table 1, the monoclonal antibody of the present invention contains heptanosialosyl ganglioside (0M4.0M3.0
M2 and GMI), and neutral glycolipids (GlcCer
, LacCer, GgOs, Cer, GgOs, Ce
r, Gg0s3Cer and GbOs4Car).
即ち、Ａ１．２６７はＮｅｕＡｃ ａ ２ →８Ｎｓｕ
Ａｃ ａ ２−） ３Ｇａｌという三糖構造を持つＧＤ
３、ＧＤ２． ＧＤｌｂ、 ＧＴｌａ、 ＧＴｌｂおよ
びＧＱｌｂと反応し、他のガングリオシドとは反応しな
い。最も強く反応するのがＧＤ２であり逆に最も反応が
弱いのはＧＴｌｂである。ＧＤ２にガラスダース１個が
付加されたＧＤｌｂは反応性が弱くなり、ＧＤＩｂに更
にシアル酸が末端ガラスドース残基に付加されたＧＴｌ
ｂはもっと反応性が落ちる。この知見からシアル酸が°
末端のガラクトース残基（ＧＤｌｂ）や末端のシアン酸
残基（ＧＴｌｂ）に付加することによって、抗原決定基
であるＮｅｕＡｃα２→８ＮｅｕＡｃα２→３Ｇａｌは
マスクされることが示唆される。一方、ＧＴｌｂの末端
シアル酸残基にシアル酸が付加したＧＱｌｂはＧＤｌｄ
のレベルまで反応性が回復する。ＧＴｌａは反応性はＧ
Ｑｌｂと同程度であるから、ＧＱｌｂの認識部位は末端
の三糖であるはずである。以上から、Ａ１．２６７はガ
ラクトース残基（場所は内部、末端を問わない）に結合
したジシアロシル残基ｒＮｅｕＡｃα２→８ＮａｕＡｃ
α２→３」を認識していることが判明した。That is, A1.267 is NeuAc a 2 →8Nsu
Ac a 2-) GD with a trisaccharide structure called 3Gal
3.GD2. Reacts with GDlb, GTla, GTlb and GQlb, but does not react with other gangliosides. GD2 reacts most strongly, and conversely, GTlb reacts the weakest. GDlb, in which one glass dose is added to GD2, has weaker reactivity, whereas GTl, in which sialic acid is further added to the terminal glass dose residue, is GDIb.
b is even less reactive. This finding suggests that sialic acid
It is suggested that the antigenic determinant NeuAcα2→8NeuAcα2→3Gal is masked by addition to the terminal galactose residue (GDlb) or the terminal cyanate residue (GTlb). On the other hand, GQlb, which has sialic acid added to the terminal sialic acid residue of GTlb, is GDld.
Reactivity is restored to the level of GTla has a reactivity of G
Since it is similar to Qlb, the recognition site of GQlb should be the terminal trisaccharide. From the above, A1.267 is a disialosyl residue rNeuAcα2→8NauAc bound to a galactose residue (the location does not matter whether it is internal or terminal).
It turned out that it recognized "α2→3".
次に４種類のＧＤ３（ＧＤ３（ＮａｕＡｃ、 ＮｅｕＡ
ｃ）、ＧＤ３（ＮｅｕＡｃ、 ＮａｕＧｃ）、 ＧＤ３
（ＮａｕＧｃ、 ＮｅｕＡｃ）、およびＧＤ３（Ｎｅｕ
Ｇｃ、 ＮｅｕＧｃ））とモノクローナル抗体のＴＬＣ
プレート上で酵素免疫染色法の結果は第２図に示されて
いる。Next, four types of GD3 (GD3 (NauAc, NeuA
c), GD3 (NeuAc, NauGc), GD3
(NauGc, NeuAc), and GD3 (Neu
Gc, NeuGc)) and TLC of monoclonal antibodies
The results of enzyme immunostaining on the plate are shown in FIG.
第２図（Ａ）は４種類のＧＤ３をＴＬＣにかけ、レゾル
シノールで染色したものである。左からガングリオシド
純品の混合物（ＧＭ３＋ＧＭ１＋ＧＤ３）、レーン番号
１はＧＤ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）、レーン番号
２はＧＤ３（ＮｅｕＡｃ、 ＮｅｕＧｃ）、レーン番号
３はＧＤ３ （ＮｅｕＧｃ 。FIG. 2(A) shows four types of GD3 subjected to TLC and stained with resorcinol. From the left, a mixture of pure gangliosides (GM3+GM1+GD3), lane number 1 is GD3 (NeuAc, NeuAc), lane number 2 is GD3 (NeuAc, NeuGc), and lane number 3 is GD3 (NeuGc).
ＮｅｕＡｃ）、レーン番号４はＧＤ３（ＮｅｕＧｃ、
ＮｅｕＧｃ）である。第２図（Ｂ）はＡ１．２６７によ
るＴＬＣの酵素免疫染色の結果である。レーン番号とＧ
Ｄ３の種類は第２図（Ａ）と同じである。NeuAc), lane number 4 is GD3 (NeuGc,
NeuGc). FIG. 2(B) shows the results of TLC enzyme immunostaining with A1.267. Lane number and G
The type of D3 is the same as in FIG. 2(A).
Ａ１．２６７が反応したのはＧＤ３（ＮｅｕＡｃ、 Ｎ
ｅｕＡｃ）とＧＤ３（ＮｅｕＧｃ、 ＮｅｕＡｃ）であ
る。この結果はＡ１．２６７のガングリオシドの認識に
は三糖の内部シアル酸が必須であり、かつ該シアル酸は
ＮｅｕＡｃ残基でなければならないことを示している。A1.267 reacted with GD3 (NeuAc, N
euAc) and GD3 (NeuGc, NeuAc). This result indicates that the internal sialic acid of the trisaccharide is essential for recognition of the ganglioside of A1.267, and that the sialic acid must be a NeuAc residue.
内部シアル酸がＮａｕＡｃでなく、ＮｅｕＧｃの時は′
Ｌ２ｐ識されていない。−方、末端シアル酸残基はＮｅ
ｕＧｃ、 ＮｅｕＡｃのいずれでも＾１．２６７に認識
されている。すると、 Ａ１．２６７の抗原決定基はＳ
ｉａ ａ ２−＋８ＮｅｕＡｃ ａ ２−’）３Ｇａｌ
残基となるはずである。When the internal sialic acid is not NauAc but NeuGc'
Not aware of L2p. -, the terminal sialic acid residue is Ne
Both uGc and NeuAc are recognized by ^1.267. Then, the antigenic determinant of A1.267 is S
ia a 2-+8NeuAc a 2-')3Gal
It should be a residue.
以上の結果をまとめると、モノクローナル抗体Ａ Ｉ
、 ２６７の細胞表面ガングリオシドに対する認識の特
異性は。To summarize the above results, monoclonal antibody A I
, the specificity of recognition of 267 for cell surface gangliosides.
ＧＤ２＞ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）＋ ＧＤ
３（ＮｅｕＧｃ、 ＮｅｕＡｃ）。GD2>GD3(NeuAc, NeuAc)+GD
3 (NeuGc, NeuAc).
ＧＤｌｂ、 ＧＴｌａ、 ’ＧＱ１ｂ＞ＧＴＩであると
考えられる。GDlb, GTla, 'GQ1b>GTI.
この特異性のパターンは本発明者が先に出願したＰＣＴ
／ＪＰ８７１００６９０の実施例に記載のモノクローナ
ル抗体ＭｏＡ２と全く同じである。但しＭｏＡ２は免疫
グロブリンのクラスが（ＩｇＭ、に）であって、Ａ１．
２６７のクラス（ＩｇＧ、に）とは異なっている。This pattern of specificity is based on the PCT patent application previously filed by the present inventor.
It is exactly the same as the monoclonal antibody MoA2 described in the Example of /JP87100690. However, MoA2 belongs to the immunoglobulin class (IgM), and A1.
It is different from the 267 class (IgG, ni).
去傭＃１４ （％ ／ ’ｙ ｏ ：ｊ」ｌ買はＡ１．
４１０ｑ８１ Ｎ）Ｂａｌｂ／ｃ系のヌードマウスの腹
腔にブリステン０．５ｍＱ投与し２７日後に実施例１区
より得られたハイブリドーマＡ１．．４１０（ＦＥＲＭ
Ｐ−９！１９３ ）約５〜１０ＸＩＯ’細胞を腹腟内
接種して腹水化を行なった。Leave #14 (% / 'y o :j'l purchase is A1.
