JPH0428719B2 - - Google Patents
Info
- Publication number
- JPH0428719B2 JPH0428719B2 JP56172910A JP17291081A JPH0428719B2 JP H0428719 B2 JPH0428719 B2 JP H0428719B2 JP 56172910 A JP56172910 A JP 56172910A JP 17291081 A JP17291081 A JP 17291081A JP H0428719 B2 JPH0428719 B2 JP H0428719B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- avidin
- chain
- complex
- antibody
- 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.)
- Expired - Lifetime
Links
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- 108090000623 proteins and genes Proteins 0.000 claims description 105
- 108090001008 Avidin Proteins 0.000 claims description 65
- 230000014616 translation Effects 0.000 claims description 42
- 230000002401 inhibitory effect Effects 0.000 claims description 40
- 239000012634 fragment Substances 0.000 claims description 21
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 21
- 125000002228 disulfide group Chemical group 0.000 claims description 20
- 238000004132 cross linking Methods 0.000 claims description 17
- 108010053187 Diphtheria Toxin Proteins 0.000 claims description 12
- 102000016607 Diphtheria Toxin Human genes 0.000 claims description 12
- 239000004472 Lysine Substances 0.000 claims description 8
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 7
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- 108010039491 Ricin Proteins 0.000 description 25
- 238000000034 method Methods 0.000 description 24
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 21
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- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 4
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- XTHFKEDIFFGKHM-UHFFFAOYSA-N Dimethoxyethane Chemical compound COCCOC XTHFKEDIFFGKHM-UHFFFAOYSA-N 0.000 description 2
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- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
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- MUKYLHIZBOASDM-UHFFFAOYSA-N 2-[carbamimidoyl(methyl)amino]acetic acid 2,3,4,5,6-pentahydroxyhexanoic acid Chemical compound NC(=N)N(C)CC(O)=O.OCC(O)C(O)C(O)C(O)C(O)=O MUKYLHIZBOASDM-UHFFFAOYSA-N 0.000 description 1
- 125000004105 2-pyridyl group Chemical group N1=C([*])C([H])=C([H])C([H])=C1[H] 0.000 description 1
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- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- HAEPBEMBOAIUPN-UHFFFAOYSA-L sodium tetrathionate Chemical compound O.O.[Na+].[Na+].[O-]S(=O)(=O)SSS([O-])(=O)=O HAEPBEMBOAIUPN-UHFFFAOYSA-L 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000003573 thiols Chemical group 0.000 description 1
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229940047183 tribulus Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Peptides Or Proteins (AREA)
Description
本発明は新規な蛋白複合体とその製造法に関す
る。更に詳しくは、本発明は、蛋白生合成阻害活
性蛋白をアビジンに結合せしめて得られる蛋白複
合体とその製造法に関するものである。
種々の細胞群の混合体の中から成る特定の細胞
群を選択的に破壊することは、種々の生化学的操
作、例えば、細胞レベルでの免疫応答機構の解
明、薬物の作用機作の解明、或いは臨床検査にお
いて必要とされる場合が多い。このような目的の
ために、従来、破壊すべき細胞群を認識する抗体
を補体とともに用いて、補体依存性細胞溶解反応
を起こさせる方法が用いられて来た。しかしなが
らこの方法では、例えば、ヒトIgG2，ヒトIgG4，
ラツトIgG3，マウスIgG3，モルモツトIgG1など
の如く、用いる抗体のクラス、サブクラスによつ
ては補体結合性がなく、細胞溶解反応を起こし得
ないし、又、補体結合性抗体であつても、用いる
補体の動物種によつては補体依存性の細胞溶解反
応をおこしにくい場合がある。更に、補体源とし
て用いる血清中には、補体以外の各種の血清成
分、例えば、アルプミン、免疫グロブリン、リン
ホカイン等が含まれているので、これらの成分が
目的とする細胞の選択的溶解反応を阻げる場合が
ある。
本発明者らは、これらの欠点を有しない、殺す
べき特定の細胞群を選択的に効率よく破壊する強
力な殺細胞法、に用い得る試剤を開発すべく鋭意
研究の結果、本発明に到達した。
即ち、本発明は、アビジンと蛋白生合成阻害活
性蛋白とをジスルフイド結合を含む共有結合によ
つて架橋することにより得られる蛋白複合体であ
る。
本発明において、アビジンとは卵白中に依存す
る分子量約68000の塩基性糖蛋白質であり、ビタ
ミンＨとして知られるビオチンと極めて高い親和
性（親和性定数1015M-1）を有することが知られ
ている。アビジンは、４個のサブユニツトから成
ることが知られているが、本発明のアビジンとは
これらのサブユニツトも含む。
本発明において用いられる蛋白生合成阻害活性
蛋白としては、蛋白生合成阻害活性を有する蛋白
質であればいかなるものでもよいが、特に望まし
いのは、リシンのＡ鎖、ジフテリア毒素のフラグ
メントＡ、ツルレイシより抽出される蛋白生合成
阻害活性蛋白、アメリカヤマゴボウより抽出され
る蛋白生合成阻害活性蛋白である。
リシンとは、植物ヒマ（Ricinus communis）
の種子より公知の方法、例えばオルスンズとピー
ル〔S.Olsnes and A.Pihl，バイオケミストリー
（Biochemistry）12巻、3121〜3126頁，1973年〕
の方法によつて抽出精製することができる蛋白毒
素をいう。リシンは分子量32000のサブユニツト
Ａと分子量34000のサブユニツトＢから成つてお
り、一本のジスルフイド結合によつて結ばれてい
る〔第１図のイ〕。リシンを還元剤で処理すると
第１図のロに示した如く各々少なくとも１個のメ
ルカプト基（−SH）を有するサブユニツトＡ（Ａ
鎖）とサブユニツトＢ（Ｂ鎖）とに分離する。切
断前のリシンは動物に対する非常な毒性を有して
いるが、Ａ鎖はそのままでは弱い毒性を有するの
みであり、またＢ鎖は若干の毒性を有するのみで
ある。リシンは蛋白の生合成を、ペプチド鎖の伸
長に不可欠な成分を不活化することによつて阻害
し、細胞毒性を発揮すると考えられている。Ａ鎖
は無細胞系での蛋白生合成阻害活性を有している
のに対し、Ｂ鎖はそのような活性を有しないが、
Ａ鎖には見られない細胞のリセプターとの結合能
を有している。本発明においてはＡ鎖が用いられ
る。
本発明においてジフテリア毒素とはジフテリア
菌又はその変異株が産生する蛋白毒素をいう。例
えば、ジフテリア菌が産生するジフテリア毒素
は、分子量約62000〜63000の単一のポリペプチド
鎖からなり、これはインタクトトキシンと呼ばれ
ている。インタクトトキシンは第２図のイに模式
的に示した如く、その分子内にジスルフイド結合
（−Ｓ−Ｓ−結合）を２個有している。このイン
タクトトキシンを、例えば、トリプシンのような
蛋白分解酵素で温和な条件で処理すると、アミノ
基末端に近い側のジスルフイド結合の間の特定の
部位に分割がおこり、第２図のロに示すごときニ
ツクドトキシンとなる。このニツクドトキシンを
還元剤で処理すると、第２図のハに示した如く分
子量約24000のフラグメントＡと約38000〜39000
のフラグメントＢに分離する。
ジフテリア毒素に於いてこのフラグメントＢに
相当する部分（Ｂ部分）は毒素が細胞と結合する
ための部分であり、フラグメントＡに相当する部
分（Ａ部分）はＢ部分の細胞との結合を介して細
胞内に入り蛋白合成を阻害し細胞を死に到らしめ
る部分である。かかるジフテリア毒素のフラグメ
ントＡは本発明における蛋白合成阻害活性蛋白と
して用いることができる。ジフテリア変異株、例
えばC7（β30）やC7（β45）が産生する、例えば
CRM30やCRM45のごとき無毒変異蛋白（分子中
にジスルフイド結合を１個有している）や、これ
らを例えばチオール試薬等の還元剤の存在下でト
リプシンで温和に処理して得られるフラグメント
も、本発明におけるフラグメントＡに含まれる。
本発明において、ツルレイシ（Momordica
charantia）より得られる蛋白生合成阻害活性を
有する蛋白（MOM）とは、植物ツルレイシの種
子より公知の方法、例えばバルビエリら（L.
