JPH06327491A - Hamster monoclonal antibody against mouse cell surface antigen - Google Patents
Hamster monoclonal antibody against mouse cell surface antigenInfo
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- JPH06327491A JPH06327491A JP5123905A JP12390593A JPH06327491A JP H06327491 A JPH06327491 A JP H06327491A JP 5123905 A JP5123905 A JP 5123905A JP 12390593 A JP12390593 A JP 12390593A JP H06327491 A JPH06327491 A JP H06327491A
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Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、マウス細胞表面抗原に
対するモノクローナル抗体に関する。FIELD OF THE INVENTION The present invention relates to a monoclonal antibody against a mouse cell surface antigen.
【0002】[0002]
【従来技術】哺乳類の細胞の発生の各段階での細胞の死
滅の多くがいわゆるアポトーシスと呼ばれる「計画的な
死」によることが知られており、このアポトーシスと緊
密な関係にある細胞表面の物質(ポリペプチド)が「細
胞表面抗原(FasまたはFas抗原)」である。アポトー
シスは、種々の細胞の交替、死滅に際して観察されてい
る[ウイリーら(Wyllie et al.)Int. Rev. Cytol. 6
8: 251-306(1980)]。これはまた、免疫細胞(胸腺細
胞)の死や腫瘍細胞の死滅の形態であることから、生理
学および医学分野で注目されている。例えば、胸腺にお
いて、Fasは、前駆体T細胞がT細胞になる過程での該
前駆体細胞の死滅に関与する可能性が指摘されている
[ワタナベ−フクナガら(Watanabe-Fukunaga,R.) Natu
re 356: 314-317 (1992)2. Description of the Related Art It is known that many deaths of cells at each stage of development of mammalian cells are due to "planned death" called so-called apoptosis, and cell surface substances closely related to this apoptosis. (Polypeptide) is "cell surface antigen (Fas or Fas antigen)". Apoptosis has been observed upon alternation and death of various cells [Wyllie et al. Int. Rev. Cytol. 6
8 : 251-306 (1980)]. It is also of interest in the fields of physiology and medicine as it is a form of immune cell (thymocyte) death or tumor cell death. For example, in the thymus, it has been pointed out that Fas may be involved in the death of precursor T cells in the process of becoming T cells [Watanabe-Fukunaga, R. Natu.
re 356 : 314-317 (1992)
【0003】また、TNFとFas抗原とはダウンレギュ
レーションの関係にあり、TNFの細胞毒性作用の発現
にFas抗原が加担していることが推測されている。とこ
ろで、細胞表面のTNFレセプター(I型およびII
型)、神経成長因子(NGF)レセプター、B細胞抗原
CD40およびT細胞抗原OX40等は、すべて生理学
的に重要な細胞表面膜タンパク質群を構成しておりNG
FR/TNFRファミリーと称される。Fas抗原もこ
のファミリーに属している。[イトウら(Itoh, N., Ce
ll 66:233-243 (1991); ワタナベ−フクナガら(Watanab
e-Fukunaga,R., J.Immunol. 148: 1274-1279 (199
2)]。Further, TNF and Fas antigen have a down-regulating relationship, and it is speculated that Fas antigen plays a role in the expression of the cytotoxic action of TNF. By the way, cell surface TNF receptors (type I and II)
Type), nerve growth factor (NGF) receptor, B cell antigen CD40, T cell antigen OX40, etc. all constitute a physiologically important cell surface membrane protein group.
It is called the FR / TNFR family. Fas antigens also belong to this family. [Itoh, N., Ce
ll 66 : 233-243 (1991); Watanab-Fukunaga et al.
e-Fukunaga, R., J. Immunol. 148 : 1274-1279 (199
2)].
【0004】さらに本発明者らは先の研究でマウスのリ
ンパ増殖性突然変異(lpr)をコードする構造遺伝子
は、Fas抗原遺伝子であることを示した[ワタナベ−
フクナガ(Watanabe-Fukunaga,R) Nature 356:314-317
(1982)]。また、悪性B−細胞およびT−細胞系で高度
に発現されているAPO−1抗原がFas抗原と同定され
た[オームら(Oehm,A., J.Biol.Chem. 267: 10709-1071
5 (1992)]。Furthermore, the present inventors have shown in a previous study that the structural gene encoding the murine lymphoproliferative mutation (lpr) is the Fas antigen gene [Watanabe.
Fukunaga (Watanabe-Fukunaga, R) Nature 356: 314-317
(1982)]. Also, the APO-1 antigen highly expressed in malignant B-cell and T-cell lines was identified as Fas antigen [Oehm, A., J. Biol. Chem. 267 : 10709-1071.