410q81 N) 0.5 mQ of Blisten was administered into the peritoneal cavity of Balb/c nude mice, and 27 days later, hybridoma A1. ．． 410 (FERM
P-9!193) Approximately 5 to 10XIO' cells were inoculated intraperitoneally to induce ascites.
１〜２週間後に腹水を採取し、３．００Ｏｒ、ｐ、＋ａ
、、１５分間の遠心により上清上層部分のブリステンと
沈澱物である細胞塊を取り除いたモノクローナル抗体が
含まれる中間層の部分を回収した。1 to 2 weeks later, ascites was collected and 3.00 Or, p, +a
By centrifugation for 15 minutes, the upper layer of the supernatant and the cell aggregates as precipitates were removed, and the middle layer containing the monoclonal antibody was recovered.
に記により回収された溶液１０＠Ｑに硫酸アンモニウム
を最終濃度５０％となるように加えて、塩析した。これ
を１０，０００ｒ、ｐ、’ｌｆ１．．３０分間の遠心に
かけ、沈澱した分画をＰＢＳ（ｐ１１７．２の０．０１
Ｍ燐Ｍｉ１衝液に０．１５Ｍ ＮａＣＱを加えた燐酸緩
衝塩溶液） １０ｍＭに溶かした。この溶液をＰＢＳ
１，０００ｍ１２に対して３回透析を繰り返し、免疫グ
ロブリン分画とした。Ammonium sulfate was added to the solution 10@Q recovered as described above to give a final concentration of 50% for salting out. This is 10,000r, p, 'lf1. ．． Centrifugation was performed for 30 minutes, and the precipitated fraction was dissolved in PBS (0.01 of p117.2).
(Phosphate buffered salt solution prepared by adding 0.15M NaCQ to M phosphorus Mi1 solution) Dissolved at 10mM. This solution was added to PBS.
Dialysis was repeated three times against 1,000 m12 to obtain an immunoglobulin fraction.
次いで該免疫グロブリン分画を、０，０３Ｍ ＮａＣＱ
を含むｐＨ８，０の燐酸Ｈ１１Ｂ液に対して十分に透析
した。The immunoglobulin fraction was then treated with 0.03M NaCQ.
The sample was thoroughly dialyzed against a phosphoric acid H11B solution containing pH 8.0.
別にこれと同じ緩衝液で平衡化しておいたイオン交換樹
脂ＤＥ５２カラム（ワットマン社製）に該透析液をかけ
、ＤＥ５２カラムに吸着されなかった両分を回収した。Separately, the dialysate was applied to an ion exchange resin DE52 column (manufactured by Whatman) that had been equilibrated with the same buffer, and both fractions that were not adsorbed to the DE52 column were collected.
この回収された両分をＰＢＳに対して透析を行なったも
のを精製ＩｇＧ抗体とし、モノクローナル抗体をＡ１．
４１０を得た。Both recovered fractions were dialyzed against PBS to obtain purified IgG antibodies, and monoclonal antibodies were obtained from A1.
I got 410.
実遣−夕Ｌ５（モノ先且二ｊ）吃疲体岐、４１０の杵天
４ｙモノクローナル抗体Ａ１．４１０が認識しているガ
ングリオシドを決定するために、薄層クロマトグラフィ
ー（以下ＴＬＣと称す）上での酵素免疫染色法を行なっ
た。In order to determine the ganglioside recognized by the Kiten 4y monoclonal antibody A1.410 of 410, it was analyzed by thin layer chromatography (hereinafter referred to as TLC). Enzyme immunostaining was performed.
（１）ヒトメラノーマ株Ｍ１４のガングリオシド分画と
糖脂質純品の調製
Ｍ１４のガングリオシド分画の調製はＴａｉ、 Ｔ、
ｅｔａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、、 Ａｃａｄ、
Ｓｃｉ、 ＵＳＡ ８０．５３９２−５３９６（１９８
３）に従った。(1) Preparation of ganglioside fraction and pure glycolipid of human melanoma strain M14 Ganglioside fraction of M14 was prepared by Tai, T.
etal, Proc, Natl, Acad,
Sci, USA 80.5392-5396 (198
3) was followed.
ガングリオシドＧＭＩ、 ＧＤｌａ、ＧＤＩｂ、 ＧＴ
ｌｂはウシの脳から調製し、Ｋａｎｆｅｒ、 Ｊ、 Ｎ
、 Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ。Gangliosides GMI, GDla, GDIb, GT
lb was prepared from bovine brain and Kanfer, J, N.
, Methods Enzymol.
旦、 ６６０−６６４（１９６９）に従って精製した。660-664 (1969).
ＧＯＩｂはヤトロン社から購入した。ヒト脳のＧＭ３は
スウェーデンのＧｏｔｅｂｏｒｇ大学の１４． Ｓｖｅ
ｎｎｅｒｈｏ１ｍ博士から入手した。０Ｍ２とＧＤ２は
それぞれ０Ｍ２とＧＤｌｂから、ウシ來丸β−ガラクト
シダーゼ（アメリカのミシガン大学のＪ、 Ｗ、 Ｊｏ
ｕｒｄｉａｎ博士から入手）を用いて調製した。ヒトミ
ニリンの０Ｍ４とヒト脳のＧＴｌａは東京都臨床医学総
合研究所の有賀博士から入手した。クマ赤血球のＧＤ３
（ＮｅｕＡｃ、 ＮｅｕＡｅ）、ＧＤ３（ＮｅｕＡｃ。GOIb was purchased from Yatron. Human brain GM3 was developed by Goteborg University, Sweden, at 14. Sve
Obtained from Dr. nnerho1m. 0M2 and GD2 were obtained from 0M2 and GDlb, respectively, using Ushiraimaru β-galactosidase (J, W, Jo, University of Michigan, USA).
(obtained from Dr. Urdian). Human minirin 0M4 and human brain GTla were obtained from Dr. Ariga of the Tokyo Metropolitan Institute of Clinical Medicine. GD3 of bear red blood cells
(NeuAc, NeuAe), GD3 (NeuAc.
ＮｅｕＧｃ）、ＧＤ３（ＮｅｕＧｃ、 ＮｅｕＡｃ）、
およびＧＤ３ （ＮｅｕＧｃ ＋ＮｅｕＧｃ）は東京都
臨床医学総合研究所の橋本博士と鈴木明身博士から入手
した。ＬａｃＣｅｒ、 ＧｇＯｓ、Ｃｅｒ。NeuGc), GD3 (NeuGc, NeuAc),
and GD3 (NeuGc +NeuGc) were obtained from Dr. Hashimoto and Dr. Akimi Suzuki of the Tokyo Metropolitan Institute of Clinical Medicine. LacCer, GgOs, Cer.
およびＧｇ０ｓ４ＣｅｒはそれぞれＧＭ３．０Ｍ２．
ＧＭＩから弱酸で処理し、イアトロビーズカラムクロマ
トグラフィーで精製して調製した＊ Ｇ］、ｃＣｅｒは
Ｔａｉ、 Ｔ。and Gg0s4Cer are GM3.0M2.
*G], cCer is Tai, T.
ａｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、 Ａｃａｄ、
Ｓｃｉ、 ＩＪｓＡ ８０．５３９２−５３９６（１９
８３）に従って調製した。 ＧｂＯｓ、ＣｅｒとＧｂＯ
ｓ、Ｃｅｒは５ｕｐｅｌｃｏ社（アメリカ、ペンシルバ
ニア州、ベラフオンテ）から購入した。at al, Proc, Natl, Acad,
Sci, IJsA 80.5392-5396 (19
83). GbOs, Cer and GbO
S and Cer were purchased from 5upelco (Bellajuonte, Pennsylvania, USA).