Barbieri et al.，バイオケミカル、ジヤーナル
（Biohem.J.），第186巻、443〜452頁、1980年参
照）の方法によつて抽出精製することができる分
子量約23000の蛋白であり、ウサギ網状赤血球の
ライゼート（lysate）による蛋白合成に対して強
力な阻害活性をもつているが、細胞表面に結合性
を示すリシンのＢ鎖相当部分を元々持たないので
MOM単独では低い細胞毒性しか示さない。
本発明において、アメリカヤマゴボウ（phy−
tolacca americana）より得られる蛋白生合成阻
害活性を有する蛋白（PAP）とは、植物アメリ
カヤマゴボウの葉より公知の方法、例えばアービ
ン（J.D.Irvin，アーカイブス オブ バイオケ
ミストリー アンド バイオフイジクス
（Archives of Biochemistry and Biohysics），
第169巻，522〜528頁，1975年参照）の方法によ
つて抽出精製することができる、分子量約27000
の単一のポリペプチド鎖からなる蛋白であり、
MOMと同様にウサギ網状赤血球のライゼート
（lysate）による蛋白合成に対して強力な阻害活
性をもつているが、細胞表面に結合性を示すリシ
ンＢ鎖相当部分を元々持たないのでPAP単独で
は低い細胞毒性しか示さない。PAPは葉からだ
けでなく、アメリカヤマゴボウの種子、根等から
も抽出精製できる。
本発明に於いて用いられるビオチニル化抗体
は、抗体を公知の方法によりビオチンの活性エス
テル体と反応せしめることにより得ることができ
る。ビオチニル化に用いる抗体は、破壊すべき細
胞またはその膜抗原をサル、ウマ、ウシ、ヤギ、
ヒツジ、ウキギ等の動物に過免疫した血清から、
コーンのエタノール分画法、硫安分画法、イオン
交換クロマトグラフイー法等の公知の手段によつ
て調製され、さらに必要によつては各種細胞を用
いて吸収、吸着操作をほどこして得ることができ
る。また破壊すべき細胞またはその膜抗原で免疫
した動物から得たリンパ球を、例えば骨髄腫と細
胞融合させ、目的とする抗体産生性のクローンを
選別して融合細胞（ハイブリドーマ）を作製し、
これの培養液から、またはこれを動物に接種して
その血清または腹腔液からも抗体を得ることがで
きる。抗体（免疫グロプリン）には、IgG，IgA，
IgM，IgD，IgEの５種類があることが知られて
いるが、そのどれでも本発明に用いることができ
る。
本発明に於いて抗体はそのまゝでも、或いは抗
原と結合し得る部分（図３のFab）を含む限りに
おいては、そのフラグメント（例えば、Fab，
Fab部分と図３中の斜線で示されたいわゆるヒン
ジ部分とからなるFab′，Fab′の２量体Ｆ（ab′）2）
でもビオチニル化して用いることができる。本発
明でいうビオチニル化抗体には、かかる抗体のフ
ラグメントをビオチニル化したものも含まれる。
本発明のアビジンと蛋白生合成阻害活性蛋白よ
り成る蛋白複合体は、両蛋白を共有結合により架
橋することによつて製造される。共有結合によつ
て架橋する方法としては、例えば、シクロヘキシ
ルカルボジイミドや１−エチル−３−（３−ジメ
チルアミノプロピル）カルボジイミド塩酸塩等の
カルボジイミド試薬（縮合剤）を用いて、両蛋白
を直接結合させてもよいし、あるいは、分子中に
複数個の官能基を有する架橋剤、例えば、グルタ
ルアルデヒド、トルエンジイソシアネート、２，
2′−ジカルボキシ−４，4′−アゾフエニレンジイ
ソシアネート，ジエチルマロンイミデート二塩酸
塩、Ｎ−サクシンイミジルｍ−（Ｎ−マレイミド）
ベンゾエートを用いて、両蛋白を結合させてもよ
い。共有結合としては、ジスルフイド結合を含有
する結合が特に望ましく、ジスルフイド結合を形
成する２個の硫黄原子は両蛋白に元々存在してい
た硫黄原子であつても、また外部から架橋剤によ
つて導入された硫黄原子であつてもよい。本発明
のアビジンと蛋白生合成阻害活性蛋白がジスルフ
イド結合により架橋された蛋白複合体を製造する
には、例えば、蛋白複合体を構成するアビジンま
たはそのサブユニツトと蛋白生合成阻害活性蛋白
のどちらか（任意の一方の蛋白をP1で他方の蛋
白をP2で表わす）に活性ジスルフイドを導入し
ておき、他方にチオール基を導入あるいは生成さ
せておき、両者をジスルフイド結合で架橋する方
法をとることができる。すなわち、P1に例えば
下記式（）、
〔Ｙは結合している硫黄原子Ｓと共に活性ジル
フイド基を形成し得る１価の有機基を、Ｒは２価
の有機基をＺは活性エステルのアルコール残基を
表わす。〕
で表わされる架橋剤、あるいは下記式（）、
〔ＹおよびＲの定義は式（）の場合に同じで
ある。Ｑはイミドエステルのアルコール残基を、
Ｘはハロゲン原子を表わす。〕
で表わされる架橋剤を反応せしめ〔反応(1)，(2)〕
るか、
式（Ｖ）、
〔ＲおよびＺの定義は式（）の場合に、Ｘの
定義は式（）の場合に同じである。ｎは１〜３
の整数、以下同じ。〕
で表わされる架橋剤を反応させた後、生成物
（）をチオ亜硫酸イオンで処理〔反応(3)〕する
か、
あるいは、P1を式（）あるいは（）
で表わされる架橋剤（それぞれ、２−イミノチオ
ラクトン、Ｎ−アセチルホモシステイン）と反応
せしめて生成した式（Ｘ）または（XI）で表わ
される蛋白、またはP1を式（）
で表わされる架橋剤（Ｓ−アセチルメルカプトコ
ハク酸無水物）と反応せしめて生成した蛋白を脱
アセチル化して得られる式（Ｘ）で表わされる
蛋白を、チオール基を活性ジスルフイド基に変換
する試薬〔例えば、２，2′−ジピリジルジスルフ
イド
The present invention relates to a novel protein complex and a method for producing the same. More specifically, the present invention relates to a protein complex obtained by binding a protein biosynthesis-inhibiting activity protein to avidin, and a method for producing the same. Selective destruction of a specific cell group from a mixture of various cell groups can be achieved through various biochemical operations, such as elucidation of the immune response mechanism at the cellular level and elucidation of the action mechanism of drugs. , or is often required in clinical tests. For this purpose, a method has conventionally been used in which an antibody that recognizes the cell group to be destroyed is used together with complement to cause a complement-dependent cytolytic reaction. However, in this method, for example, human IgG2, human IgG4,
Some classes and subclasses of antibodies used, such as rat IgG3, mouse IgG3, and guinea pig IgG1, do not have complement-fixing properties and cannot cause cytolytic reactions; Depending on the animal species that require complement, it may be difficult to cause complement-dependent cytolytic reactions. Furthermore, since the serum used as a source of complement contains various serum components other than complement, such as albumin, immunoglobulin, lymphokines, etc., these components can be used to selectively lyse target cells. may be prevented. The present inventors have arrived at the present invention as a result of intensive research to develop a reagent that does not have these drawbacks and can be used in a powerful cell killing method that selectively and efficiently destroys specific cell groups to be killed. did. That is, the present invention is a protein complex obtained by crosslinking avidin and a protein with a protein biosynthesis inhibitory activity through a covalent bond containing a disulfide bond. In the present invention, avidin is a basic glycoprotein with a molecular weight of approximately 68,000 that is dependent on egg white, and is known to have an extremely high affinity (affinity constant: 10 15 M -1 ) with biotin, which is known as vitamin H. ing. Avidin is known to be composed of four subunits, and the avidin of the present invention also includes these subunits. The protein biosynthesis-inhibiting protein used in the present invention may be any protein as long as it has protein biosynthesis-inhibiting activity, but particularly desirable are ricin A chain, diphtheria toxin fragment A, and extracts from vine This is a protein biosynthesis-inhibiting protein extracted from the American pokeweed. Ricin is derived from the plant castor bean (Ricinus communis).
For example, S. Olsnes and A. Pihl [Biochemistry 12, pp. 3121-3126, 1973].
A protein toxin that can be extracted and purified by the following method. Lysine consists of subunit A with a molecular weight of 32,000 and subunit B with a molecular weight of 34,000, which are connected by a single disulfide bond [Fig. 1 A]. When lysine is treated with a reducing agent, subunits A (A
chain) and subunit B (chain B). Ricin before cleavage is highly toxic to animals, but the A chain as it is is only weakly toxic, and the B chain is only slightly toxic. Ricin is thought to inhibit protein biosynthesis by inactivating components essential for peptide chain elongation, thereby exerting cytotoxicity. The A chain has protein biosynthesis inhibitory activity in a cell-free system, whereas the B chain does not have such activity.
It has the ability to bind to cell receptors that is not found in the A chain. In the present invention, the A chain is used. In the present invention, diphtheria toxin refers to a protein toxin produced by B. diphtheriae or a mutant strain thereof. For example, diphtheria toxin produced by the diphtheria bacterium consists of a single polypeptide chain with a molecular weight of about 62,000 to 63,000, and is called an intact toxin. Intact toxin has two disulfide bonds (-S-S- bonds) in its molecule, as schematically shown in Figure 2A. When this intact toxin is treated with a proteolytic enzyme such as trypsin under mild conditions, cleavage occurs at a specific site between the disulfide bonds near the terminal amino group, as shown in Figure 2 (b). It becomes Nikdotoxin. When this nickdotoxin is treated with a reducing agent, fragment A with a molecular weight of about 24,000 and fragment A with a molecular weight of about 38,000 to 39,000
is separated into fragment B. In diphtheria toxin, the part corresponding to fragment B (part B) is the part for the toxin to bind to cells, and the part corresponding to fragment A (part A) is the part that allows the toxin to bind to cells through the binding of part B to cells. This is the part that enters cells and inhibits protein synthesis, leading to cell death. Such fragment A of diphtheria toxin can be used as a protein with protein synthesis inhibiting activity in the present invention. Diphtheria mutant strains, such as C 7 (β30) and C 7 (β45), produce e.g.
This book also includes nontoxic mutant proteins such as CRM30 and CRM45 (which have one disulfide bond in the molecule) and fragments obtained by mildly treating them with trypsin in the presence of a reducing agent such as a thiol reagent. Included in fragment A in the invention. In the present invention, Momordica
A protein with protein biosynthesis inhibitory activity (MOM) obtained from the plant Vittoria charantia can be obtained by known methods such as Barbieri et al. (L.
It is a protein with a molecular weight of approximately 23,000 that can be extracted and purified by the method of Barbieri et al., Biochem. Although it has a strong inhibitory activity against protein synthesis by lysate of red blood cells, it does not originally have a portion corresponding to the lysine B chain that binds to the cell surface.
MOM alone exhibits low cytotoxicity. In the present invention, American pokeweed (phy-
A protein with protein biosynthesis inhibitory activity (PAP) obtained from P. tolacca americana can be obtained using known methods such as JDIrvin, Archives of Biochemistry and Biohysics.
169, pp. 522-528, 1975), with a molecular weight of approximately 27,000.
A protein consisting of a single polypeptide chain of
Like MOM, it has a strong inhibitory activity against protein synthesis by rabbit reticulocyte lysate, but since it does not originally have a portion corresponding to the lysine B chain that binds to the cell surface, PAP alone has a low inhibitory activity on protein synthesis. Shows only toxicity. PAP can be extracted and purified not only from the leaves but also from the seeds and roots of the American pokeweed. The biotinylated antibody used in the present invention can be obtained by reacting the antibody with an active ester of biotin by a known method. Antibodies used for biotinylation target cells to be destroyed or their membrane antigens from monkeys, horses, cows, goats,
From serum from hyperimmune animals such as sheep and sea breams,
It can be prepared by known means such as Cohn's ethanol fractionation method, ammonium sulfate fractionation method, and ion exchange chromatography method, and if necessary, it can also be obtained by absorption and adsorption operations using various cells. can. In addition, lymphocytes obtained from an animal immunized with the cells to be destroyed or their membrane antigens are fused with, for example, myeloma, and clones producing the desired antibody are selected to produce fused cells (hybridoma).