5 (1992)].
【0005】一方、ヒト免疫不全ウイルス(HIV)感
染細胞が抗Fasモノクローナル抗体の細胞死滅作用に対
して高い感受性を有していること、並びにインターフェ
ロン−γ(INF−γ)で処理されたヒト結腸がんHT
−29細胞には細胞表面Fas抗原が誘導され、該腫瘍細
胞の抗Fas抗体の細胞毒性作用に対する感受性が高めら
れることが示されている。これらの研究結果はFas抗原
に特異的なモノクローナル抗体の、HIV感染症治療お
よび結腸がん治療における有用性を強く示唆するもので
ある。On the other hand, human immunodeficiency virus (HIV) -infected cells are highly sensitive to the cell-killing effect of anti-Fas monoclonal antibody, and human colon treated with interferon-γ (INF-γ). Cancer HT
It has been shown that cell surface Fas antigen is induced in −29 cells and the sensitivity of the tumor cells to the cytotoxic effect of anti-Fas antibody is increased. The results of these studies strongly suggest the usefulness of Fas antigen-specific monoclonal antibodies in the treatment of HIV infection and colon cancer.
【0006】上記のことから、Fas抗原に特異的な抗体
を用いてFas抗原を発現する細胞を特異的にアポトーシ
スにより死滅させることが可能であり、その臨床面での
有用性が予測される。これに関連し、Fas抗原に対する
作動性抗体(agonistic antibody)を、EB−ウイルス
誘発性リンパ増殖性病巣[ファークら(Falk,M.H.) Blo
od 79: 3300-3306 (1992)]、HTLV−1関連悪性疾
患[デバチンら(Debatin,K-M.) Lancet 335: 497-500
(1990)]またはエイズ患者[コバヤシら(Kobayashi,N.)
Proc.Natl.Acad.Sci.USA 87: 9620ー9624 (1990)]に
投与することが提案された。From the above, it is possible to specifically kill cells expressing Fas antigen by apoptosis using an antibody specific to Fas antigen, and its clinical utility is predicted. In this connection, an agonistic antibody against the Fas antigen was labeled with an EB-virus-induced lymphoproliferative lesion [Falk, MH) Blo.
od 79 : 3300-3306 (1992)], HTLV-1-related malignant disease [Debatin, KM. Lancet 335 : 497-500.
(1990)] or AIDS patients [Kobayashi, N.)
Proc. Natl. Acad. Sci. USA 87 : 9620-9624 (1990)].
【0007】しかしながら、安全で有効な治療を達成す
るには、臨床適用の前にマウスなどを用いた十分な動物
実験を重ねる必要がある。既に、ヒトFas抗原に対する
マウスモノクローナル抗体は得られているが、それは、
ヒトFas抗原を発現している細胞に対してのみ溶解作用
を示し、マウスFas抗原を発現している細胞には作用し
ないことが分かっている[ヨネハラら、J. Exp. Med. 1
69: 1747-1756(1989)]。従って、上記の目的を達成する
には、マウスFas抗原に対するハムスターモノクローナ
ル抗体を得る必要がある。However, in order to achieve safe and effective treatment, it is necessary to repeat sufficient animal experiments using mice etc. before clinical application. A mouse monoclonal antibody against human Fas antigen has already been obtained.
It has been shown that it has a lytic effect only on cells expressing the human Fas antigen and does not act on cells expressing the mouse Fas antigen [Yonehara et al., J. Exp. Med. 1
69 : 1747-1756 (1989)]. Therefore, in order to achieve the above object, it is necessary to obtain a hamster monoclonal antibody against mouse Fas antigen.
【0008】[0008]
【課題を解決するための手段】本発明者らは、マウスF
as抗原に特異的なハムスターモノクローナル抗体を供給
することを目的として研究を重ね、マウスFas抗原と特
異的に反応するモノクローナル抗体の開発に成功した。
以下、本明細書中、単に抗Fas抗体というときは、本発
明のマウス細胞表面抗原に特異的なハムスターモノクロ
ーナル抗体を指すものとする。本発明のモノクローナル
抗体はマウスミエローマ細胞とマウス細胞表面抗原を発
現するW4形質転換細胞で免疫された哺乳類の脾臓細胞
とを融合させ、目的の抗体を生産する融合細胞をクロー
ン化することにより得られるハイブリドーマJo2を培
養することにより製造される。[Means for Solving the Problems]
With the aim of supplying a hamster monoclonal antibody specific to the as antigen, the inventors have conducted repeated research and succeeded in developing a monoclonal antibody that specifically reacts with the mouse Fas antigen.