（２） ＴＬＣの方法
メルク社製のシリカゲルを塗布済みのＴＬＣプレート「
シリカゲル６０」（厚さ２００μｍ）と同じくメルク社
製のシリカゲルを塗布済みのＴＬＣプラスチックシート
を使用した。クロマトグラムの展開溶媒はクロロフォル
ム／メタノール１０．２２％ＣａＣ１，水溶液を容積比
５５／４５／１０としたものを用いた。 ＴＬＣプレ
ート上に展開されたガングリオシドの染色にはレゾルシ
ノール（ｒｅｓｏｒｃｉｎｏｌ ）染色を、中性糖脂質
の染色にはオルシノール（ｏｒｃｉｎｏｌ）染色を用い
た。(2) TLC method TLC plate coated with Merck's silica gel
A TLC plastic sheet coated with silica gel manufactured by Merck and Co., Ltd., as well as "Silica Gel 60" (thickness 200 μm), was used. The developing solvent for the chromatogram was chloroform/methanol 10.22% CaCl, an aqueous solution with a volume ratio of 55/45/10. Resorcinol staining was used to stain gangliosides developed on the TLC plate, and orcinol staining was used to stain neutral glycolipids.
（３） ＴＬＣプレート上での酵素免疫染色法ガングリ
オシドのＴＬＣ終了後、ＴＬＣプレートをウシ血清アル
ブミン１％とポリビニールピロリドン（ｐｏｌｙｖｉｎ
ｙｌｐｙｒｏｌｉｄｏｎｅ） １％とを含有する燐酸緩
衝塩溶液に浸した。空気中で乾燥させた後、プレートを
モノクローナル抗体溶液（１０Ｍｇ／ｍＱ）と２時間２
５℃で反応させた。次に燐酸緩衝塩溶液（以下ＰＢＳと
称す）を５回交換しつつその中でクロマトグラムを洗浄
した。そして西洋ワサビペルオキシダーゼを結合させた
ヤギ抗マウスＩｇＧおよびＩｇＭ抗体をこのクロマ１−
グラムと２５℃で２時間反応させた。(3) Enzyme immunostaining method on TLC plate After TLC of gangliosides, the TLC plate was treated with 1% bovine serum albumin and polyvinyl pyrrolidone (polyvinyl pyrrolidone).
ylpyrolidone) in a phosphate buffered saline solution containing 1%. After drying in air, the plate was soaked with monoclonal antibody solution (10 Mg/mQ) for 2 hours.
The reaction was carried out at 5°C. Next, the chromatogram was washed in phosphate buffered saline solution (hereinafter referred to as PBS) with five changes. Then, horseradish peroxidase-conjugated goat anti-mouse IgG and IgM antibodies were added to this chroma1-
gram at 25° C. for 2 hours.
再びＰＢＳを５回交換しつつその中でクロマトグラムを
洗浄した。染色に当たっては４００μｇ／ａＱ（１）０
−フェニレンジアミンと０．１２％の過酸化水素を含む
クエン酸−燐酸緩衝液（ｐＨ５，０，８０ｍＭ）を用い
、１５分間反応させた。その役、水に浸して反応を停止
させた。The chromatogram was washed again in 5 changes of PBS. 400μg/aQ(1)0 for staining
A reaction was carried out for 15 minutes using a citric acid-phosphate buffer (pH 5, 0, 80 mM) containing -phenylenediamine and 0.12% hydrogen peroxide. For that purpose, the reaction was stopped by soaking it in water.
（４） Ｍ１４のガングリオシドのモノクローナル抗体
とを反応させたＴＬＣプレート上での酵素免疫染色法の
結果
第１図は左のレーンから、ガングリオシド純品の混合物
（ＧＭ３４ＧＭ２＋Ｇ旧＋ＧＤ３＋ＧＤ２）をＴＬＣに
かけてレゾルシノールで染色したもの、ヒトメラノーマ
細胞株Ｍ１４のガングリオシド分画をＴＣＬにかけてレ
ゾルシノールで染色したもの、Ｍ１４のガングリオシド
分画をＴＣＬにかけた後Ａ１．４１０で酵素免疫染色を
行なったものである。（レーン番号５）この図のレーン
番号５で示されるように、モノクロナール抗体Ａ１．４
１０はＧＤ２にのみ反応している。(4) Results of enzyme immunostaining on a TLC plate reacted with M14 ganglioside monoclonal antibody Figure 1 shows, starting from the left lane, a mixture of pure gangliosides (GM34GM2+Gold+GD3+GD2) was subjected to TLC and stained with resorcinol. The ganglioside fraction of human melanoma cell line M14 was subjected to TCL and stained with resorcinol. The ganglioside fraction of M14 was subjected to TCL and then subjected to enzyme immunostaining using A1.410. (Lane number 5) As shown in lane number 5 of this figure, monoclonal antibody A1.4
10 reacts only to GD2.
その他のガングリオシド純品および中性糖脂質純品につ
いても各々ＴＣＬにかけた後モノクローナル抗体Ａ１．
４１０で酵素免疫染色を行なった。これらの結果につい
ては図には示さず、第１図の内容と合わせて、Ａ１．４
１０との反応性を表２に示す。Other pure ganglioside products and pure neutral glycolipids were also subjected to TCL, followed by monoclonal antibody A1.
Enzyme immunostaining was performed with 410. These results are not shown in the figure, but together with the contents of Figure 1, A1.4
Table 2 shows the reactivity with 10.
表 ２
ガングリオシド反応性 ガングリオシド反応性ＧＤ３
０Ｍ３ −ＧＤ２
＋＋＋ ０Ｍ２ −ＧＤｌａ
ＧＭＩ −ＧＤｌｂ
ＧｌｃＣｅｒ −ＧＴ １３
ＬａｃＣａｒ −ＧＴｌｂ
ＧｇＯｓ：１Ｃｅｒ −ＧＱｌｂ
Ｇｇ０ｓ４Ｃｅｒ −ＧＴ３
ＧｂＯｓ、Ｃｅｒ −ＧＴ２
Ｇｇ０ｓ４Ｃａｒ −ＧＭ４
−
表２から分かるように本発明のモノクローナル抗体ＡＩ
、４１０はモノ−シアロシル−ガングリオシド（ＧＭ４
．０Ｍ３．０Ｍ２およびＧＭＩ）、および中性糖脂質（
Ｇ１．ｃＣｅｒ、 ＬａｃＣｅｒ、 ０ｇ０ｓ３Ｃａ
ｒ％ Ｇｇ０ｓ４Ｃａｒ、 ＧｂＯｓ、Ｃｅｒおよび
ＧｂＯｓ、Ｃｅｒ）とは反応しない。Table 2 Ganglioside reactivity Ganglioside reactivity GD3
0M3-GD2
+++ 0M2 -GDla
GMI-GDlb
GlcCer-GT13
LacCar-GTlb
GgOs:1Cer-GQlb
Gg0s4Cer-GT3
GbOs, Cer-GT2
Gg0s4Car-GM4
- Monoclonal antibody AI of the invention as seen from Table 2
, 410 is mono-sialosyl-ganglioside (GM4
．． 0M3.0M2 and GMI), and neutral glycolipids (
G1. cCer, LacCer, 0g0s3Ca
r% Gg0s4Car, GbOs, Cer and GbOs, Cer).
次に４種類のＧＤ３（ＧＤ３（ＮａｕＡｃ、 ＮｅｕＡ
ｃ）、ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＧｃ）、ＧＤ３（
ＮｅｕＧｃ、 ＮｅｕＡｃ）、およびＧＤ３（ＮｅｕＧ
ｃ、ＮｅｕＧｃ））とモノクローナル抗体Ａ１．４１０
のＴＬＣプレート上で酵素免疫染色法の結果は第２図の
（Ｃ）に示されている。Ａ１．４１０はこれらの何れと
も反応しなかった。Next, four types of GD3 (GD3 (NauAc, NeuA
c), GD3 (NeuAc, NeuGc), GD3 (
NeuGc, NeuAc), and GD3 (NeuG
c, NeuGc)) and monoclonal antibody A1.410
The results of enzyme immunostaining on TLC plates are shown in FIG. 2(C). A1.410 did not react with any of these.
以上のように、少なくとも反応性試験を行なったガング
リオシドの範囲内ではモノクローナル抗体Ａ１．４ＬＯ
は細胞表面ガングリオシドに対してはＧＤ２とのみ反応
した。As mentioned above, at least within the range of gangliosides for which reactivity tests were conducted, monoclonal antibody A1.4LO
reacted only with GD2 for cell surface gangliosides.