Antibodies can be obtained from the culture solution thereof, or from the serum or peritoneal fluid of an animal inoculated with it. Antibodies (immunoglobulins) include IgG, IgA,
It is known that there are five types, IgM, IgD, and IgE, and any of them can be used in the present invention. In the present invention, the antibody can be used as it is, or as a fragment thereof (for example, Fab,
Fab', Fab' dimer F(ab') 2 ) consisting of the Fab part and the so-called hinge part indicated by diagonal lines in Figure 3
However, it can be biotinylated and used. The biotinylated antibody as used in the present invention includes biotinylated fragments of such antibodies. The protein complex of the present invention comprising avidin and a protein with protein biosynthesis inhibitory activity is produced by covalently crosslinking both proteins. As a method for cross-linking by covalent bonds, for example, a carbodiimide reagent (condensing agent) such as cyclohexylcarbodiimide or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is used to directly bond both proteins. Alternatively, a crosslinking agent having multiple functional groups in the molecule, such as glutaraldehyde, toluene diisocyanate, 2,
2'-dicarboxy-4,4'-azophenylene diisocyanate, diethylmalonimidate dihydrochloride, N-succinimidyl m-(N-maleimide)
Benzoate may be used to link both proteins. As a covalent bond, a bond containing a disulfide bond is particularly desirable, and the two sulfur atoms forming the disulfide bond may be sulfur atoms originally present in both proteins, or they may be introduced from the outside by a cross-linking agent. It may also be a sulfur atom. In order to produce a protein complex in which avidin and a protein biosynthesis inhibitory protein of the present invention are cross-linked by a disulfide bond, for example, either avidin or its subunit constituting the protein complex and a protein biosynthesis inhibitory protein ( It is possible to introduce an active disulfide into one protein (represented by P1 and the other protein by P2), introduce or generate a thiol group into the other protein, and then cross-link the two with a disulfide bond. . In other words, for P1, for example, the following formula (), [Y represents a monovalent organic group capable of forming an active dilphide group together with the bonded sulfur atom S, R represents a divalent organic group, and Z represents an alcohol residue of the active ester. ] A crosslinking agent represented by the following formula (), [The definitions of Y and R are the same as in the case of formula (). Q is the alcohol residue of imidoester,
X represents a halogen atom. [Reaction (1), (2)]
Ruka, Formula (V), [The definitions of R and Z are the same in the case of formula (), and the definition of X is the same as in the case of formula (). n is 1 to 3
integer, the same applies below. ] After reacting the crosslinking agent represented by, the product () is treated with thiosulfite ions [reaction (3)], or Alternatively, P1 can be expressed as () or () A protein represented by formula (X) or (XI) produced by reacting with a crosslinking agent represented by (2-iminothiolactone, N-acetylhomocysteine, respectively), or P1 by formula () [ For example, 2,2'-dipyridyl disulfide
【式】４，4′−ジ
ピリジルジスルフイド
[Formula] 4,4'-dipyridyl disulfide
【式】５，5′−ジチ
オビス（２−ニトロ安息香酸）
〕で処理して〔反応(4)，(5)または(6)〕，活性ジス
ルフイド基
〔Ｙの定義は式（）の場合に同じである。〕
〔Ｙの定義は式（）の場合に同じである。〕
〔Ｙの定義は式（）の場合に同じである。〕
が導入されたP1〔式（），（），（−１），（
−１），（−２）または（−３）で表わされる
蛋白〕を得る。他方、もう一方の蛋白P2より上
記式(1)，(2)または(3)の如くして作つた、下記式
（Ｘ），（XV）または（Ｘ−１）で表わされ
るジスルフイド基が導入された蛋白のジスルフイ
ド基を、例えば２−メルカプトエタノールまたは
ジチオスレイト−ル等のチオール試薬で還元する
〔反応(7)，(8)または(9)〕か、または
反応式(4)または(5)の第一段階の反応または反応
式(6)の第一及び二段階の反応と同様にしてチオー
ル基が導入されたP2〔式（Ｘ），（Ｘ），（Ｘ
−１），（Ｘ−１）または（Ｘ−２）で表わさ
れる蛋白〕を得る。
〔式（），（）または（−１）中のＲと式
（Ｘ）または（Ｘ）中のＲは同一でもまたは
互に異なつていてもよい〕。例えば、上記の如く
して製造したチオール基が導入されたP2を、同
じく上記の如くして製造した活性ジスルフイド基
が導入されたP1と反応させしめて、本発明のア
ビジンと蛋白生合成阻害活性蛋白が活性ジスルフ
イド基で架橋された蛋白複合体を製造することが
できる。なお、ジスルフイド結合によつて架橋さ
れた本発明の蛋白複合体を得るため、一方の蛋白
がシスチン残基の一部としてのジスルフイド結合
をもつているときには、場合によつてスルホ化分
解を行うことによつて、或いは蛋白がチオール基
を持つている場合には、上に記した如きチオール
基を活性ジスルフイド基に変換する試薬で処理す
ることによつて、直接に活性ジスルフイド基をも
つた蛋白に導くことができ、かかる蛋白もまたチ
オール基を発生または導入された他方の蛋白と反
応せしめて本発明の蛋白複合体を製造することが
できる。また、一方の蛋白が元々チオール基をも
つている場合には、そのまゝ、またシスチン残基
の一部としてのジスルフイド結合もつているとき
には、場合によつてジスルフイド結合より還元等
の操作によりチオール基を発生させた後、直接
に、活性ジスルフイド結合をもつた他方の蛋白と
反応せしめて本発明の蛋白複合体を製造すること
ができる。
Ｙで表わされる、結合している硫黄原子と共に
活性ジスルフイド基を形成し得る１価の有機基の
具体例としては、２−ピリジル基
[Formula] 5,5'-dithiobis(2-nitrobenzoic acid) [Reaction (4), (5) or (6)] to form an active disulfide group. [The definition of Y is the same as in the case of formula (). ] [The definition of Y is the same as in the case of formula (). ] [The definition of Y is the same as in the case of formula (). ] was introduced P1 [formula (), (), (-1), (
-1), (-2) or (-3)] is obtained. On the other hand, a disulfide group represented by the following formula (X), (XV) or (X-1) prepared according to the above formula (1), (2) or (3) is introduced from the other protein P2. The disulfide groups of the resulting protein are reduced with a thiol reagent such as 2-mercaptoethanol or dithiothreitol [reactions (7), (8) or (9)], or Thiol group-introduced P2 [formula (X), (X), (X
-1), (X-1) or (X-2)] is obtained. [R in formula (), () or (-1) and R in formula (X) or (X) may be the same or different from each other]. For example, P2 produced as described above into which a thiol group has been introduced is reacted with P1 produced similarly above and introduced with an active disulfide group, and the avidin and protein biosynthesis inhibiting active protein of the present invention are reacted. Protein complexes can be produced in which the conjugates are cross-linked with active disulfide groups. In addition, in order to obtain the protein complex of the present invention cross-linked by disulfide bonds, when one of the proteins has a disulfide bond as part of a cystine residue, sulfonation and decomposition may be performed depending on the case. or, if the protein has a thiol group, by treatment with a reagent that converts the thiol group into an active disulfide group as described above. Such a protein can also be reacted with another protein in which a thiol group has been generated or introduced to produce the protein complex of the present invention. In addition, if one of the proteins originally has a thiol group, if it has a disulfide bond as part of the cystine residue, the thiol group may be removed from the disulfide bond by reduction or other operations in some cases. After generating the group, the protein complex of the present invention can be produced by directly reacting with another protein having an active disulfide bond. Specific examples of the monovalent organic group represented by Y that can form an active disulfide group together with the bonded sulfur atom include 2-pyridyl group.
【式】４−ピリジル基[Formula] 4-pyridyl group
【式】３−カルボキシ−４−ニト ロフエニル基[Formula] 3-carboxy-4-nito Lofenyl group
【式】等を挙げ
ることができる。Ｒで表わされる２価の有機基
は、化学的に不活性であれば特に限定されない
が、一般的には分岐を有するか有しないアルキレ
ン基，フエニレン基等から適宜選ばれる。Ｚで表
わされる活性エステルのアルコール残基の具体例
としては２，４−ジニトロフエノキシ基
[Formula] etc. can be mentioned. The divalent organic group represented by R is not particularly limited as long as it is chemically inert, but is generally appropriately selected from alkylene groups, phenylene groups, etc., which may or may not have branches. A specific example of the alcohol residue of the active ester represented by Z is 2,4-dinitrophenoxy group.