Hereinafter, in the present specification, the term "anti-Fas antibody" refers to a hamster monoclonal antibody specific to the mouse cell surface antigen of the present invention. The monoclonal antibody of the present invention is obtained by fusing mouse myeloma cells with mammalian spleen cells immunized with W4 transformed cells expressing a mouse cell surface antigen and cloning the fused cells producing the desired antibody. It is produced by culturing the hybridoma Jo2.
【0009】本発明の抗Fas抗体は以下の特性を有す
る。 (1)マウス細胞表面抗原を発現する形質転換細胞に対
して用量依存性の細胞溶解作用を示すが、マウス細胞表
面抗原を発現しない細胞には細胞溶解作用を示さず、
(2)マウス胸腺細胞のサブセットの内CD4+CD
8+、CD4+CD8-およびCD4-CD8+を免疫化学
的に認識するがCD4-CD8-を認識せず、(3)マウ
スに腹腔内投与した時、正常なマウスの胸腺および肝臓
の細胞の細胞表面抗原を免疫化学的に認識し、アポトー
シスに基づく肝細胞の死をもたらし、数時間以内にマウ
スを死に至らしめるが、細胞表面抗原を発現しないMR
L−lpr/lprマウスに腹腔内投与した時には、そのよう
な細胞死および個体死をもたらさない。The anti-Fas antibody of the present invention has the following characteristics. (1) shows a dose-dependent cytolytic effect on transformed cells expressing a mouse cell surface antigen, but does not show a cytolytic effect on cells that do not express a mouse cell surface antigen,
(2) CD4 + CD in a subset of mouse thymocytes
Immunochemically recognizes 8 + , CD4 + CD8 − and CD4 − CD8 + but does not recognize CD4 − CD8 − , and (3) when administered intraperitoneally to mice, the cells of thymus and liver of normal mice were detected. MR that immunochemically recognizes cell surface antigens, causes apoptosis-based hepatocyte death, and kills mice within hours, but does not express cell surface antigens
When administered intraperitoneally to L- lpr / lpr mice, it does not cause such cell death and individual death.
【0010】本発明のモノクローナル抗体を生産するハ
イブリドーマはFERM P−13635の下で茨城県
筑波市の通商産業省工業技術院生命工学工業技術研究所
に寄託されているハイブリドーマJo2を培養すること
により得られる。The hybridoma producing the monoclonal antibody of the present invention is obtained by culturing the hybridoma Jo2 deposited under the FERM P-13635 at the Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry, Tsukuba City, Ibaraki Prefecture. To be
【0011】本発明のハイブリドーマは、免疫原として
マウスFas抗原を発現するW4形質転換細胞で免疫化し
た哺乳類(ホスト)から得た抗体産生細胞とミエローマ
細胞とを常法に従って融合させ、目的のモノクローナル
抗体を産生するハイブリッド融合細胞をクローン化する
ことにより得られる。W4細胞は、WR19L細胞(A
TCC,TIB52)を、マウスFas抗原cDNA[ワ
タナベ-フクナガ(Watanabe-Fukunaga, R., J.Immunol.1
48, 1274-1279(1992)]を保持するpEF−BOS発現
ベクター[ミズシマとナガタ(Mizushima and Nagata),
Nucleic.Acids Res. 18, 5322(1990)]で形質転換して
得られる。The hybridoma of the present invention is prepared by fusing a myeloma cell with an antibody-producing cell obtained from a mammal (host) immunized with a W4-transformed cell that expresses mouse Fas antigen as an immunogen according to a conventional method to obtain the desired monoclonal antibody. It is obtained by cloning a hybrid fused cell producing an antibody. W4 cells are WR19L cells (A
TCC, TIB52) and mouse Fas antigen cDNA [Watanabe-Fukunaga, R., J. Immunol.
48, 1274-1279 (1992)]-containing pEF-BOS expression vector [Mizushima and Nagata,
Nucleic. Acids Res. 18, 5322 (1990)].
【0012】免疫動物(ホスト)としてはハムスター、
ラットなどの哺乳類が使用可能である。ホスト動物を免
疫するには、免疫原であるW4細胞の懸濁液(好ましく
は2000rad照射処理したもの)を動物の皮下あるい
は腹腔内に注射する。接種量は動物、目的とする免疫化
の程度によって異なるが、通常、1回あたり106〜1
07細胞/動物で、1〜2週間ごとに数回、好ましくは
1週間ごとに6回行う。最終感作の1〜5日後に脾臓を
摘出し、抗体産生細胞として用いる。抗体産生細胞と融
合させる骨髄腫細胞も当業者に既知であり、マウス、ラ
ット、ヒトなどに由来するものを用いることができる。
そのようなミエローマ細胞は市販されており、例えばP
3X63Ag8U.1がある。As an immunized animal (host), a hamster,
Mammals such as rats can be used. In order to immunize a host animal, a suspension of W4 cells (preferably 2000 rad irradiation treatment) as an immunogen is injected subcutaneously or intraperitoneally into the animal. The inoculum varies depending on the animal and the degree of immunization intended, but is usually 10 6 to 1 per dose.