スー施朋６ モノクローナル Ａ１．４２５の ｙ
Ｂａｌｂ／ｃ系のヌードマウスの腹腔にブリステン０．
５ｍΩ投与し、７日後に実施例１により得られたハイブ
リドーマＡ１．４２５（Ｆ！ＥＲＭ Ｐ−９９９４）約
５〜１０ｘｉｏ’細胞を腹腟内接種して腹水化を行なっ
た。Sue Shiho 6 Monoclonal A1.425 y
Blisten 0.0.
After 7 days, about 5 to 10 xio' cells of hybridoma A1.425 (F!ERM P-9994) obtained in Example 1 were inoculated intraperitoneally to induce ascites.
１〜２週間後に腹水を採取し、３ 、０ＯＯｒ、３．ｍ
、、１５分間の遠心により上清上層部分のブリステンと
沈澱物である細胞塊を取り除いたモノクローナル抗体が
含まれる中間層の部分を回収した。After 1 to 2 weeks, ascites was collected and treated with 3,0OOr,3. m
By centrifugation for 15 minutes, the upper layer of the supernatant and the cell aggregates as precipitates were removed, and the middle layer containing the monoclonal antibody was recovered.
上記により回収された溶液１０ｍ１２に硫酸アンモニウ
ムを最終濃度５０％となるように加えて、塩析した。こ
れを１０，０ＯＯｒ、ｐ、＋ａ、、３０分間の遠心にか
け、沈澱した分画をＰＢＳ（ｐＨ７，２の０．０１Ｍ燐
酸緩衝液に０．１５Ｍ ＮａＣ４を加えた燐酸緩衝塩溶
液） １０＋＋＋Ｑに溶かした。この溶液をＰＢＳ １
，０００ｍＭに対して３回透析を繰り返し、免疫グロブ
リン分画とした。Ammonium sulfate was added to 10 ml of the solution recovered above to give a final concentration of 50% for salting out. This was centrifuged for 30 minutes at 10,0OOr, p, +a, and the precipitated fraction was dissolved in PBS (a phosphate buffered salt solution prepared by adding 0.15M NaC4 to 0.01M phosphate buffer, pH 7.2) 10+++Q. Ta. This solution was dissolved in PBS 1
,000mM was repeated three times to obtain an immunoglobulin fraction.
次いで該免疫グロブリン分画を、０，０３Ｍ ＮａＣＱ
を含むＰＩ＋８．０の燐酸緩衝液に対して十分に透析し
た。The immunoglobulin fraction was then treated with 0.03M NaCQ.
The sample was thoroughly dialyzed against a phosphate buffer containing PI+8.0.
別にこれと同じ緩衝液で平衡化しておいたイオン交換樹
脂ＤＨ５２カラム（ワットマン社製）に該透析液をかけ
、 ＤＥ５２カラムに吸着されなかった両分を回収した
。この回収された両分をＰＢＳに対して透析を行なった
ものを精製ＩｇＧ抗体とし、モノクローナル抗体Ａ１．
４２５を得た。Separately, the dialysate was applied to an ion exchange resin DH52 column (manufactured by Whatman) that had been equilibrated with the same buffer, and both fractions that were not adsorbed to the DE52 column were collected. Both recovered fractions were dialyzed against PBS to obtain purified IgG antibodies, and monoclonal antibodies A1.
I got 425.
失週−例７（モノクローナル抗体Ａ１．４２５（１）＃
踵（’ｌ）−モノクローナル抗体４１．４２５が認識し
ているガングリオシドを決定するために、薄層クロマト
グラフィー（以下ＴＬＣと称す）上での酵素免疫染色法
を行なった。Week loss - Case 7 (monoclonal antibody A1.425 (1) #
In order to determine the ganglioside recognized by the heel ('l) monoclonal antibody 41.425, enzyme immunostaining on thin layer chromatography (hereinafter referred to as TLC) was performed.
（１）ヒトメラノーマ株Ｍ１４のガングリオシド分画と
糖脂質純品の調製
Ｍ１４のガングリオシド分画の調製はＴａｉ、 Ｔ、
ｅｔ’ａ１．、Ｐｒｏｃ、 Ｎａむ１． Ａｃａｄ
、 Ｓｃｉ、 ＬＩＳＡ ８０． ５３９２−５
３９６（１９８３）に従った。(1) Preparation of ganglioside fraction and pure glycolipid of human melanoma strain M14 Ganglioside fraction of M14 was prepared by Tai, T.
et'a1. , Proc, Nam1. Acad
, Sci, LISA 80. 5392-5
396 (1983).
ガングリオシドＧＭ１、ＧＤｌａ、 ＧＤｌｂ、 ＧＴ
ｌｂはウシの脳から調製し、Ｋａｎｆｅｒ、 Ｊ、 Ｎ
、 Ｍｅｔｈｏｄｓ Ｅｎｚｙｍｏｌ。Ganglioside GM1, GDla, GDlb, GT
lb was prepared from bovine brain and Kanfer, J, N.
, Methods Enzymol.
旦、 ６６０−６６４（１，９６９）に従って精製した
。 ＧＱｌｂはヤトロン社から購入した。ヒト脳のＧＭ
３はスウェーデンのＧｏｔａｂｏｒｇ大学のり、 Ｓｖ
ｅｎｎｅｒｈｏ１ｍ博士から入手した。６阿２とＧＤ２
はそれぞれ０Ｍ２とＧＤｌｂから、ウシ來丸β−ガラク
トシダーゼ（アメリカのミシガン大学のＪ、 ＩＩ、
Ｊｏｕｒｄｉａｎ博士から入手）を用いて調製した。ヒ
トミニリンの０Ｍ４とヒト脳のＧＴｌａは東京都臨床医
学総合研究所の有賀博士から入手した。クマ赤血球のＧ
Ｄ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）、 ＧＤ３（Ｎｅｕ
Ａｃ。660-664 (1,969). GQlb was purchased from Yatron. GM of the human brain
3 is from Gotaborg University in Sweden, Sv
Obtained from Dr. Ennerho1m. 6A2 and GD2
were obtained from 0M2 and GDlb, respectively, using Ushiraimaru β-galactosidase (J, II, University of Michigan, USA).
(obtained from Dr. Jourdian). Human minirin 0M4 and human brain GTla were obtained from Dr. Ariga of the Tokyo Metropolitan Institute of Clinical Medicine. bear red blood cell G
D3 (NeuAc, NeuAc), GD3 (Neu
Ac.
ＮｅｕＧｃ）、ＧＤ３（ＮａｕＧｃ、 ＮａｕＡｃ）、
およびＧＤ３ （ＮｅｕＧｃ 。NeuGc), GD3 (NauGc, NauAc),
and GD3 (NeuGc.
ＮｅｕＧｃ）は東京都臨床医学総合研究所の橋本博士と
鈴木明身博士から入手したｅ ＬａｃＣｅｒ＋ ＧｇＯ
ｓ、Ｃｅｒ。NeuGc) was obtained from Dr. Hashimoto and Dr. Akimi Suzuki of the Tokyo Metropolitan Institute of Clinical Medicine.
s, Cer.
およびＧｇ０８４ＣｅｒはそれぞれＧＭ３．０Ｍ２．
ＧＭＩから弱酸で処理し、イアトロビーズカラムクロマ
トグラフィーで精製して調製した。ＧｌｃＣｅｒはＴａ
ｉ、 Ｔ。and Gg084Cer are GM3.0M2.
It was prepared from GMI by treatment with a weak acid and purification by Iatrobead column chromatography. GlcCer is Ta
i, T.
ｅｔ ａｌ、 Ｐｒｏｃ、 Ｎａｔｌ、Ａｃａｄ、 Ｓ
ｃｉ、 ＵＳＡ ８０．５３９２−５３９６（１９１３
３）に従って調製した。ＧｂＯｓ３ＣｅｒとＧｂＯｓ、
Ｃｅｒは５ｕｐｅｌｃｏ社（アメリカ、ペンシルバニア
州、ベラフォンテ）から購入した。et al, Proc, Natl, Acad, S
ci, USA 80.5392-5396 (1913
3). GbOs3Cer and GbOs,
Cer was purchased from 5upelco (Belafonte, Pennsylvania, USA).