【式】サクシンイミドキシ 基[Formula] Succinimidoxy base
【式】等を挙げることができ
る。Ｑで表わされるイミドエステルのアルコール
残基の具体例としてはメトキシ、エトキシ基等を
挙げることができる。Ｘで表わされるハロゲン原
子の具体例としては塩素、臭素等を挙げることが
できる。
架橋剤の具体例としては、式（）で表わされ
る架橋剤として、Ｎ−サクシンイミジル３−（２
−ピリジルジチオ）プロピオネート，２，４−ジ
ニトロフエニル３−（４−ピリジルジチオ）プロ
ピオネートを、式（）で表わされる架橋剤とし
て、メチル３−（２−ピリジルジチオ）プロピオ
ンイミデート塩酸塩を、式（Ｖ）で表わされる架
橋剤として、Ｎ−サクシンイミジル３−ブロモプ
ロピオネートを挙げることができる。
上記のアビジンと蛋白生合成阻害活性蛋白の架
橋反応において、アビジン或いは蛋白生合成阻害
活性蛋白に架橋剤を反応させる場合は、反応せし
める蛋白１モルに対し、架橋剤を１〜20モル用い
るのが好ましい。反応はアビジン或いは蛋白生合
成阻害活性蛋白の、PH５〜９の緩衝液中蛋白濃度
が0.5〜100mg／ml（より好ましくは１〜20mg／
ml）になるように調製された溶液に、０〜50℃で
攪拌しながら架橋剤の水溶液または架橋剤が水に
溶けない場合には、架橋剤を少量の有機溶媒、例
えば、Ｎ，Ｎ−ジメチルホルムアミド，ジメチル
スルホキシド，１，２−ジメトキシエタン，メタ
ノール，エタノール，アセトン等に溶かした溶液
を添加して行なわれる。反応時間は反応スケー
ル、反応条件によるが、一般に２日間以内であ
る。反応終了後、透析または分子ふるいのカラム
クロマトグラフイーにより、未反応の架橋剤及び
低分子生成物を除いた後、得られた架橋剤が導入
された蛋白溶液に複合体のもう一方の構成々分で
ある蛋白（または架橋剤により架橋用官能基が導
入された蛋白）のPH５〜９の緩衝液の溶液（好ま
しい蛋白濃度の範囲は上に記載したのと同じであ
る）を添加して、０〜50℃で反応せしめる。複合
体の反応混合物からの分離、精製は通常用いられ
る操作、例えば分子ふるいのカラムクロマトグラ
フイーによつて行なうことができる。なお、複合
体の一方の成分の蛋白の溶液に、架橋剤が導入さ
れた他方の成分の蛋白の溶液を添加して複合体を
製造することもできる。さらに、架橋剤が導入さ
れた蛋白を、低分子の試薬で処理して特定の架橋
用官能基を有する蛋白に変換する場合（例えば架
橋剤によつて導入された活性ジスルフイド基をチ
オール基に変換する場合）、或いは、アビジン或
いは蛋白生合成阻害活性蛋白を低分子試薬を用い
直接活性化誘導体に変換する場合（例えば、リシ
ンのフラグメントＡのチオール基を活性ジスルフ
イドに変換する場合）の反応条件も上記の蛋白に
架橋剤を反応せしめる場合の反応条件と同様であ
る。
本発明で得られるアビジンと蛋白生合成阻害活
性蛋白とを共有結合によつて架橋することによつ
て得られる蛋白複合体は、特定の細胞群を認識す
るビオチニル化抗体と、アビジンのビオチンに対
する強い親和性を利用して、結合せしめることに
より、抗体が認識する特定細胞群を選択的に破壊
するために使用することができる。
即ち、本発明者らは、蛋白生合成阻害活性蛋白
を、アビジンとビオチンの強い親和力に基づく非
共有的結合を介して、抗体と結合させることによ
つて、蛋白生合成阻害活性蛋白が抗体が認識する
特定細胞群に対し細胞毒性を発揮するに到るとい
うことを見い出した。かかる特定細胞群破壊シス
テムの優れた点の一つは、既に述べた通り、補体
依存性細胞溶解反応が、抗体のクラス、サブクラ
スによつては補体との結合性がないために、或い
は補体結合性がある場合でも用いる動物種によつ
ては補体依存性細胞溶解反応が起こり難いため
に、適用できない場合でも本発明のシステムは適
用できることである。また、選択的補体依存性細
胞溶解反応が、補体源として用いる血清中の補体
以外の成分で阻げられる場合があるのに対し、本
発明方法ではそのようなことがない。さらに本発
明方法の特徴は、蛋白生合成阻害活性蛋白として
アビジンとの蛋白複合体を作るために好適に用い
られるリシンのＡ鎖、ジフテリア毒素のフラグメ
ントＡ、ツルレイシより得られる蛋白生合成阻害
活性蛋白及びアメリカヤマゴボウより得られる蛋
白生合成阻害活性蛋白が、いずれもそれ自体で
は、細胞表面と結合する部位を欠いているがため
に、極めて弱い細胞毒性しか示さないことと、一
旦抗体をキヤリアーとして、細胞内へ導入される
と極めて強い細胞毒性を発揮する点にある。加え
て、ビオチニル化抗体上には、一般にアビジンと
結合する部位が複数個依存するので、本発明方法
の如く、蛋白生合成阻害蛋白をアビジン−ビオチ
ン結合体を介して抗体に結合させた場合、抗体１
分子当り、複数個の蛋白生合成阻害活性蛋白が付
くことになり、細胞破壊効果の増幅が得られるこ
とも本発明方法の特長として挙げることができ
る。
本発明方法によつて特定細胞群を破壊する望ま
しい実施態様は、破壊したい細胞群を含む細胞集
団を先ず、その細胞群を選択的に認識する抗体の
ビオチニル化体（ビオチニル化抗体）溶液と、０
〜37℃で５〜60分接触反応させ、細胞培養培地も
しくは緩衝化食塩水で洗うことにより過剰の未反
応抗体を除去した後、さらに、アビジンと蛋白生
合成阻害活性蛋白とを共有結合によつて架橋する
ことにより得られる蛋白複合体を加えて、０〜37
℃で１〜30分接触反応させ、細胞培養培地もしく
は緩衝化食塩水で洗つて過剰の蛋白複合体を除去
した後、細胞培養する方法である。細胞培養は通
常の条件、即ち５％CO2加湿雰囲気下37℃で通常
２時間〜５日間行えばよい。なお、ビオチニル化
抗体と、アビジンと蛋白生合成阻害活性蛋白の複
合体は、同時に細胞に加えてもよく、また予めビ
オチニル化抗体と本発明の蛋白複合体とを接触さ
せてアビジン・ビオチン結合体を作らせた後、そ
の結合体で細胞集団を処理してもよい。
以下、実施例により本発明を詳述する。
実施例 １
アビジンとリシンＡ鎖とをジスフイド結合を含
む共有結合によつて架橋することにより得られる
蛋白複合体
(イ) アビジンへの活性ジスルフイド基の導入3.6
mgアビジンを0.1M塩化ナトリウムを含む0.1M
リン酸緩衝液PH7.5（以下Ａ液と略す。）１mlに
溶解し、これに、エタノール中に18.4mMのＮ
−サクシンイミジル３−（２−ピリジルジチオ）
プロピオネート（以下SPDPと略す。）を含む
溶液0.02mlを加え、室温で30分間反応させた。
この反応液を、0.14M塩化ナトリウムと1mM
エチレンジアミン四酢酸ナトリウム（以下
EDTAと略す。）とを含む5mM酢酸緩衝液PH
5.5（以下Ｂ液と略す。）で平衡化したセフアデ
ツクスG25カラムクロマトグラフイーにかけ
て、未反応物と低分子生成分を除き活性ジスル
ヒド基が導入されたアビジンを得た。
(ロ) リシンＡ鎖の調製法
ヒマ（Rieinus communis）のマメよりリシン
を精製し、リシンよりそのＡ鎖を分離する方法は
オルスンズとピール〔S.Olsnes and A.Pihl，バ
イオケミストリー（Biochem.），12巻、3121〜
3126頁，1973年〕の報告に基いて行なつた。得ら
れたＡ鎖の溶液には２−メルカプトエタノールが
含まれているので、使用直前に上記(イ)のセフアデ
ツクスG25カラムクロマトグラフイーにより除去
した。このＡ鎖には、ドデシル硫酸ナトリウム−
ポリアクリルアミドゲル電気泳動（以下SDS−
PAGEと略す）においてＢ鎖もインタクトリシン
も認められなかつた。
(ハ) 活性ジスルフイド基が導入されたアビジンと
リシンＡ鎖との架橋及び蛋白複合体の精製上記
(イ)項で得られた、活性ジスルフイド基が導入さ
れたアビジンを含むＢ液（280nmの吸光度は
3.5であつた。）1.7mlと、リシンＡ鎖（チオー
ル基を有する）を含むＢ液（280nmの吸光度は
2.4であつた。）1.8mlと、10mM EDTAを含む
0.4Mリン酸緩衝液PH7.5（以下Ｃ液と略す。）
0.34mlを混合し、室温で一晩反応させた。
この反応液を生理食塩水で平衡化したセフアデ
ツクスG150スーパーフアイン・カラムクロマト
グラフイー（カラムサイズ1.3cm×95cm）にかけ
た。第４図の如く、分子量９万ないし16万くらい
の流出位置に大きなピークが認められた。この分
画21番から26番まで（図の斜線部）をプールし
た。こうして得られた蛋白溶液をドデシル硫酸ナ
トリウム−ポリアクリルアミドゲル電気泳動にか
けた。第５図のデイスク１はリシンＡ鎖、デイス
ク２はアビジン、デイスク３は架橋、精製された
蛋白複合体、デイスク４はその蛋白複合体を２−
メルカプトエタノールで還元した生成物をかけた
ものである。デイスク３から、分子量約1.5万
（右端）くらいのアビジン・サブユニツトの他に
分子量約4.7万と7.9万（左端）くらいの生成物が
認められる。これらの分子量はアビジンサブユニ
ツト１分子、サブユニツト１分子とリシンＡ鎖１
分子の和及びサブユニツト１分子とリシンＡ鎖２
分子の和にそれぞれ一致する（系中にドデシル硫
酸ナトリウムが存在しているため、アビジンはサ
ブユニツトに分離して検出される。）実際、これ
らの生成物を還元すると、デイスク４の如く、か
なりの量のリシンＡ鎖が生成する。従つて得られ
た蛋白複合体は第６図の如く、４つのアビジン・
サブユニツトのいくつかがリシンＡ鎖１個と結合
した（２個結合したものもわずかに存在する）構
造をとつている。
実施例 ２
アビジンとジフテリア毒素のフラグメントＡと
をジスルフイド結合を含む共有結合によつて架橋
することにより得られる蛋白複合体
(イ) ジフテリア毒素のフラグメントＡの調製ジフ
テリア毒素210mgを含む0.05Mトリス・塩酸−
2mMエチレンジアミン四酢酸水溶液（PH8.3）
18.5mlに、0.1mg／ml濃度のトリプシン溶液を
0.15ml加えて、25℃で50分間分解し、その後
0.5mg／ml濃度の大豆トリプシン阻害物質溶液
0.3mlを加えて反応を停止した。この分解生成
物に尿素（最終濃度6M）、亜硫酸ナトリウム
（最終濃度0.168M）およびテトラチオン酸ナト
リウム（最終濃度0.042M）を加えて、37℃で
２時間Ｓ−スルホ化分解を行なつた。得られた
反応液を、6M尿素−0.03M酢酸緩衝液（PH
5.3）中でセフアデツクスG150カラムクロマト
グラフイー（カラムサイズ3.5cm×112cm）にか
けて、遅れて流出するフラグメントＡ画分のみ
を取り出し、更に蒸留水に透析することによつ
て純粋なフラグメントＡ溶液を得た（Ｓ−スル
ホ基を１個有する）。得られたＳ−スルホ化フ
ラグメントＡ溶液をトリス塩酸緩衝液（PH8.4）
に対して透析後、２−メルカプトエタノール
（最終濃度10mM）で37℃で１時間処理し、実
施例１(イ)項記載のセフアデツクスG25カラムク
ロマトグラフイーに対し、チオール基を１個有
するジフテリア毒素のフラグメントＡを得た。
(ロ) 活性ジスルフイド基が導入されたアビジンと
ジフテリア毒素のフラグメントＡとの架橋及び
蛋白複合体の精製
上記(イ)項の如くして得たジフテリア毒素のフラ
グメントＡと、実施例１の(イ)項の如くして得た活
性ジスルフイド基が導入されたアビジンとを用
い、実施例１の(ハ)項の場合と同様の操作により、
アビジンとジフテリア毒素フラグメントＡとがジ
スルフイド結合を含む共有結合で架橋された蛋白
複合体を得た。
実施例 ３
アビジンとツルレイシより得られる蛋白生合成
阻害活性蛋白とを、ジスルフイド結合を含む共有
結合によつて架橋することにより得られる蛋白複
合体
(イ) ツルレイシの種子より蛋白生合成阻害活性を
有する蛋白（以下MOMという）の抽出・精製
ツルレイシの種子9.2ｇを乳バチですりつぶ
し、エーテルで脂質を除去した。こうして得た
脱脂粉体に、5mMリン酸緩衝液−0.2M塩化ナ
トリウム溶液（PH7.2）50mlを加えて、４℃で
一晩攪拌を続けた。その後、遠心分離によつて
不溶物を除去し、硫安を30％飽和になるように
加えた。０℃で１時間攪拌後、遠心分離によつ
てその上清を取り、この上清にさらに硫安を加
えて70％飽和として、０℃で２時間攪拌後、遠
心分離によつてその沈澱を分離した。沈澱物に
５mlの5mMリン酸緩衝液（PH6.5）を加えて溶
解し、同じリン酸緩衝液に対して十分に透析し
た。その透析内液を、同じリン酸緩衝液で平衡
化したセフアデツクスG150スーパーフアイ
ン・カラムクロマトグラフイー（カラム・サイ
ズ84cm×2.5cm）にかけて、分子量約３万の位
置に流出する蛋白をプールした。これをCMセ
フアデツクスＣ−50カラムクロマトグラフイ
（カラム・サイズ1.6cm×34cm）にかけた。レジ
ンは5mMリン酸緩衝液（PH6.5）に平衡化され
ているが、0.05Mリン酸緩衝液（PH6.5），0.1M
リン酸緩衝液（PH6.5）さらに0.2Mリン酸緩衝
液（PH6.5）という具合に塩濃度を上げていく