0 7 cells / animal several times every 1-2 weeks, preferably 6 times per week. 1 to 5 days after the final sensitization, the spleen is removed and used as antibody-producing cells. Myeloma cells to be fused with antibody-producing cells are also known to those skilled in the art, and those derived from mouse, rat, human, etc. can be used.
Such myeloma cells are commercially available, eg P
There is 3X63Ag8U.1.
【0013】細胞融合は文献記載の周知の方法[サンチ
ェ−マドリードら(Sanchez-Madrid,F.) J. Immunol.
130: 309-312 (1983);ヒラタら(Hirata,Y),J.Immuno
l. 143: 2900-2906 (1989)等]に従って行うことができ
る。通常、融合促進剤として50%ポリエチレングリコ
ールを含有するRPMI 1640等の培地で融合さ
せ、細胞融合によって得られた細胞を選択し、クローニ
ングし、目的のハイブリドーマを得る。通常、細胞をH
AT等の選択培地を含むマイクロタイタープレート内で
培養し、適宜選択培地を交換して培養を続け、骨髄腫細
胞を死滅させる。ハイブリドーマが成育したウエルの上
清における抗体の存在をスクリーニングするには、様々
な方法が使用可能であるが、本明細書の実施例では、マ
ウスFas抗原を発現するJ6細胞の細胞表面のマウスF
as抗原との結合活性をFACS(Fluorescent Activate
d Cell Sorter)分析法で調べて該スクリーニングを行っ
ている。しかし、これに限定されるものではない。最後
に抗体産生の認められたウエルの細胞を限界希釈法等で
クローニングを行い、目的のモノクローナル抗体を生産
する安定なハイブリドーマを得る。このハイブリドーマ
によるモノクローナル抗体の製造は、当業者既知のイン
ビトロまたはインビボの方法で行うことができる。Cell fusion is a well-known method described in the literature [Sanchez-Madrid, F.] J. Immunol.
130 : 309-312 (1983); Hirata, Y, J. Immuno
l. 143 : 2900-2906 (1989)]]. Usually, fusion is performed in a medium such as RPMI 1640 containing 50% polyethylene glycol as a fusion promoter, and cells obtained by cell fusion are selected and cloned to obtain a target hybridoma. Normally, cells
The myeloma cells are killed by culturing in a microtiter plate containing a selective medium such as AT, exchanging the selective medium appropriately and continuing the culturing. Although various methods can be used to screen for the presence of antibodies in the supernatants of hybridoma-grown wells, in the examples herein, mouse Fs on the cell surface of J6 cells expressing mouse Fas antigen are used.
FACS (Fluorescent Activate)
The screening is carried out by investigating by the d Cell Sorter) analysis method. However, it is not limited to this. Finally, cells in wells in which antibody production is observed are cloned by the limiting dilution method or the like to obtain stable hybridomas that produce the desired monoclonal antibody. Production of the monoclonal antibody by this hybridoma can be performed by an in vitro or in vivo method known to those skilled in the art.
【0014】例えば、血清不含培地(ALM−V培地、
Gibco)などの適当な培地で培養し、その上清から精製マ
ウス抗Fas抗体を得ることができる。精製は、例えば、
培養培地または腹水に硫安を加えて分画し、プロテイン
A−アガロース(Pharmacia)を用いた、アフィニティー
クロマトグラフィー等を用いて行う。For example, serum-free medium (ALM-V medium,
Gibco) and the like, and the purified mouse anti-Fas antibody can be obtained from the supernatant. Purification, for example,
Ammonium sulphate is added to the culture medium or ascites to fractionate, and affinity chromatography is performed using protein A-agarose (Pharmacia).
【0015】以下に実施例を挙げ、本発明をさらに詳し
く説明するが、これらの実施例は本発明を制限するもの
ではない。実施例1 マウスFas抗原に対するハムスターモノクロ
ーナル抗体の製造 1. 材料 免疫動物(ホスト):8週令雌性アルメニア(Armenia
n)ハムスター(日本クレア) 骨髄腫細胞:P3X63Ag8U 1 免疫原:W4細胞 免疫原である、マウスFas抗原を発現する形質転換体
(W4)の調製は以下の方法で行う。The present invention will be described in more detail with reference to examples below, but these examples do not limit the present invention. Example 1 Hamster monochrome against mouse Fas antigen
Production of monoclonal antibody 1. Material Immunized animal (host): 8-week-old female Armenia (Armenia)
n) Hamster (Clair Japan) Myeloma cell: P3X63Ag8U 1 Immunogen: W4 cell An immunogen, a transformant (W4) expressing mouse Fas antigen is prepared by the following method.