（２） ＴＬＣの方法
メルク社製のシリカゲルを塗布済みのＴ１．Ｃプレート
「シリカゲル６０」（厚さ２００μｍ）と同じくメルク
社製のシリカゲルを塗布済みのＴＬＣプラスチックシー
トを使用した。クロマトグラムの展開溶媒はクロロフォ
ルム／メタノール１０．２２％ＣａＣ１□水溶液を容積
比５５／４５／１０としたものを用いた。ＴＬＣプレー
ト上に展開されたガングリオシドの染色にはレゾルシノ
ール（ｒｅｓｏｒｃｉｎｏｌ ）染色を、中性糖脂質の
染色にはオルシノール（ｏｒｃｉｎｏｌ）染色を用いた
。(2) TLC method T1. which has been coated with Merck's silica gel. A TLC plastic sheet coated with silica gel manufactured by Merck Co., Ltd., like the C plate "Silica Gel 60" (thickness 200 μm), was used. The developing solvent for the chromatogram was a chloroform/methanol 10.22% CaCl□ aqueous solution with a volume ratio of 55/45/10. Resorcinol staining was used to stain gangliosides developed on the TLC plate, and orcinol staining was used to stain neutral glycolipids.
（３） ＴＬＣプレート上での酵素免疫染色法ガングリ
オシドのＴＬＣ終了後、ＴＬＣプレートをウシ血清アル
ブミン１％とポリビニールピロリドン（ｐｏｌｙｖｉｎ
ｙｌｐｙｒｏｌｉｄｏｎｅ） １％とを含有する燐酸緩
衝塩溶液に浸した。空気中で乾燥させた後、プレートを
モノクローナル抗体溶液（１０μｇ／ｍＱ）と２時間２
５℃で反応させた０次に燐酸緩衝塩溶液（以下ＰＢＳと
称す）を５回交換しつつその中でクロマトグラムを洗浄
した。そして西洋ワサビベルオキシダ゛−ゼを結合させ
たヤギ抗マウスＩｇＧおよび丁ｇＭ抗体をこのクロマト
グラムと２５℃で２時間反応させた５再びＰＢＳを５回
交換しつつその中でクロマトグラムを洗浄した。染色に
当たっては４００μｇ／ｍＱのＯ−フェニレンジアミン
と０．１２％の過酸化水素を含むクエン酸−燐酸緩衝液
（ｐ１１５．ｏ、８０ｎ＋Ｍ）を用い、１５分間反応さ
せた。その後、水に浸して反応を停止させた。(3) Enzyme immunostaining method on TLC plate After TLC of gangliosides, the TLC plate was treated with 1% bovine serum albumin and polyvinyl pyrrolidone (polyvinyl pyrrolidone).
ylpyrolidone) in a phosphate buffered saline solution containing 1%. After drying in air, the plate was incubated with monoclonal antibody solution (10 μg/mQ) for 2 hours.
The chromatogram was washed in a zero-order phosphate buffered saline solution (hereinafter referred to as PBS) which had been reacted at 5° C. and was exchanged five times. The chromatogram was then reacted with horseradish peroxidase-conjugated goat anti-mouse IgG and anti-gM antibodies at 25°C for 2 hours.5 The chromatogram was washed again in PBS with 5 changes of PBS. . For staining, a citric acid-phosphate buffer (p115.o, 80n+M) containing 400 μg/mQ O-phenylenediamine and 0.12% hydrogen peroxide was used, and the reaction was carried out for 15 minutes. Thereafter, the reaction was stopped by immersing it in water.
（４） ８１４のガングリオシドのモノクローナル抗体
とを反応させたＴＬＣプレート上での酵素免疫染色法の
結果
第１図は左のレーンから、ガングリオシド純品の混合物
（ＧＭ３÷ａｉｚ÷ＧＭ１＋ＧＤ３＋ＧＤ２）をＴＬＣ
にかけてし。(4) Results of enzyme immunostaining on a TLC plate in which 814 ganglioside monoclonal antibodies were reacted. Figure 1 shows, from the left lane, a mixture of pure gangliosides (GM3 ÷ aiz ÷ GM1 + GD3 + GD2) by TLC.
I put it on.
ゾルシノールで染色したもの、ヒトメラノーマ細胞株Ｍ
１４のガングリオシド分画を丁ＣＬにかけてレゾルシノ
ールで染色したもの、Ｍ１４のガングリオシド分画をＴ
ＣＬにかけた後Ａ１．４２５で酵素免疫染色を行なった
ものである。（レーン番号６）この図のレーン番号６で
示されるように、モノクロナール抗体Ａ１．４２５はＧ
Ｄ２にのみ反応している。Stained with zorcinol, human melanoma cell line M
The ganglioside fraction of M14 was subjected to TCL and stained with resorcinol, and the ganglioside fraction of M14 was stained with TCL.
After being subjected to CL, enzyme immunostaining was performed using A1.425. (Lane number 6) As shown in lane number 6 of this figure, monoclonal antibody A1.425 was
It reacts only to D2.
その他のガングリオシド純品および中性糖脂質純品につ
いても各々ＴＣＬにかけた後モノクローナル抗体Ａ１．
４２５で酵素免疫染色を行なった。これらの結果につい
ては図には示さず、第１図の内容と合わせて、Ａ１．４
２５との反応性を表３に示す。Other pure ganglioside products and pure neutral glycolipids were also subjected to TCL, followed by monoclonal antibody A1.
Enzyme immunostaining was performed with 425. These results are not shown in the figure, but together with the contents of Figure 1, A1.4
Table 3 shows the reactivity with 25.
表 ３
ガングリオシド 反応性 ガングリオシド 反応性Ｇ
Ｄ３ ＧＭ３’ −Ｇ
Ｄ２ ＋＋＋ ６Ｍ２
−ＧＤｌａ −ＧＭＩ −ＧＤｌｂ
ＧｌｃＣａｒ −ＧＴｌ
ａ ＬａｃＣｅｒ −ＧＴ
ｌｂ ＧｇＯｓ、Ｃｅｒ −
ＧＱｌｂ Ｇｇ０ｓ４Ｃｅｒ
−ＧＴ３ ＧｂＯｓ、Ｃｅｒ
−ＧＴ２ Ｇｇ０ｓ４Ｃ
ｅｒ −０Ｍ４ −
表３から分かるように本発明のモノクローナル抗体Ａ１
．４２５はモノ−シアロシル−ガングリオシド（０Ｍ４
、ＧＭ３．６Ｍ２およびＧＭＩ）、および中性糖脂質（
ＧｌｃＣｅｒ、 ＬａｃＣｅｒ、 Ｇｇ０ｓ３Ｃａｒ、
０ｇ０ｓ４Ｃａｒ％ＧｂＯｓ、ＣｅｒおよびＧｇ０ｓ
３Ｃａｒ）とは反応しない。Table 3 Ganglioside Reactivity Ganglioside Reactivity G
D3 GM3'-G
D2 +++ 6M2
-GDla -GMI -GDlb
GlcCar-GTl
a LacCer-GT
lb GgOs, Cer −
GQlb Gg0s4Cer
-GT3 GbOs, Cer
-GT2 Gg0s4C
er -0M4 - As can be seen from Table 3, monoclonal antibody A1 of the present invention
．． 425 is mono-sialosyl-ganglioside (0M4
, GM3.6M2 and GMI), and neutral glycolipids (
GlcCer, LacCer, Gg0s3Car,
0g0s4Car%GbOs, Cer and Gg0s
3Car).
次に４種類のＧＤ３（ＧＤ３（ＮａｕＡｃ、 ＮｅｕＡ
ｃ）、ＧＤ３（ＮｅｕＡｃ、 ＮｅｕＧｃ）、ＧＤ３（
ＮｅｕＧｃ、 ＮｅｕＡｃ）、およびＧＤ３（ＮｅｕＧ
ｃ、 ＮｅｕＧｃ））とモノクローナル抗体ＡＩ、４２
５のＴＬＣプレート上で酵素免疫染色法の結果は第２図
（Ｃ）に示されている。Next, four types of GD3 (GD3 (NauAc, NeuA
c), GD3 (NeuAc, NeuGc), GD3 (
NeuGc, NeuAc), and GD3 (NeuG
c, NeuGc)) and monoclonal antibody AI, 42
The results of enzyme immunostaining on TLC plates of 5 are shown in Figure 2 (C).