と、0.10MのところでMOMが流出してきた。
この分画の蛋白は、分子量約27000であり、他
の蛋白を含まぬことはSDS−PAGEによつて確
認された。
(ロ) チオール基が導入されたMOMの調製上記(イ)
項の如くして抽出精製されたMOMを12.9mg／
mlの濃度で含むＡ液４mlに、20mMのSPDPの
エタノール溶液0.19mlを加えて室温で30分間反
応させた。この反応液に10mM EDTA、
200mM2−メルカプトエタノールを含む
500mMトリス塩酸緩衝液（PH8.5）を0.1ml加え
て、室温で15分間反応させ、Ｂ液で平衡化した
セフアデツクスＧ−25カラムクロマトグラフイ
ーにかけてチオール基が導入されたMOMを得
た。
(ハ) 活性ジスルフイド基が導入されたアビジン
と、チオール基が導入されたMOMとの架橋及
び蛋白複合体の精製
実施例１の(イ)項で得られた活性ジスルフイド
基の導入されたアビジンを含むＢ液（280nmの
吸光度は3.0であつた。）2.0mlと、上記(ロ)項で
得られたチオール基の導入されたMOMを含む
Ｂ液（280nmの吸光度は9.8であつた。）0.6ml
と、Ｃ液0.26mlを混合し、室温で一晩反応させ
た。
この反応液を生理食塩水で平衡化したセフア
デツクスＧ−150スーパーフアインクロマトグ
ラフイー（カラムサイズ1.3×95cm）にかけた。
第７図がそのクロマトグラフイーの図である。
分画28番から33番、34番から39番までをそれぞ
れプールして分画，とした。この，分
画をドデシル硫酸ナトリウム−ポリアクリルア
ミドゲル電気泳動にかけ第８図の如き結果を得
た。第８図のデイスク１はMOM、デイスク２
はアビジン、デイスク３は分画、デイスク４
は分画、デイスク５，６はそれぞれ分画，
を２−メルカプトエタノールで還元した生成
物をかけたものである。
デイスク３より分画は分子量約1.5万（右
端）のアビジンサブユニツトのほかに分子量約
4.5万，６万と7.5万（左端）の生成物が認めら
れ、これらの分子量はそれぞれアビジンサブユ
ニツト１分子とMOM1分子，アビジンサブユ
ニツト２分子とMOM1分子そしてアビジンサ
ブユニツト１分子とMOM2分子の分子量の和
に一致し、またデイスク４よりの分画は分子
量約4.5万と６万の生成物が認められ、これら
はそれぞれ１分画の分子量4.5万，６万の生成
物と同様のものである。そしてこれらの生成物
を還元するとデイスク５，６のごとくMOMと
アビジンサブユニツトだけとなる。従つて得ら
れた複合体は、アビジンの４つのサブユニツト
のいくつかがMOM1個と結合した。（２個結合
したものもわずかに存在する）構造をとつてい
ることがわかる。
実施例 ４
アビジンとアメリカヤマゴボウより得られる蛋
白生合成阻害活性蛋白を、ジスルフイド結合を有
する共有結合によつて架橋することにより得られ
る蛋白複合体
(イ) アメリカヤマゴボウの葉より蛋白生合成阻害
活性を有する蛋白（以下PAPという）の抽出
精製
アメリカヤマゴボウの葉500ｇを、500mlの水
を加えて、ミキサーでホモゲナイズした。この
ホモジエネイトより、アービン（Irvin）の方
法（アーカイブス オブ バイオケミストリー
アンド バイオフイジクス（Archives of
Biochemistry and Biophysics），第169巻，
522〜528頁，1975年）に基づいて、硫安分画、
DEAEセルロー ス・クロマトグラフイーおよ
びホスホセルロース・クロマトグラフイーによ
つてPAPを精製した。このPAPはSDS−
PAGEにおいて、分子量約27000の位置に一つ
のバンドを形成した。
なお、アメリカヤマゴボウの種子、根からも同
様に抽出、精製し、PAPが取れることをSDS−
PAGEで確認した。以下の実験では葉より精製し
たPAPを用いた。
(ロ) チオール基が導入されたPAPの調製
上記の(イ)項の如く抽出精製されたPAP8.1mg
を含む、0.02Mリン酸緩衝液−0.14M塩化ナト
リウム−1mM EDTA水溶液（PH7.5）1.6mlに、
9mMのSPDPを含むエタノール溶液を0.09ml加
え、室温で30分間反応させた。
この反応液に10mM EDTA、200mM2−メ
ルカプトエタノールを含む500mMトリス塩酸
緩衝液（PH8.5）を0.04ml加えて、37℃で30分
間反応させ、反応液を、Ｂ液で平衡化したセフ
アデツクスＧ−25カラムクロマトグラフイーに
かけてチオール基が導入されたPAPを得た。
(ハ) 活性ジスルフイド基が導入されたアビジンと
チオール基が導入されたPAPとの架橋及び蛋
白複合体の精製
実施例１の(イ)項の如くして得られた活性ジス
ルフイド基が導入されたアビジンと、上記(ロ)項
の如くして得られたチオール基が導入された
PAPより、実施例３の(ハ)項と同様にしてアビ
ジンとPAPがジスルフイド結合を含む共有結
合によつて架橋された蛋白複合体を得た。
実施例 ５
膜表面にイムノグロプリンをもつたリンパ球Ｂ
細胞に対する細胞傷害（その１）
(イ) 抗体（IgG）のビオチニル化
0.1M NaHCO3に透析したウサギ抗マウスIgG
抗体溶液（１mg／ml）１mlと、Ｎ−ヒドロキシサ
クシニミドビオチンのＮ，Ｎ−ジメチルホルムア
ミド溶液（１mg／ml）0.1mlを混合し、室温で４
時間反応させた。反応後、10mM−リン酸塩緩衝
化食塩水（PH7.2）に透析して過剰のＮ−ヒドロ
キシサクシニミドビオチンを除き、ビオチニル化
ウサギ抗マウスIgG抗体（ビオチニル化抗体）を
得た。
(ロ) ビオチニル化抗体とアビジン・リシンＡ鎖複
合体を用いる、表面イムノグロブリンを持つた
Ｂ細胞に対する細胞傷害
ジニトロフエニル（DNP）−キーホール・リン
ペツト・ヘモシアニン（KLH）で感作した
BALB／Ｃマウスの脾細胞（DNPに対する表面
イムノグロブリン産生前駆Ｂ細胞）２×106個に、
ビオチニル化ウサギ抗マウスIgG抗体（１mg／
ml）50μlを加えて氷中30分反応させ（マウスのＢ
細胞にビオチニル化抗体が結合する）、冷ハンク
ス溶液で２回洗つた後、更にアビジン・リシンＡ
鎖複合体（可変濃度）50μlを加えて氷中15分反応
させ（ビオチニル化抗体にアビジン・リシンＡ鎖
複合体が結合する）、冷ハンクス溶液で３回洗つ
た。こうして前処置した脾細胞に、DNP−KLH
（抗原刺激のため）（40ng／ml）２−メルカプト
エタノール（50μM）及び10％ウシ胎児血清を含
むRPMI 1640培地600μlを加え、200μlずつ３つ
に分けてそれぞれ５％CO2加湿雰囲気下37℃で５
日間培養した後、Jerneのプラーク法〔ヤーン及
びノルデイン（N.K.Jerne and A.A.Nordin）サ
イエンス（Science），第140巻、405頁、1963年参
照〕によつて、抗DNP抗体産生細胞（PFC）を
数えた。
比較例も含めて結果を第９図に示した。図よ
り、抗原（DNP−KLH）の添加により、PFC
が、著しくビオチニル化体するが（グループ２）、
予め脾細胞をビオチニル化抗体で処理し、更にア
ビジン・リシンＡ鎖複合体で処理すると、PFC
数が有意に減少することが分つた。即ち、抗
DNP抗体産生前駆細胞（Ｂ細胞）が、ビオチニ
ル化抗マウスIgG抗体とこれに結合しうるアビジ
ン・リシンＡ鎖複合体の殺細胞効果によつて除去
されたことを示している。また殺細胞効果を得る
には、ビオチニル化抗体とアビジン・リシンＡ鎖
複合体の両者が共に必要であることを示してい
る。
実施例 ６
膜表面にイムノグロプリンをもつたリンパ球Ｂ
細胞に対する細胞傷害（その２）
DNP−KLH感作脾細胞を実施例５と同様に、
ビオチニル化抗マウスIgG抗体とアビジン・
MOM複合体で前処理した後、抗体産生能を調べ
た。
第１０図に示されるように、ビオチニル化抗体
またはアビジン・MOM複合体単独では、いずれ
もPFC数を有意に減少させなかつたが、両者を
共に用いるとPFC数が著しく減少し、抗体が認
識する抗DNP抗体産生前駆Ｂ細胞群に細胞傷害
作用があつたことがわかる。
実施例 ７
Ｔ細胞代替因子（TRF）受容体をもつたＢ細
胞に対する細胞傷害
(イ) 抗BALB／cB細胞抗体のビオチニル化Ｆ
（ab′）2の調製
ジニトロフエニル（DNP）−キーホール・リ
ンペツト・ヘモシアニン（KLH）で免疫した
BALB／ｃマウスの脾細胞を抗Thy1.2抗体と
ウサギ補体で処理しＴ細胞を除き、DNP−
KLH感作Ｂ細胞を得た。次にこのＢ細胞を水
酸化アルミニウムゲル４mgと百日咳菌
（Bordetella pertussis）１×109個と共に
（DBA／2Ha×BALB／ｃ）（DC）F1雄性マウ
スに腹腔内投与し、更にＢ細胞だけで一週間毎
に６回免疫した後、その最終免疫の一週間後か
ら、毎週採血した。得られた抗血清はプールし
て、硫安分画並びにDEAE−セルロースカラム
クロマトグラフイーによりIgG画分を得、ペプ
シン処理及びセフアデツクスＧ−150カラムク
ロマトグラフイーによりＦ（ab′）2を調製した。
Ｆ（ab′）2のビオチニル化は実施例５のイ項の如
くして行つた。
(ロ) ビオチニル化抗体とアビジン・リシンＡ鎖複
合体を用いる、TRF受容体をもつた細胞に対
する細胞傷害
DNP−KLH感作脾細胞を、抗Thy1.2抗体及
びウサギ補体で処理してＴ細胞を除き、次に実
施例５と同様に、ビオチニル化抗BALB／cB
細胞抗体のＦ（ab′）2とアビジン・リシンＡ鎖複
合体で前処理した後、DNP−卵白アルブミン
（OVA）（40nｇ／ml），２−メルカプトエタノ
ール（50μM），TRF（50容量％）及び10％ウシ
胎児血清を含むRPMI1640培地600μを添加し
て、５％CO2加湿雰囲気下37℃５日間培養し
PFC反応を調べた。
第１１図に示す通り、脾細胞から、Ｔ細胞を
除いてしまうと、抗原（DNP−OVA）刺激だ
けではPFC反応がおこらなくなるが（グルー
プ１）、培地中にTRFを添加するとPFC数が著
しく回復した。しかし、脾細胞を予めビオチニ
ル化抗BALB／cB細胞抗体とアビジン・リシ
ンＡ鎖で処理することにより、TRFのPFC回
復効果が、抑制された。この結果はDCF1雄性
マウスをBALB／ｃのＢ細胞で免疫すると
TRF受容体に対する抗体ができ、かかる抗体
のビオチニル化体とアビジン、リシンＡ鎖複合
体によつてＢ細胞を処理すると、TRF受容体
を有するＢ細胞群が除去されたことを示してい
る。[Formula] etc. can be mentioned. Specific examples of the alcohol residue of the imidoester represented by Q include methoxy and ethoxy groups. Specific examples of the halogen atom represented by X include chlorine and bromine. As a specific example of the crosslinking agent, N-succinimidyl 3-(2
-pyridyldithio)propionate, 2,4-dinitrophenyl 3-(4-pyridyldithio)propionate as a crosslinking agent represented by the formula (), methyl 3-(2-pyridyldithio)propionimidate hydrochloride, the formula ( As the crosslinking agent represented by V), N-succinimidyl 3-bromopropionate can be mentioned. In the above crosslinking reaction between avidin and protein biosynthesis inhibiting protein, when reacting avidin or protein biosynthesis inhibiting protein with a crosslinking agent, it is recommended to use 1 to 20 moles of the crosslinking agent per mole of protein to be reacted. preferable. The reaction is carried out using avidin or protein biosynthesis inhibiting protein at a protein concentration of 0.5 to 100 mg/ml (more preferably 1 to 20 mg/ml) in a buffer solution with a pH of 5 to 9.