【0016】1)発現ベクターpEFMF1の構築 2μgのプラスミドpMF1[ワタナベ−フクナガら,
(Watanabe-Fukunaga,R. J.Immunol. 148, 1274-1279(1
992)]からFascDNAを含有する1.5kbXhoI
断片を調製し、それを哺乳類発現プラスミドpEF−B
OS[ミズシマ(Mizusima)およびナガタ(Nagata) Nucl
eic Acids Res. 18: 5322 (1990)]のBstXIサイト
にBstXIアダプターを用いて導入し、ヒトペプチド
鎖延長因子1α遺伝子のプロモーターのコントロール下
にFasをコードするcDNAを含有する発現ベクター
pEFMF1を構築した。1) Construction of expression vector pEFMF1 2 μg of plasmid pMF1 [Watanabe-Fukunaga et al.
(Watanabe-Fukunaga, RJImmunol. 148, 1274-1279 (1
992)] to 1.5 kb XhoI containing Fas cDNA
A fragment was prepared and used as a mammalian expression plasmid pEF-B.
OS [Mizusima and Nagata Nucl
eic Acids Res. 18: 5322 (1990)] was introduced into the BstXI site using a BstXI adapter to construct an expression vector pEFMF1 containing a cDNA encoding Fas under the control of the promoter of the human peptide chain elongation factor 1α gene. .
【0017】2)形質転換 マウスT−細胞リンパ腫WR19L細胞の形質転換は以
下の方法で行った。10%FCS含有RPMI1640
培地で増殖させたWR19L細胞(ATCC TIB5
2)[キネブチ(T. Kinebuchi) Tokyo Institute for
Immunopharmacology, Inc. より供与]1×107個
(0.8ml中)を、ネオマイシン耐性付与遺伝子を含
有するpSTneoB(0.2μg)およびApaLI
消化pEFMF1(25μg/ml)で、電気穿孔法
[ポッターら(Potter et al.) Proc. Natl.Acad. Sci.
USA 81: 7161-7165(1984)]により同時形質転換した。
[290ボルト、キャパシタンス:950μ F;Gene
Pulser(Bio-Rad)]。 細胞を96ウエルのマイクロタイタ
ープレートを用い増殖培地(0.1ml/ウエル)で2
日間培養し、最終濃度900μg/mlでG−418を
含有する培地でネオマイシン耐性クローンを選択した。
9日後、個々のG−418耐性形質転換体におけるFa
s抗原の発現をウサギ抗マウスFasポリクロナール抗
体(後述)を用いてサイトフルオロメーターで分析し、
Fas陽性細胞株(W4)を限界希釈法でクローン化し
た。2) Transformation Transformation of mouse T-cell lymphoma WR19L cells was performed by the following method. RPMI1640 containing 10% FCS
WR19L cells grown in culture medium (ATCC TIB5
2) [T. Kinebuchi] Tokyo Institute for
Donated by Immunopharmacology, Inc.] 1 × 10 7 cells (in 0.8 ml) were treated with pSTneoB (0.2 μg) and ApaLI containing a neomycin resistance-conferring gene.
Digested pEFMF1 (25 μg / ml) with electroporation [Potter et al. Proc. Natl. Acad. Sci.
USA 81: 7161-7165 (1984)].
[290V, Capacitance: 950μF; Gene
Pulser (Bio-Rad)]. Cells were grown in 96 well microtiter plates in growth medium (0.1 ml / well) for 2
After culturing for a day, neomycin resistant clones were selected in a medium containing G-418 at a final concentration of 900 μg / ml.
After 9 days Fa in individual G-418 resistant transformants
The expression of s antigen was analyzed by a cytofluorometer using a rabbit anti-mouse Fas polyclonal antibody (described later),
The Fas-positive cell line (W4) was cloned by the limiting dilution method.