第２図（Ａ）は４種類のＧＤ３をＴＬＣにかけ、レゾル
シノールで染色したものである。左からガングリオシド
純品の混合物（ＧＭ３＋ＧＭ ｌ＋ＧＤ １ ）、レー
ン番号１はＧＤ３（ＮｅｕＡｃ、’ ＮｅｕＡｃ）、レ
ーン番号２はＧＤ３（ＮｅｕＡｃ、 ＮａｕＧｃ）、レ
ーン番号３はＧＤ３（ＮｅｕＧｃ、 ＮｅｕＡｃ）、レ
ーン番号４はＧＤ３（ＮｅｕＧｃ、 ＮｅｕＧｃ）であ
る。第２図（Ｃ）はＡ１．４２５によるＴＬＣの酵素免
疫染色の結果である。レーン番号とＧＤ３の種類は第２
図（Ａ）と同じである。この図の（Ｃ）から判るように
、 Ａ１．４２５はこれらの何れとも反応しなかった。FIG. 2(A) shows four types of GD3 subjected to TLC and stained with resorcinol. From the left: a mixture of pure gangliosides (GM3 + GM l + GD 1 ), lane number 1 is GD3 (NeuAc, 'NeuAc), lane number 2 is GD3 (NeuAc, NauGc), lane number 3 is GD3 (NeuGc, NeuAc), lane number 4 is GD3 (NeuGc, NeuGc). FIG. 2(C) shows the results of TLC enzyme immunostaining with A1.425. Lane number and GD3 type are second
This is the same as in Figure (A). As can be seen from (C) of this figure, A1.425 did not react with any of these.
以上のように、少なくとも反応性試験を行なったガング
リオシドの範囲内ではモノクローナル抗体Ａｔ、４２５
は細胞表面ガングリオシドに対してはＧＤ２とのみ反応
した。As mentioned above, at least within the range of gangliosides for which reactivity tests were conducted, monoclonal antibodies At, 425
reacted only with GD2 for cell surface gangliosides.
８ モノクローナルＪｉＡ１．２６７の実験動物として
はＣ５７［ＩＬ／６マウスを対照群は１４匹、試験群は
モノクローナル抗体の投与量により２群に分け、各々５
匹使用した。移植腫瘍にはＣ５７／ＢＬ６と同系（ｓｙ
ｎｇｅｎｅｉｃ）のＴ細胞白血病細胞株ＥＬ４を使用し
、２Ｘ１０’個腹腟内に接種した。翌日燐酸緩衝塩類液
０ 、２ｍＲに溶かした１００μｇ又は５００μｇのモ
ノクローナル抗体Ａ１．２６７を試験群に、対照群には
同量の鱗酸緩衝塩類液のみを腹腔内に投与し、マウスの
生死を６０日１ａｍした。8 The experimental animals for monoclonal JiA1.267 were C57[IL/6 mice, the control group was 14 mice, and the test group was divided into 2 groups depending on the dose of monoclonal antibody, each with 5 mice.
I used two. The transplanted tumor contains C57/BL6 and syngeneic (sy)
The T-cell leukemia cell line EL4 (geneic) was used and inoculated intravaginally into 2×10′ cells. The next day, 100 μg or 500 μg of monoclonal antibody A1.267 dissolved in 0.2 mR of phosphate buffered saline was intraperitoneally administered to the test group, and the same amount of scaly acid buffered saline alone was administered intraperitoneally to the control group. It was 1am on the day.
結果を表４に示す。なお、抗腫瘍効果の判定にはＭＳＤ
（Ｍｅｄｉａｎ ５ｕｒｖｉｖａｌ Ｄａｙ；生存日数
中央値）とＩＬＳ（Ｉｎｃｒｅａｓｅｄ Ｌｉｆｅ ５
ｐａｎ＝ （Ｔ−Ｃ）／Ｃ：延命率）を用いた。The results are shown in Table 4. In addition, for determining the antitumor effect, MSD
(Median 5urvival Day) and ILS (Increased Life 5
pan=(TC)/C: life extension rate) was used.
また第３図には対称群とＡ１．２６７を５００μｇ投与
群について、腫瘍細胞移植後の日数経過に対して生残し
ているマウスの割合をグラフに示す６表 ４
（接種細胞数２Ｘ１０’）
対照群 ０ ２２．０ ０ ０／１４采
丈立１１−
対照群の動物数を９匹、接種細胞数をｌ Ｘ １０’個
、接種方法を皮下移植、モノクローナル抗体の投与量を
５００μｇだけとした他は実施例８と同じ条件で実験を
行なった６
結果を表５に示す。抗腫瘍効果の判定は実施例８と同じ
値を使用した。In addition, Fig. 3 shows a graph of the percentage of surviving mice as a function of the number of days after tumor cell implantation for the control group and the group administered with 500 μg of A1.267. Group 0 22.0 0 0/14 Kajotachi 11- The number of animals in the control group was 9, the number of inoculated cells was 1 x 10', the inoculation method was subcutaneous implantation, and the dose of monoclonal antibody was only 500 μg. An experiment was conducted under the same conditions as in Example 8. The results are shown in Table 5. The same values as in Example 8 were used to evaluate the antitumor effect.
第４図は第３図と同様に対照群とＡ１．２６７を５００
μｇ投与群について、腫瘍細胞移植後の日数経過に対し
て生残しているマウスの割合をグラフに示したものであ
る。Figure 4 shows the control group and A1.267 at 500% as in Figure 3.
This is a graph showing the percentage of surviving mice with respect to the number of days after tumor cell transplantation for the μg administration group.
表 ５
（接種細胞数ｌＸ１０’）
対照群 ０ ２２．５ ０ ０／９投与
群 ５００ ＞６０ ＞１６６．７ ４１５
実施例８および実施例９の結果から判るように、モノク
ローナル抗体Ａ１．２６７の投与によりマウスＴ細胞白
血病の増殖は著しく抑制され、或は移植腫瘍は拒絶され
るに至ったことが示された。Table 5 (Number of inoculated cells l x 10') Control group 0 22.5 0 0/9 administration group 500 >60 >166.7 415
As can be seen from the results of Examples 8 and 9, it was shown that administration of monoclonal antibody A1.267 significantly suppressed the proliferation of murine T-cell leukemia or led to rejection of the transplanted tumor.
実験動物としてはＣ５７ＢＬ／６マウスを対照群は１４
匹、試験群はモノクローナル抗体の投与量により２群に
分け、各々５匹使用した。移植腫瘍にはＣ５７／Ｂｌ、
６と同系（ｓｙｎｇｅｎｅｉｃ）のＴ細胞白血病細胞株
１’：１４を使用し、２ｘｌＯ’個腹腔内に接種した。The experimental animals were C57BL/6 mice, and the control group was 14
The test group was divided into two groups depending on the dose of monoclonal antibody, and five animals were used in each group. C57/Bl for the transplanted tumor;
The T-cell leukemia cell line 1':14, which is syngeneic to 6, was used and 2xlO' cells were inoculated intraperitoneally.
翌日燐酸緩衝塩類液０ 、２ｍ（ｌに溶かした１００μ
ｇ又は５００μｇのモノクローナル抗体Ａｔ、４１０を
試験群に、対照群には同量の燐酸緩衝塩類液のみを腹腔
内に投与し、マウスの生死を６０日間観察した。The next day, 100μ dissolved in 0.2ml (l) of phosphate buffered saline solution was added.
g or 500 μg of the monoclonal antibody At, 410 was intraperitoneally administered to the test group, and the same amount of phosphate buffered saline alone was intraperitoneally administered to the control group, and the survival or death of the mice was observed for 60 days.
結果を表６に示す。なお、抗腫瘍効果の判定にはＭＳＤ
（Ｍｅｄｉａｎ ５ｕｒｖｉｖａｌ Ｄａｙ；生存日数
中央値）とＩＬＳ（Ｉｎｃｒｅａｓｅｄ Ｌｉｆｅ ５
ｐａｎ＝（Ｔ−Ｃ）／Ｃ；延命率）を用いた。The results are shown in Table 6. In addition, for determining the antitumor effect, MSD
(Median 5urvival Day) and ILS (Increased Life 5
pan=(TC)/C; life extension rate) was used.
また第５図には対称群とＡ１．４１０を５００μｇ投与
群について、腫瘍細胞移植後の日数経過に対して生残し
ているマウスの割合をグラフに示す。Furthermore, FIG. 5 is a graph showing the percentage of surviving mice versus the number of days after tumor cell implantation for the control group and the group administered with 500 μg of A1.410.