ml) into an aqueous solution of the crosslinking agent or, if the crosslinking agent is not soluble in water, add a small amount of an organic solvent such as N,N- This is carried out by adding a solution dissolved in dimethylformamide, dimethylsulfoxide, 1,2-dimethoxyethane, methanol, ethanol, acetone, etc. The reaction time depends on the reaction scale and reaction conditions, but is generally within 2 days. After the reaction is complete, unreacted crosslinking agent and low-molecular products are removed by dialysis or molecular sieve column chromatography, and the other constituents of the complex are added to the protein solution into which the resulting crosslinking agent has been introduced. Adding a solution of a protein (or a protein into which a cross-linking functional group has been introduced by a cross-linking agent) in a buffer with a pH of 5 to 9 (the preferred protein concentration range is the same as described above), React at 0-50°C. Separation and purification of the complex from the reaction mixture can be carried out by commonly used operations, such as molecular sieve column chromatography. Note that the complex can also be produced by adding a solution of the protein, the other component, into which a crosslinking agent has been introduced, to a solution of the protein, the other component of the complex. Furthermore, when a protein into which a cross-linking agent has been introduced is treated with a low-molecular reagent to convert it into a protein having a specific cross-linking functional group (for example, an active disulfide group introduced by a cross-linking agent is converted to a thiol group). or when converting avidin or protein biosynthesis inhibitory active protein directly into an activated derivative using a low molecular reagent (for example, when converting the thiol group of lysine fragment A into an activated disulfide), the reaction conditions are also The reaction conditions are the same as those for reacting the above-mentioned protein with a crosslinking agent. A protein complex obtained by covalently cross-linking avidin obtained in the present invention and a protein with a protein biosynthesis inhibitory activity is a biotinylated antibody that recognizes a specific cell group, and avidin's strong resistance to biotin. By utilizing affinity and binding, it can be used to selectively destroy specific cell groups recognized by antibodies. That is, the present inventors have demonstrated that the protein biosynthesis-inhibiting protein is bound to an antibody through a non-covalent bond based on the strong affinity between avidin and biotin. It has been discovered that this method can exert cytotoxicity against specific cell groups that it recognizes. One of the advantages of such a system for destroying specific cell groups is that, as already mentioned, the complement-dependent cell lysis reaction may not be able to bind to complement depending on the antibody class or subclass. The system of the present invention can be applied even in cases where the complement-dependent cell lysis reaction is difficult to occur depending on the animal species used even when the animal species has complement binding ability. Furthermore, while selective complement-dependent cell lysis reaction may be inhibited by components other than complement in serum used as a complement source, this does not occur in the method of the present invention. Furthermore, the method of the present invention is characterized by the A chain of ricin, fragment A of diphtheria toxin, and the protein biosynthesis inhibiting protein obtained from Tribulus chinensis, which is preferably used to form a protein complex with avidin as a protein biosynthesis inhibiting protein. The protein biosynthesis-inhibiting protein obtained from the and American pokeweed, by itself, lacks a site that binds to the cell surface, so it exhibits only extremely weak cytotoxicity, and once an antibody is used as a carrier, It exhibits extremely strong cytotoxicity when introduced into cells. In addition, since biotinylated antibodies generally have multiple sites that bind to avidin, when a protein biosynthesis-inhibiting protein is bound to an antibody via an avidin-biotin conjugate as in the method of the present invention, Antibody 1
Another feature of the method of the present invention is that a plurality of protein biosynthesis-inhibiting active proteins are attached to each molecule, thereby amplifying the cell-destroying effect. A preferred embodiment of destroying a specific cell group by the method of the present invention is to first prepare a cell group containing the cell group to be destroyed using a solution of a biotinylated antibody (biotinylated antibody) that selectively recognizes the cell group; 0
After contact reaction at ~37°C for 5 to 60 minutes and removing excess unreacted antibody by washing with cell culture medium or buffered saline, avidin and protein biosynthesis inhibiting protein were further bonded by covalent bonding. Add the protein complex obtained by crosslinking with 0 to 37
In this method, the cells are subjected to a contact reaction for 1 to 30 minutes at ℃, washed with a cell culture medium or buffered saline to remove excess protein complexes, and then cultured. Cell culture may be carried out under normal conditions, ie, at 37° C. in a humidified atmosphere of 5% CO 2 for usually 2 hours to 5 days. The biotinylated antibody and the complex of avidin and protein biosynthesis inhibitory activity protein may be added to cells at the same time, or the avidin-biotin conjugate may be prepared by contacting the biotinylated antibody and the protein complex of the present invention in advance. The cell population may then be treated with the conjugate. Hereinafter, the present invention will be explained in detail with reference to Examples. Example 1 Protein complex obtained by cross-linking avidin and ricin A chain through a covalent bond containing a disulfide bond (a) Introduction of active disulfide group into avidin 3.6
mg avidin 0.1M containing 0.1M sodium chloride
Dissolve in 1 ml of phosphate buffer PH7.5 (hereinafter referred to as solution A), and add 18.4 mM N in ethanol to this.
-succinimidyl 3-(2-pyridyldithio)
0.02 ml of a solution containing propionate (hereinafter abbreviated as SPDP) was added, and the mixture was allowed to react at room temperature for 30 minutes.
This reaction solution was mixed with 0.14M sodium chloride and 1mM
Sodium ethylenediaminetetraacetate (hereinafter
Abbreviated as EDTA. ) and 5mM acetate buffer PH
The mixture was subjected to Sephadex G25 column chromatography equilibrated with 5.5 (hereinafter referred to as Solution B) to remove unreacted substances and low-molecular-weight products to obtain avidin into which active disulfide groups had been introduced. (b) Method for preparing ricin A chain A method for purifying ricin from castor beans (Rieinus communis) and separating the A chain from ricin is described by S. Olsnes and A. Pihl [Biochem. , Volume 12, 3121~
3126 pages, 1973]. Since the obtained A chain solution contained 2-mercaptoethanol, it was removed by the Sephadex G25 column chromatography described in (a) above immediately before use. This A chain contains sodium dodecyl sulfate-
Polyacrylamide gel electrophoresis (hereinafter referred to as SDS-
PAGE) neither B chain nor intactin was detected. (c) Crosslinking of avidin into which an active disulfide group has been introduced and ricin A chain and purification of the protein complex as described above
Solution B (absorbance at 280 nm is
It was 3.5. ) 1.7 ml and B solution containing ricin A chain (with thiol group) (absorbance at 280 nm is
It was 2.4. ) contains 1.8ml and 10mM EDTA
0.4M phosphate buffer PH7.5 (hereinafter abbreviated as solution C)
0.34 ml was mixed and reacted overnight at room temperature. This reaction solution was subjected to Sephadex G150 Superfine column chromatography (column size 1.3 cm x 95 cm) equilibrated with physiological saline. As shown in Figure 4, a large peak was observed at the outflow position with a molecular weight of about 90,000 to 160,000. These fractions No. 21 to No. 26 (shaded area in the figure) were pooled. The protein solution thus obtained was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Figure 5, disk 1 is the ricin A chain, disk 2 is the avidin, disk 3 is the cross-linked and purified protein complex, and disk 4 is the protein complex with 2-
The product is reduced with mercaptoethanol. From disk 3, in addition to the avidin subunit with a molecular weight of approximately 15,000 (right end), products with molecular weights of approximately 47,000 and 79,000 (left end) are recognized. These molecular weights are 1 molecule of avidin subunit, 1 molecule of subunit and 1 molecule of ricin A chain.
Sum of molecules and 1 subunit molecule and ricin A chain 2
(Due to the presence of sodium dodecyl sulfate in the system, avidin is detected separately into subunits.) In fact, when these products are reduced, a considerable amount of amount of ricin A chain is produced. Therefore, the obtained protein complex is composed of four avidin molecules as shown in Figure 6.