【0018】2.検出 マウスFas抗原を高発現するJurkat形質転換細胞(J6
細胞)を用いて行う。ヒトT−細胞リンパ腫 Jurkat細
胞の形質転換は以下の方法で行った。10%FCS含有
RPMI1640培地で増殖させたJurkat細胞
(ATCC TIB152) 1×107個(0.8ml
中)を、ネオマイシン耐性付与遺伝子を含有するpST
neoB(0.2μg)およびApaLI消化pEFM
F1(25μg/ml)で、電気穿孔法[ポッターら
(Potter et al.) Proc. Natl.Acad. Sci. USA 81: 716
1-7165(1984)]により同時形質転換した。290ボル
ト、キャパシタンス:950μ F;Gene Pulser(Bio-
Rad)。 細胞を96ウエルのマイクロタイタープレート
を用い、増殖培地(0.1ml/ウエル)で2日間培養
し、最終濃度900μg/mlでG−418を含有する
培地でネオマイシン耐性クローンを選択した。9日後、
個々のG−418耐性形質転換体におけるFas抗原の
発現をウサギ抗マウスFasポリクロナール抗体(後
述)を用いてサイトフルオロメーターで分析し、Fas
陽性細胞株(J6)を限界希釈法でクローン化した。2. Jurkat transformant cells (J6) that highly express the detected mouse Fas antigen
Cells). Human T-cell lymphoma Jurkat cells were transformed by the following method. 1 × 10 7 Jurkat cells (ATCC TIB152) grown in RPMI1640 medium containing 10% FCS (0.8 ml)
PST containing the neomycin resistance-conferring gene
neoB (0.2 μg) and ApaLI digested pEFM
F1 (25 μg / ml), electroporation [Potter et al. Proc. Natl. Acad. Sci. USA 81: 716.
1-7165 (1984)]. 290 V, capacitance: 950 μF; Gene Pulser (Bio-
Rad). Cells were cultivated for 2 days in growth medium (0.1 ml / well) using 96 well microtiter plates and neomycin resistant clones were selected in medium containing G-418 at a final concentration of 900 μg / ml. 9 days later,
The expression of Fas antigen in individual G-418 resistant transformants was analyzed by a cytofluorometer using a rabbit anti-mouse Fas polyclonal antibody (described later), and Fas antigen was analyzed.
The positive cell line (J6) was cloned by the limiting dilution method.
【0019】ウサギ抗マウスFas抗体(マウスFas抗原
に対する抗ペプチド抗体)は以下の方法で作成した。N
−末端に余分のシステイン残基を有するマウスFas抗原
配列(アミノ酸番号8−21)[ワタナベ−フクナガら
(J.Immunol. 148, 1274-1279(1992)]を含有するC−
末端がアミド形のペプチドを合成した。m−マレイミド
ベンゾイル−N−ヒドロキシスルホスクシンイミド(M
BS)を用い、ペプチドをウシ血清アルブミン(BS
A)にカップリングさせた。ペプチドの合成およびその
BSAへの結合はMultiple Peptide Systems Co.,(サ
ンディエゴ)により行った。雌性ニュージランドウサギ
にペプチドを皮下注射した。条件:フロインドの完全ア
ジュバント中BSAコンジュゲート(0.5mg/注射)
を14日間隔で2回。さらに、ウサギにペプチド25μ
gを静注した。条件:PBS中BSAコンジュゲートを
3日間隔で3回。最終注射の3日後にウサギの全血清を
採取し、抗ペプチド抗体(抗−mFASP)をペプチド
とコンジュゲートしたTSKゲル上でのアフィニティー
クロマトグラフィーで精製した。簡単に述べると、MB
Sを用いてペプチド(2mg)をAF Amino Toyopearl
650 (東ソー) (充填容量:0.8ml)にカップリング
し、未反応部位を、ゲルを1M Tris−Hcl(pH8.
5)により洗浄してブロックした。血清(1.5ml)を
カラム(0.5ml)に負荷し、0.005%Tween20お
よび水で広範囲に洗浄した後、0.1M グリシンーH
Cl(pH2.2)で特異抗体をゲルから溶離した。Rabbit anti-mouse Fas antibody (anti-peptide antibody against mouse Fas antigen) was prepared by the following method. N
C-containing mouse Fas antigen sequence (amino acid number 8-21) [Watanabe-Fukunaga et al. (J. Immunol. 148, 1274-1279 (1992)]] having an extra cysteine residue at the end
An amide-terminated peptide was synthesized. m-maleimidobenzoyl-N-hydroxysulfosuccinimide (M
BS) and the peptide as bovine serum albumin (BS
A) was coupled. Peptide synthesis and its binding to BSA was performed by Multiple Peptide Systems Co., (San Diego). Female New Zealand rabbits were injected subcutaneously with the peptide. Conditions: BSA conjugate in Freund's complete adjuvant (0.5 mg / injection)
Twice every 14 days. In addition, 25μ peptide to rabbit
g was injected intravenously. Conditions: BSA conjugate in PBS 3 times at 3 day intervals. Rabbit whole sera were collected 3 days after the final injection and purified by affinity chromatography on TSK gel conjugated with anti-peptide antibody (anti-mFASP) with the peptide. Simply put, MB
Peptide (2 mg) was added to S by using AF Amino Toyopearl
650 (Tosoh) (filling volume: 0.8 ml) was coupled to the unreacted site and the gel was washed with 1 M Tris-Hcl (pH 8.