表 ６
（接種細胞数２Ｘ１０’）
対照群 ０ ２２．０ ０ ０／１４対
照群の動物数を９匹、接種細胞数をｔ ｘ ｔｏ’個、
接種方法を皮下移植、モノクローナル抗体の投与量を５
００μｇだけとした他は実施例１０と同じ条件で実験を
行なった。Table 6 (Number of inoculated cells 2 x 10') Control group 0 22.0 0 0/14 The number of animals in the control group is 9, the number of inoculated cells is t x to',
Inoculation method: subcutaneous implantation, monoclonal antibody dose: 5
The experiment was conducted under the same conditions as in Example 10, except that only 00 μg was used.
結果を表７に示す。抗腫瘍効果の判定は実施例１０と同
じ値を使用した。The results are shown in Table 7. The same values as in Example 10 were used to evaluate the antitumor effect.
第６図は第５図と同様に対照群とＡ１．４１０を５００
μｇ投与群について、腫瘍細胞移植後の日数経過に対し
て生残しているマウスの割合をグラフに示したものであ
る。Figure 6 shows the control group and A1.410, similar to Figure 5.
This is a graph showing the percentage of surviving mice with respect to the number of days after tumor cell transplantation for the μg administration group.
表 ７
（接種細胞数ｌＸｌ０’）
対照群 ０ ２２．５ ０ ０／９投与
群 ５００ ６０．５ ６２．２ １１５大週
−例１２ （モノクロ二丈ｐ皿木へＪ−リリ旦９−疲腫
−済蘇孟ユ）
対照群の動物数を８匹、接種細胞数をｉ ｘ ｔｏ５個
、接種方法を皮下移植とし、モノクローナル抗体の投与
方法を静脈投与（ｉｖ）と腹Ｊ控内投与（ｉｐ）の両方
で行なった。他は実施例１０と同じ条件で実験を行なっ
た。Table 7 (Number of inoculated cells lXl0') Control group 0 22.5 0 0/9 administration group 500 60.5 62.2 115 weeks - Example 12 (Monochrome Nijo p Saraki he J - Lily Dan 9 - Exhaustion - completed (Su Mengyu) The number of animals in the control group was 8, the number of inoculated cells was ix to 5, the inoculation method was subcutaneous implantation, and the monoclonal antibody was administered both intravenously (IV) and intraperitoneally (IP). I did it. The experiment was otherwise conducted under the same conditions as in Example 10.
結果を表８に示す。抗１１！Ｔ！瘍効果の判定は実施例
１０と同じ値を使用した６
表 ８
（接種細胞数ｌＸｌ０’）
２０２６．５１８．８０１５
１ν投与 １００ ３１．５４１．３ ０１５５
００４４．５９９．６２１５
２０２５゜５１４．３０１５
ｉｐ投与 １００ ３７，５ ６８．２ ０１
５５００３９．５７７．１２１５
実施例１０、実施例１１および実施例１２の結果から判
るように、モノクローナル抗体Ａ１．４１０の投与によ
りマウスＴ細胞白血病の増殖は著しく抑制され、或は移
植腫瘍は拒絶されるに至ったことが示された。The results are shown in Table 8. Anti-11! T! The same values as in Example 10 were used to determine the tumor effect6 Table 8 (Number of inoculated cells lXl0') 2026.518.8015 1ν administration 100 31.541.3 0155
0044.599.6215 2025°514.3015 IP administration 100 37,5 68.2 01
550039.577.1215 As can be seen from the results of Example 10, Example 11, and Example 12, administration of monoclonal antibody A1.410 significantly suppresses the proliferation of mouse T cell leukemia or rejects the transplanted tumor. It was shown that this has been achieved.
実験動物としてはＣ５７ＤＬ／６マウスを対照群は１４
匹、試験群はモノクローナル抗体の投与量により２群に
分け、各々５匹使用した。移植腫瘍にはＣ５７／ＢＬ６
と同系（Ｓｙｎｇｅｎ８ｉＣ）のＴ細胞白血病細胞株［
Ｅ＋、４を使用し、２ＸｌＯ”側腹腔内に接種した。翌
［１燐酸緩衝塩類液０．２ｍυに溶かした１００μｇ又
は５００μにのモノクローナル抗体Ａ１．４２５を試験
群に、対照群には同量の燐酸緩？＃塩類液のみを腹腔内
に投与し、マウスの生死を６０日間ＩＩＦ；％した。The experimental animals were C57DL/6 mice, and the control group was 14
The test group was divided into two groups depending on the dose of monoclonal antibody, and five animals were used in each group. C57/BL6 for transplanted tumor
Syngeneic (Syngen8iC) T-cell leukemia cell line [
The test group was inoculated with monoclonal antibody A1.425 at 100 μg or 500 μg dissolved in 0.2 mυ of phosphate buffered saline, and the control group was inoculated with the same amount. Only a mild phosphoric acid saline solution was administered intraperitoneally, and the survival of the mice was determined by IIF% for 60 days.
結果を表９に示す、なお、抗腫瘍効果の判定にはＭＳＤ
（Ｍｅｄｉａｎ ５ｕｒｖｉｖａｌ Ｄａｙ；生存日数
中央値）とＩＬＳ（Ｉｎｃｒｅａｓｅｄ Ｌｉｆｅ Ｓ
ｐａ＋１＝（Ｔ−Ｃ）／Ｃ；延命率）を用いた。The results are shown in Table 9. In addition, MSD was used to determine the antitumor effect.
(Median 5urvival Day) and ILS (Increased Life S
pa+1=(TC)/C; life extension rate) was used.
また第７図には対照群とＡ１．４２５を５００μｇ投与
群について、腫瘍、細胞移植後の日数経過に対して生残
しているマウスの割合をグラフに示す。Furthermore, FIG. 7 shows a graph of the percentage of surviving mice with respect to the number of days after tumor and cell transplantation for the control group and the group administered with 500 μg of A1.425.
表 ９
（接種細胞数２Ｘ１０４）
対照群 ０ ２２．０ ０ ０／１４処
來丈攻フー）
対照群の動物数を９匹、接種細胞数をｌＸｌ０’個。Table 9 (Number of inoculated cells 2 x 104) Control group 0 22.0 0 0/14 The number of animals in the control group was 9, and the number of inoculated cells was 1 x 10'.
接種方法を皮下移植、モノクローナル抗体の投４量を５
００μｇだけとした他は実施例３と同じ条件で実験を行
なった。The inoculation method was subcutaneous implantation, and 4 doses of monoclonal antibody were administered in 5 doses.
The experiment was conducted under the same conditions as in Example 3 except that only 00 μg was used.
結果を表１０に示す。抗腫瘍効果の判定は実施例１３と
同じ値を使用した。The results are shown in Table 10. The same values as in Example 13 were used to evaluate the antitumor effect.
第８図は第７図と同様に対照群とＡ１．４２５を５００
μｇ投与群について、腫瘍細胞移植後の日数経過に対し
て生残しているマウスの割合をグラフに示したものであ
る。Figure 8 shows the control group and A1.425 at 500% as in Figure 7.
This is a graph showing the percentage of surviving mice with respect to the number of days after tumor cell transplantation for the μg administration group.
表 １０
（接種細胞数ＩＸ］、０’）
対照群 ０ ２２．５ ０ ０／９投与
群 に００ ３９．５ ７５．６ １１５実施
例１３、および実施例１／ｌの結果から判るように、モ
ノクローナル抗体Ａ１．４２５の投与によりマウスＴ細
胞白血病の増殖は著しく抑制され、或は移植腫瘍は拒絶
されるに至ったことが示された。Table 10 (Number of inoculated cells IX], 0') Control group 0 22.5 0 0/9 Administration group 00 39.5 75.6 115 As can be seen from the results of Example 13 and Example 1/l, It was shown that administration of monoclonal antibody A1.425 significantly suppressed the proliferation of murine T-cell leukemia or led to rejection of the transplanted tumor.