Some of the subunits have a structure in which they are bound to one ricin A chain (there are also a few bound to two ricin A chains). Example 2 Protein complex obtained by cross-linking avidin and fragment A of diphtheria toxin through a covalent bond containing a disulfide bond (a) Preparation of fragment A of diphtheria toxin 0.05M Tris/HCl containing 210 mg of diphtheria toxin −
2mM ethylenediaminetetraacetic acid aqueous solution (PH8.3)
Add trypsin solution at a concentration of 0.1 mg/ml to 18.5 ml.
Add 0.15ml and decompose at 25℃ for 50 minutes, then
Soybean trypsin inhibitor solution at 0.5mg/ml concentration
The reaction was stopped by adding 0.3 ml. Urea (final concentration 6M), sodium sulfite (final concentration 0.168M) and sodium tetrathionate (final concentration 0.042M) were added to this decomposition product to perform S-sulfonation decomposition at 37°C for 2 hours. The obtained reaction solution was mixed with 6M urea-0.03M acetate buffer (PH
5.3) was subjected to Sephadex G150 column chromatography (column size 3.5 cm x 112 cm), and only the fragment A fraction that flowed out with a delay was taken out, and further dialyzed against distilled water to obtain a pure fragment A solution. (Has one S-sulfo group). The obtained S-sulfonated fragment A solution was added to Tris-HCl buffer (PH8.4).
Diphtheria toxin, which has one thiol group, was treated with 2-mercaptoethanol (final concentration 10mM) at 37°C for 1 hour and subjected to Sephadex G25 column chromatography described in Example 1 (a). Fragment A was obtained. (b) Crosslinking of avidin into which an active disulfide group has been introduced and fragment A of diphtheria toxin and purification of a protein complex. By using the avidin into which an active disulfide group was introduced as obtained in section (), the procedure was carried out in the same manner as in section (c) of Example 1.
A protein complex in which avidin and diphtheria toxin fragment A were crosslinked by a covalent bond containing a disulfide bond was obtained. Example 3 A protein complex obtained by cross-linking avidin and a protein with protein biosynthesis inhibitory activity obtained from the seeds of Vinegar chinensis (A) that has a protein biosynthesis inhibitory activity from the seeds of Vinegar chinensis Extraction and purification of protein (hereinafter referred to as MOM) 9.2 g of Ganoderma seeds were ground with a milk wasp, and the lipids were removed with ether. To the thus obtained defatted powder was added 50 ml of 5mM phosphate buffer-0.2M sodium chloride solution (PH7.2), and stirring was continued at 4°C overnight. Thereafter, insoluble matter was removed by centrifugation, and ammonium sulfate was added to achieve 30% saturation. After stirring at 0°C for 1 hour, remove the supernatant by centrifugation, add ammonium sulfate to the supernatant to achieve 70% saturation, stir at 0°C for 2 hours, and separate the precipitate by centrifugation. did. The precipitate was dissolved by adding 5 ml of 5mM phosphate buffer (PH6.5), and thoroughly dialyzed against the same phosphate buffer. The dialyzed solution was subjected to Sephadex G150 Superfine column chromatography (column size 84 cm x 2.5 cm) equilibrated with the same phosphate buffer, and proteins flowing out at a position with a molecular weight of approximately 30,000 were pooled. This was subjected to CM Sephadex C-50 column chromatography (column size 1.6 cm x 34 cm). The resin is equilibrated in 5mM phosphate buffer (PH6.5), 0.05M phosphate buffer (PH6.5), 0.1M
When the salt concentration was increased from phosphate buffer (PH6.5) to 0.2M phosphate buffer (PH6.5), MOM began to flow out at 0.10M.
The protein in this fraction had a molecular weight of approximately 27,000, and it was confirmed by SDS-PAGE that it did not contain other proteins. (b) Preparation of MOM with thiol groups introduced (a) above
12.9 mg of MOM extracted and purified as described above.
0.19 ml of a 20 mM SPDP ethanol solution was added to 4 ml of solution A containing a concentration of 1 ml, and the mixture was reacted at room temperature for 30 minutes. Add 10mM EDTA to this reaction solution,
Contains 200mM 2-mercaptoethanol
0.1 ml of 500 mM Tris-HCl buffer (PH8.5) was added, the mixture was reacted for 15 minutes at room temperature, and subjected to Sephadex G-25 column chromatography equilibrated with Solution B to obtain MOM into which a thiol group had been introduced. (c) Crosslinking of avidin into which an active disulfide group has been introduced and MOM into which a thiol group has been introduced and purification of the protein complex The avidin into which an active disulfide group has been introduced obtained in Section (a) of Example 1 was 2.0 ml of solution B (absorbance at 280 nm was 3.0) and 0.6 ml of solution B containing MOM with thiol groups introduced in the above (b) (absorbance at 280 nm was 9.8). ml
and 0.26 ml of Solution C were mixed and reacted overnight at room temperature. This reaction solution was subjected to Sephadex G-150 Super Fine chromatography (column size 1.3 x 95 cm) equilibrated with physiological saline.
FIG. 7 is a diagram of the chromatography.
Fractions 28 to 33 and 34 to 39 were pooled as fractions. This fraction was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results shown in FIG. 8 were obtained. Disk 1 in Figure 8 is MOM, disk 2
is avidin, disk 3 is fraction, disk 4
is a fraction, disks 5 and 6 are fractions, respectively.
is reduced with 2-mercaptoethanol. From disk 3, in addition to the avidin subunit with a molecular weight of approximately 15,000 (far right), the fraction with a molecular weight of approximately
Products of 45,000, 60,000, and 75,000 (leftmost) were observed, and their molecular weights were 1 molecule of avidin subunit and 1 molecule of MOM, 2 molecules of avidin subunit and 1 molecule of MOM, and 1 molecule of avidin subunit and 2 molecules of MOM, respectively. The molecular weights correspond to the sum of the molecular weights, and products with molecular weights of approximately 45,000 and 60,000 were observed in the fractions from disk 4, which are similar to the products with molecular weights of 45,000 and 60,000 in the first fraction, respectively. be. When these products are reduced, only MOM and avidin subunits are left as shown in disks 5 and 6. Therefore, in the resulting complex, some of the four subunits of avidin were bound to one MOM. It can be seen that it has a structure (there is also a small number of two bonded molecules). Example 4 Protein complex obtained by cross-linking avidin and a protein with protein biosynthesis inhibitory activity obtained from pokeweed leaves using a covalent bond having a disulfide bond (a) Extraction and purification of protein (hereinafter referred to as PAP) 500 g of American pokeweed leaves were added with 500 ml of water and homogenized using a mixer. From this homogenate, Irvin's method (Archives of Biochemistry and Biophysics)
Biochemistry and Biophysics), Volume 169,
522-528, 1975), ammonium sulfate fractionation,
PAP was purified by DEAE cellulose chromatography and phosphocellulose chromatography. This PAP is SDS−
In PAGE, one band was formed at a molecular weight of approximately 27,000. In addition, the SDS-
Confirmed on PAGE. In the following experiments, PAP purified from leaves was used. (b) Preparation of PAP with thiol group introduced 8.1 mg of PAP extracted and purified as in (a) above
Into 1.6 ml of 0.02 M phosphate buffer - 0.14 M sodium chloride - 1 mM EDTA aqueous solution (PH7.5) containing
0.09 ml of an ethanol solution containing 9 mM SPDP was added, and the mixture was allowed to react at room temperature for 30 minutes. To this reaction solution, 0.04 ml of 500 mM Tris-HCl buffer (PH8.5) containing 10 mM EDTA and 200 mM 2-mercaptoethanol was added, and the reaction was allowed to proceed at 37°C for 30 minutes. 25 column chromatography to obtain PAP with a thiol group introduced. (c) Crosslinking of avidin into which an active disulfide group has been introduced and PAP into which a thiol group has been introduced and purification of the protein complex An active disulfide group obtained as in item (a) of Example 1 was introduced into the avidin. Avidin and the thiol group obtained as described in (b) above were introduced.
From PAP, a protein complex in which avidin and PAP were cross-linked by a covalent bond containing a disulfide bond was obtained in the same manner as in Example 3 (c). Example 5 Lymphocyte B with immunoglobulins on the membrane surface
Cytotoxicity to cells (Part 1) (a) Biotinylation of antibody (IgG) Rabbit anti-mouse IgG dialyzed against 0.1M NaHCO 3
Mix 1 ml of antibody solution (1 mg/ml) and 0.1 ml of N,N-dimethylformamide solution of N-hydroxysuccinimide biotin (1 mg/ml), and incubate at room temperature for 4 hours.
Allowed time to react. After the reaction, excess N-hydroxysuccinimide biotin was removed by dialysis against 10 mM phosphate buffered saline (PH7.2) to obtain a biotinylated rabbit anti-mouse IgG antibody (biotinylated antibody). (b) Cytotoxicity of surface immunoglobulin-bearing B cells using biotinylated antibodies and avidin-lysine A chain complexes sensitized with dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH)
2 x 10 splenocytes (precursor B cells producing surface immunoglobulin for DNP) of BALB/C mice,
Biotinylated rabbit anti-mouse IgG antibody (1 mg/
ml) and incubate on ice for 30 minutes (Mouse B
(biotinylated antibody binds to cells), washed twice with cold Hank's solution, and then further washed with avidin and lysine A.
50 μl of chain complex (variable concentration) was added and reacted on ice for 15 minutes (avidin-ricin A chain complex binds to biotinylated antibody), and washed three times with cold Hank's solution. DNP-KLH was added to the splenocytes pretreated in this way.
(For antigen stimulation) Add 600 μl of RPMI 1640 medium containing (40 ng/ml) 2-mercaptoethanol (50 μM) and 10% fetal bovine serum and divide into three 200 μl portions at 37°C in a humidified atmosphere of 5% CO2 . So 5
After incubation for one day, anti-DNP antibody producing cells (PFC) were counted by Jerne's plaque method (see NK Jerne and AANordin, Science, Vol. 140, p. 405, 1963). The results, including comparative examples, are shown in FIG. From the figure, the addition of antigen (DNP-KLH) results in PFC
is significantly biotinylated (group 2),
When splenocytes were previously treated with biotinylated antibody and further treated with avidin-ricin A chain complex, PFC
It was found that the number decreased significantly. That is, anti
This shows that DNP antibody-producing progenitor cells (B cells) were removed by the cell-killing effect of the biotinylated anti-mouse IgG antibody and the avidin-ricin A chain complex that can bind thereto. Furthermore, it has been shown that both the biotinylated antibody and the avidin-ricin A chain complex are required to obtain a cell-killing effect. Example 6 Lymphocyte B with immunoglobulins on the membrane surface
Cytotoxicity to cells (Part 2) DNP-KLH sensitized splenocytes were treated in the same manner as in Example 5.