Washed and blocked according to 5). Serum (1.5 ml) was loaded onto the column (0.5 ml) and washed extensively with 0.005% Tween 20 and water, then 0.1 M glycine-H was added.
The specific antibody was eluted from the gel with Cl (pH 2.2).
【0020】3. 方法 8週令雌性アルメニアンハムスターにW4細胞(1x1
07、2800rads照射処理)を1週間間隔で6回接種
した。最終免疫の3日後にハムスターから脾臓を摘出
し、脾細胞を調製し、10%のウシ胎児血清を含むダル
ベッコ変法イーグル培地に懸濁して細胞浮遊液を調製し
た。上記の脾細胞浮遊液(6×107)とマウス骨髄腫
細胞P3X63Ag8U.1(1.2×107)とを5
0%(w/v)ポリエチレングリコール1500(ベー
リンガー)を用いて融合させた。最後に400xgで5
分間遠心して得られた細胞塊を7個の96ウエルプレー
トに分注した後、HAT培地で培養し、光学顕微鏡でハ
イブリドーマの成長を監視した[サンチェーマドリード
ら(Sanchez-Madrid,F.) J. Immunol. 130:309-312 (1
983);ヒラタ(Hirata,Y.),J.Immunol. 143: 2900-2906
(1989)]。3. Method W4 cells (1 × 1) were added to 8-week-old female Armenian hamsters.
0 7 , 2800 rads irradiation treatment) was inoculated 6 times at 1-week intervals. Three days after the final immunization, the spleen was extracted from the hamster, splenocytes were prepared, and suspended in Dulbecco's modified Eagle medium containing 10% fetal bovine serum to prepare a cell suspension. The above splenocyte suspension (6 × 10 7 ) and mouse myeloma cells P3X63Ag8U. 1 (1.2 × 10 7 ) and 5
Fusion was performed with 0% (w / v) polyethylene glycol 1500 (Boehringer). Finally 5 at 400xg
The cell mass obtained by centrifugation for 7 minutes was dispensed into seven 96-well plates, cultured in HAT medium, and the growth of hybridomas was monitored by an optical microscope [Sanchez-Madrid, F.] J. Immunol. 130: 309-312 (1
983); Hirata, Y., J. Immunol. 143: 2900-2906.
(1989)].
【0021】培地をHT培地に代えて培養を続け、その
間、培養上清における抗Fasモノクローナル抗体の産生
を、J6細胞表面のマウスFas抗原との結合活性をFA
CS分析法で調べることにより、スクリーニングした。
目的の抗マウスFasモノクローナル抗体を産生するハイ
ブリドーマJo2を選択し、3ユニット/mlのヒトイ
ンターロイキン6(IL−6),10%のウシ胎児血清
を含む、RPMI 1640培地による制限希釈法で継
代を繰り返してハイブリドーマクローンJo2を確立し
た。このJo2を血清不含培地(ALM−V培地、Gibc
o)で培養すると、培地にモノクローナル抗体が分泌され
た。The culture was continued by replacing the medium with HT medium, during which the production of anti-Fas monoclonal antibody in the culture supernatant was confirmed by the FA binding activity with mouse Fas antigen on the surface of J6 cells.
Screening was done by examining CS analysis.
A hybridoma Jo2 producing the desired anti-mouse Fas monoclonal antibody was selected and subcultured by a limiting dilution method using RPMI 1640 medium containing 3 units / ml of human interleukin 6 (IL-6) and 10% fetal bovine serum. Was repeated to establish the hybridoma clone Jo2. Serum-free medium (ALM-V medium, Gibc
When cultured in o), the monoclonal antibody was secreted into the medium.
【0022】培地に50%硫酸アンモニウムを加えて沈
澱させ、プロテインA−アガロース法(Pharmacia)[ク
リガンら(Cligan,J.E.) Current Protocols in Immunol
ogy,Willey Interscience (1991)]によってハムスター
抗マウスFasモノクローナル抗体(Jo2抗体)を精製
した。50% ammonium sulfate was added to the medium for precipitation, and the protein A-agarose method (Pharmacia) [Cligan, JE Current Protocols in Immunol] was used.
Ogy, Willey Interscience (1991)], the hamster anti-mouse Fas monoclonal antibody (Jo2 antibody) was purified.