第１図は左のレーンから、、ガングリオシド純品の混合
物（ＧＭ３＋ＧＭ２＋ＧＭ１＋ＧＤ３＋ＧＤ２）をＴＬ
Ｃにかけてレゾルシノールで染色したもの、ヒトメラノ
ーマ細胞株Ｍ１４のガングリオシド分画をＴＬＣにかけ
てレゾルシノールで染色したもの、Ｍ１４のガングリオ
シド分画をＴＬＣにかけた後Ａ１．２６７、Ａ１．４１
０及びＡ１．／１２５で酵素免疫染色を行なったもので
ある。
第２図（Ａ）は４種類のＧＤ３をＴＩ、Ｃにかけ、レゾ
ルシノールで染色したものである。左からガングリオシ
ド純品の混合物（０Ｍ３＋Ｇ旧＋ＧＤ３）、レーン番号
１はＧＤ３（ＮｅｕＡｃ、 ＮｅｕＡｃ）、レーン番号
２はＧＤ３（ＮｅｕＡｃ、 ＮｅｕＧｃ）、レーン番号
３はＧＤ３（ＮａｕＧｃ。
ＮｅｕＡｃ）、レーン番号４はＧＤ３（ＮｅｕＧｃ、
ＮｅｕＧｃ）である。第２図はＡｔ、２６７、Ａ１．．
４１０及びＡ１．４２５＆、：よル’ｒＬＣの酵素免疫
染色の結果である。レーン番号とＧＤ３の種類は共通で
ある。
第３図、第５図及び第７図には、Ｃ５７／ｎＬ６マウス
を用い、移植腫瘍としてこれと同系（ｓｙｎｇｅｎｅｉ
ｃ）のＴ細胞白血病細胞株ＥＬ４を使用し、２ＸｌＯ’
個腹腟内に接種したものに対して、対照群とＡ１．２６
７、Ａ１．４１０及びＡ１．４２５をそれぞれ５００μ
ｇ投与した群について、腫瘍細胞移植後の日数経過と生
残マウスの数の関係をグラフに示す。
第４図、第６図及び第８図は、接種細胞数を１ＸＩＯ’
個、接種方法を皮下移植とした他は第３図と同じ条件で
実験を行ない、対照群とＡ１，２６７、Ａ１．４１０及
びＡ１．４２５をそれぞれ５００μ＆投与した群につい
て、腫瘍細胞移植後の日数経過と生残マウスの数の関係
をグラフに示したものである。
代理人 弁理士 戸 １）親 男
（Ａ） （Ｂ） ・・Ｓｔｃｌｓ
Ｍ１４ １ ２ ３ ４ ５’ ６経過
日数
経過日数
手続補正書（鏝）
昭和６３年 ８月１８日Figure 1 shows a mixture of pure gangliosides (GM3+GM2+GM1+GD3+GD2) starting from the left lane.
The ganglioside fraction of human melanoma cell line M14 was subjected to TLC and stained with resorcinol, and the ganglioside fraction of M14 was subjected to TLC and stained with resorcinol.A1.267, A1.41
0 and A1. Enzyme immunostaining was performed at /125. FIG. 2(A) shows four types of GD3 subjected to TI and C and stained with resorcinol. From the left: a mixture of pure gangliosides (0M3 + G old + GD3), lane number 1 is GD3 (NeuAc, NeuAc), lane number 2 is GD3 (NeuAc, NeuGc), lane number 3 is GD3 (NauGc. NeuAc), lane number 4 is GD3 (NeuGc,
NeuGc). Figure 2 shows At, 267, A1. ．．
410 and A1.425 &: Results of enzyme immunostaining of Yoru'rLC. The lane number and GD3 type are common. In Figures 3, 5, and 7, C57/nL6 mice are used, and syngeneic (syngenei) tumors are used as transplanted tumors.
c) Using the T cell leukemia cell line EL4, 2XlO'
Control group and A1.26 for those inoculated intravaginally.
7, 500μ each of A1.410 and A1.425
The graph shows the relationship between the number of surviving mice and the number of days after tumor cell transplantation for the group administered with g. Figures 4, 6, and 8 show the number of inoculated cells being 1XIO'.
The experiment was conducted under the same conditions as in Figure 3, except that the inoculation method was subcutaneous implantation. The graph shows the relationship between the progress and the number of surviving mice. Agent Patent Attorney 1) Parent Male (A) (B) ・・Stcls
M14 1 2 3 4 5' 6 Elapsed days Procedure amendment for elapsed days (trowel) August 18, 1988
Claims (1)
Ｌ４でＡ系マウスを免疫することにより得られる抗体産
生細胞とＢａ１ｂ／ｃ系マウス由来の骨髄腫細胞株ＮＳ
１とを融合させることにより得られるハイブリドーマが
産生し、免疫グロブリンクラスが（ＩｇＧ３、ｘ）であ
って、細胞表面ガングリオシドに対する認識の特異性の
強さにおいて、 ＧＤ２＞ＧＤ３（ＮｅｕＡｃ、ＮｅｕＡｃ）、ＧＤ３（
ＮｅｕＧｃ、ＮｅｕＡｃ）、ＧＤ１ｂ、ＧＴ１ａ、ＧＱ
１ｂ＞ＧＴ１ であるモノクローナル抗体Ａ１．２６７を有効成分とす
る抗腫瘍剤。 ２、Ｃ５７ＢＬ／６マウス由来のＴ細胞白血病細胞株Ｅ
Ｌ４でＡ系マウスを免疫することにより得られる抗体産
生細胞とＢａ１ｂ／ｃ系マウス由来の骨髄腫細胞株ＮＳ
１とを融合させることにより得られるハイブリドーマが
産生し、免疫グロブリンクラスが（ＩｇＧ３、ｘ）であ
って、細胞表面ガングリオシドＧＤ２を認識するモノク
ローナル抗体Ａ１．４１０を有効成分とする抗腫瘍剤。 ３、Ｃ５７ＢＬ／６マウス由来のＴ細胞白血病細胞株Ｅ
Ｌ４でＡ系マウスを免疫することにより得られる抗体産
生細胞とＢａ１ｂ／ｃ系マウス由来の骨髄腫細胞株ＮＳ
１とを融合させることにより得られるハイブリドーマが
産生し、免疫グロブリンクラスが（ＩｇＧ３、ｘ）であ
って、細胞表面ガングリオシドＧＤ２を認識するモノク
ローナル抗体Ａ１．４２５を有効成分とする抗腫瘍剤。[Claims] 1. T-cell leukemia cell line E derived from C57BL/6 mice
Antibody-producing cells obtained by immunizing A mice with L4 and myeloma cell line NS derived from Ba1b/c mice
1, the immunoglobulin class is (IgG3, (
NeuGc, NeuAc), GD1b, GT1a, GQ
An antitumor agent containing monoclonal antibody A1.267, in which 1b>GT1, as an active ingredient. 2. T-cell leukemia cell line E derived from C57BL/6 mice
Antibody-producing cells obtained by immunizing A mice with L4 and myeloma cell line NS derived from Ba1b/c mice
1. An antitumor agent produced by a hybridoma obtained by fusion of A1.410 with A1.410, which has an immunoglobulin class of (IgG3, x) and which recognizes cell surface ganglioside GD2, as an active ingredient. 3. T-cell leukemia cell line E derived from C57BL/6 mice
Antibody-producing cells obtained by immunizing A mice with L4 and myeloma cell line NS derived from Ba1b/c mice
1. An antitumor agent produced by a hybridoma obtained by fusion of A1.425 and A1.425, which has an immunoglobulin class of (IgG3, x) and which recognizes cell surface ganglioside GD2, as an active ingredient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63095724A JPH01268646A (en) | 1988-04-20 | 1988-04-20 | Antitumor agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63095724A JPH01268646A (en) | 1988-04-20 | 1988-04-20 | Antitumor agent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01268646A true JPH01268646A (en) | 1989-10-26 |
Family
ID=14145422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63095724A Pending JPH01268646A (en) | 1988-04-20 | 1988-04-20 | Antitumor agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01268646A (en) |
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|---|---|---|---|---|
| WO1997045140A1 (en) * | 1996-05-24 | 1997-12-04 | Glaxo Group Limited | Concentrated antibody preparation |
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-
1988
- 1988-04-20 JP JP63095724A patent/JPH01268646A/en active Pending
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|---|---|---|---|---|
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| US7740851B2 (en) | 2000-01-27 | 2010-06-22 | Medimmune, Llc | Ultra high affinity neutralizing antibodies |
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