Biotinylated anti-mouse IgG antibody and avidin
After pretreatment with MOM complex, antibody production ability was examined. As shown in Figure 10, neither the biotinylated antibody nor the avidin/MOM complex alone significantly decreased the number of PFCs, but when both were used together, the number of PFCs decreased significantly, and the antibody recognized It can be seen that there was a cytotoxic effect on the anti-DNP antibody-producing precursor B cell group. Example 7 Cytotoxicity to B cells with T cell replacement factor (TRF) receptor (a) Biotinylation of anti-BALB/cB cell antibody F
Preparation of (ab′) 2 Immunized with dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH)
BALB/c mouse splenocytes were treated with anti-Thy1.2 antibody and rabbit complement to remove T cells, and DNP-
KLH-sensitized B cells were obtained. Next, these B cells were intraperitoneally administered to F1 male mice (DBA/2Ha×BALB/c) (DC) along with 4 mg of aluminum hydroxide gel and 1×10 9 Bordetella pertussis, and then only the B cells were administered intraperitoneally to F1 male mice. After 6 immunizations every week, blood was collected every week from 1 week after the final immunization. The obtained antisera were pooled, an IgG fraction was obtained by ammonium sulfate fractionation and DEAE-cellulose column chromatography, and F(ab') 2 was prepared by pepsin treatment and Sephadex G-150 column chromatography.
Biotinylation of F(ab') 2 was carried out as in Example 5, Section A. (b) Cytotoxicity against TRF receptor-bearing cells using biotinylated antibodies and avidin-ricin A chain complex DNP-KLH sensitized splenocytes were treated with anti-Thy1.2 antibody and rabbit complement to Cells were removed, and then biotinylated anti-BALB/cB was added in the same manner as in Example 5.
After pretreatment with cell antibody F(ab') 2 and avidin-ricin A chain complex, DNP-ovalbumin (OVA) (40 ng/ml), 2-mercaptoethanol (50 μM), and TRF (50% by volume) were added. and 600μ of RPMI1640 medium containing 10% fetal bovine serum, and cultured at 37°C for 5 days in a 5% CO2 humidified atmosphere.
The PFC response was investigated. As shown in Figure 11, when T cells are removed from splenocytes, antigen (DNP-OVA) stimulation alone does not cause a PFC reaction (group 1), but when TRF is added to the medium, the number of PFC increases significantly. I have recovered. However, by pre-treating splenocytes with biotinylated anti-BALB/cB cell antibody and avidin/ricin A chain, the PFC recovery effect of TRF was suppressed. This result was confirmed when DCF 1 male mice were immunized with BALB/c B cells.
It has been shown that when an antibody against the TRF receptor was generated and B cells were treated with a biotinylated antibody, avidin, and a ricin A chain complex, the B cell group having the TRF receptor was removed.
第１図のイはリシン、ロはそのＡ鎖とＢ鎖の構
造を示す模式図である。第２図はジフテリア毒素
の模式図であり、イはインタクトトキシン、ロは
ニツクドトキシン、ハはフラグメントＡとフラグ
メントＢの構造を示す。第３図は、抗体（免疫グ
ロブリン）の基本構造を示す模式図である。第４
図は、活性ジスルフイド基が導入されたアビジン
とリシンＡ鎖の反応生成物のセフアデツクス
G150スーパーフアインカラムクロマトグラフイ
ーでの流出パターンであり、斜線部は蛋白複合体
としてプールして用いた部分を示す。第５図は、
ドデシル硫酸ナトリウム−ポリアクリルアミドゲ
ル電気泳動（SDS−PAGE）のパターンであり、
デイスク１はリシンＡ鎖を、デイスク２はアビジ
ンを、デイスク３はアビジンとリシンＡ鎖の架
橋・精製された蛋白複合体を、デイスク４は蛋白
複合体を２−メルカプトエタノールで還元した生
成物を泳動させたパターンを示す。第６図は、ア
ビジンとリシンＡ鎖の蛋白複合体の模式図であ
る。第７図は、活性ジスルフイド基が導入された
アビジンとチオール基が導入されたMOMの反応
生成物のセフアデツクスG150スーパーフアイン
カラムクロマトグラフイーでの流出パターンであ
る。第８図は、SDS−PAGEのパターンであり、
デイスク１はMOMを、デイスク２はアビジン
を、デイスク３は第７図の分画を、デイスク４
は第７図の分画を、デイスク５，６はそれぞれ
分画，を２−メルカプトエタノールで還元し
た生成物を泳動させたパターンを示す。第９図
は、ビオチニル化抗体とアビジン。リシンＡ鎖複
合体を用いる、膜表面イムノグロプリンを持つた
Ｂ細胞に対する細胞傷害実験の結果を示す。第１
０図は、ビオチニル化抗体とアビジン・MOM複
合体を用いる、膜表面イムノグロプリンを持つた
Ｂ細胞に対する細胞傷害実験の結果を示す。第１
１図は、ビオチニル化抗体とアビジン・リシンＡ
鎖複合体を用いる、Ｔ細胞代替因子（TRF）受
容体をもつたＢ細胞に対する細胞傷害実験の結果
を示す。第９〜１１図に於いて、（＋）及び（−）
はそれぞれ、ビオチニル化抗体、蛋白複合体、抗
原またはTRFを系に加えたことと加えなかつた
ことを示す。
In FIG. 1, A is lysine, and B is a schematic diagram showing the structure of its A chain and B chain. FIG. 2 is a schematic diagram of diphtheria toxin, in which A shows the structure of intact toxin, B shows nickdotoxin, and C shows the structure of fragment A and fragment B. FIG. 3 is a schematic diagram showing the basic structure of an antibody (immunoglobulin). Fourth
The figure shows a cephadex of the reaction product of avidin and ricin A chain into which an active disulfide group has been introduced.
This is an outflow pattern obtained by G150 superfine column chromatography, and the shaded area indicates the portion that was pooled and used as a protein complex. Figure 5 shows
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern,
Disk 1 contains ricin A chain, Disc 2 contains avidin, Disc 3 contains the cross-linked and purified protein complex of avidin and ricin A chain, and Disc 4 contains the product obtained by reducing the protein complex with 2-mercaptoethanol. The pattern of electrophoresis is shown. FIG. 6 is a schematic diagram of a protein complex of avidin and ricin A chain. FIG. 7 shows the flow pattern of the reaction product of avidin introduced with an active disulfide group and MOM introduced with a thiol group in Sephadex G150 Superfine column chromatography. Figure 8 is the SDS-PAGE pattern,
Disk 1 contains MOM, Disk 2 contains Avidin, Disk 3 contains the fraction shown in Figure 7, and Disk 4 contains MOM.
shows the fraction of FIG. 7, and disks 5 and 6 show the electrophoresis pattern of the product obtained by reducing the fractions with 2-mercaptoethanol. Figure 9 shows biotinylated antibody and avidin. The results of a cytotoxicity experiment using a ricin A chain complex against B cells having membrane surface immunoglobulins are shown. 1st
Figure 0 shows the results of a cytotoxicity experiment against B cells with membrane surface immunoglobulins using a biotinylated antibody and an avidin/MOM complex. 1st
Figure 1 shows biotinylated antibodies and avidin/ricin A.
The results of cytotoxicity experiments against B cells with T cell replacement factor (TRF) receptors using chain complexes are shown. In Figures 9 to 11, (+) and (-)
indicate that biotinylated antibody, protein complex, antigen, or TRF was added to the system and not, respectively.
Claims (1)
ルフイド結合を含む共有結合によつて架橋するこ
とにより得られる蛋白複合体。 ２ 蛋白生合成阻害活性蛋白がリシンのＡ鎖であ
る特許請求の範囲第１項記載の蛋白複合体。 ３ 蛋白生合成阻害活性蛋白がジフテリア毒素の
フラグメントＡである特許請求の範囲第１項記載
の蛋白複合体。 ４ 蛋白生合成阻害活性蛋白がツルレイシより得
られる蛋白生合成阻害活性蛋白である特許請求の
範囲第１項記載の蛋白複合体。 ５ 蛋白生合成阻害活性蛋白がアメリカヤマゴボ
ウより得られる蛋白生合成阻害活性蛋白である特
許請求の範囲第１項記載の蛋白複合体。 ６ アビジンと蛋白生合成阻害活性蛋白のいずれ
か一方の蛋白に導入した活性ジスルフイド基に、
他方の蛋白に発生または導入したチオール基を反
応せしめることを特徴とする蛋白複合体の製造
法。[Scope of Claims] 1. A protein complex obtained by crosslinking avidin and a protein with protein biosynthesis inhibitory activity through a covalent bond containing a disulfide bond. 2. The protein complex according to claim 1, wherein the protein biosynthesis-inhibiting active protein is lysine A chain. 3. The protein complex according to claim 1, wherein the protein biosynthesis inhibiting protein is fragment A of diphtheria toxin. 4. The protein complex according to claim 1, wherein the protein biosynthesis-inhibiting protein is a protein biosynthesis-inhibiting protein obtained from A. chinensis. 5. The protein complex according to claim 1, wherein the protein biosynthesis-inhibiting protein is a protein biosynthesis-inhibiting protein obtained from pokeweed. 6 Into the active disulfide group introduced into either avidin or protein biosynthesis inhibitory protein,
A method for producing a protein complex, which comprises reacting a thiol group generated or introduced into another protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56172910A JPS5874614A (en) | 1981-10-30 | 1981-10-30 | Conjugate protein, its preparation, and selective cellucidal process with the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56172910A JPS5874614A (en) | 1981-10-30 | 1981-10-30 | Conjugate protein, its preparation, and selective cellucidal process with the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5874614A JPS5874614A (en) | 1983-05-06 |
| JPH0428719B2 true JPH0428719B2 (en) | 1992-05-15 |
Family
ID=15950606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56172910A Granted JPS5874614A (en) | 1981-10-30 | 1981-10-30 | Conjugate protein, its preparation, and selective cellucidal process with the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5874614A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4664911A (en) * | 1983-06-21 | 1987-05-12 | Board Of Regents, University Of Texas System | Immunotoxin conjugates employing toxin B chain moieties |
| JPS6028997A (en) * | 1983-07-26 | 1985-02-14 | Sumitomo Chem Co Ltd | Novel polypeptide and its preparation |
| JP2646048B2 (en) * | 1991-10-24 | 1997-08-25 | キユーピー 株式会社 | Lysozyme-containing pharmaceutical composition |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5549321A (en) * | 1978-09-28 | 1980-04-09 | Clin Midy | Cellular toxin product formed by covalent bond of lysine a chain with antibody |
-
1981
- 1981-10-30 JP JP56172910A patent/JPS5874614A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5549321A (en) * | 1978-09-28 | 1980-04-09 | Clin Midy | Cellular toxin product formed by covalent bond of lysine a chain with antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5874614A (en) | 1983-05-06 |
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