【0023】実験例1 Jo2抗体とマウス胸腺細胞の
細胞表面抗原との反応 野生型マウス由来の胸腺細胞およびFas抗原を殆ど発現
しないlprマウス(リンパ増殖変異体)由来の胸腺細胞
と、Jo2抗体との免疫化学的反応を以下の方法で検討
した。 (1)フローサイトメトリー 3−4週令のマウス(Balb/c、MRL−+/+およ
びMRL−lpr/lpr)の胸腺から単一細胞浮遊液を調製
し、1x106細胞をJo2抗体、次いでフィコエリス
リン(PE)−結合ヤギ抗ハムスターIgG(F(ab')
2)フラクション,CATALOG)により染色した
(図1における斜線部分)。図1の空白部分は、第2抗
体のみで染色された胸腺のFACS像である。図1から
明らかに、野生型マウスの胸腺から得た細胞の殆どがF
as抗原陽性であるのに対し、MRL−lpr/lprマ
ウスの胸腺由来の細胞にはFas抗原が殆ど検出されな
い。 Experimental Example 1 Jo2 antibody and mouse thymocytes
Reaction with Cell Surface Antigen The immunochemical reaction between the thymocyte derived from wild type mouse and the thymocyte derived from lpr mouse (lymphoproliferative mutant) that hardly expresses Fas antigen and the Jo2 antibody was examined by the following method. (1) Flow cytometry A single cell suspension was prepared from the thymus of 3-4 week old mice (Balb / c, MRL-+ / + and MRL- lpr / lpr ), and 1 × 10 6 cells were added to Jo2 antibody, followed by Phycoerythrin (PE) -conjugated goat anti-hamster IgG (F (ab ')
2 ) Staining with a fraction, CATALOG) (hatched portion in FIG. 1). The blank part in FIG. 1 is a FACS image of the thymus stained with only the second antibody. As is clear from FIG. 1, most of the cells obtained from the thymus of wild-type mice were F
Fas antigen is hardly detected in the cells derived from the thymus of MRL-lpr / lpr mice, while being positive for as antigens.
【0024】(2)3色フローサイトメトリー Balb/cおよびMRL−lpr/lprマウスの胸腺細胞のサ
ブセット[CD4+CD8+(DP)、CD4+CD8
-(CD4SP)、CD4-CD8+(CD8SP)およ
びCD4-CD8-(DN)]の細胞各5000個を用
い、これらサブセットにおけるFas抗原の発現状態を、
FACScan(Beckton Dickinson)によるCD4、CD
8およびFas抗原に対する3色フローサイトメトリーで
分析した。用いたモノクローナル抗体はビオチン−標識
抗CD4(RM−4−5,PharMingen)およびStreptav
idin TRI−COLOR(CATALOG)、蛍光性
イソチオシアナート(FITC)−標識抗CD8/Ly
t−2(53−6.7,Beckton Dickinson)、本発明の
Jo2抗Fas抗体およびPE−標識ヤギ抗ハムスターI
gGである。結果を図2に示す。左側パネルにCD4お
よびCD8発現のためのマウス胸腺を二次元的に示し
た。 右側パネルには、Fas抗原の発現状態を示す。図
2から明らかに、Balb/cマウスの胸腺細胞の内、2
重陽性の胸腺細胞サブセット(CD4+CD8+)、単一
陽性の胸腺細胞サブセット(CD4+CD8-およびCD
4-CD8+)はFas抗原を発現しているが、2重陰性の
胸腺細胞サブセット(CD4-CD8-)の大多数はFas
抗原を発現していないことが分かる。他方、MRL−lp
r/lprマウスの胸腺細胞は、CD4およびCD8抗原の
分布はBalb/cマウスと同様であるが、どの胸腺細胞
サブセットもFas抗原を発現していない。(2) Three-color flow cytometry Balb / c and MRL- lpr / lpr mouse thymocyte subsets [CD4 + CD8 + (DP), CD4 + CD8]
- the (DN)] cells each 5000 was used for the expression state of Fas antigen in these subsets, - (CD4SP), CD4 - CD8 + (CD8SP) and CD4 - CD8
CD4 and CD by FACScan (Beckton Dickinson)
Analyzed by 3-color flow cytometry against 8 and Fas antigens. The monoclonal antibodies used were biotin-labeled anti-CD4 (RM-4-5, PharMingen) and Streptav.
idin TRI-COLOR (CATALOG), fluorescent isothiocyanate (FITC) -labeled anti-CD8 / Ly
t-2 (53-6.7, Beckton Dickinson), Jo2 anti-Fas antibody of the invention and PE-labeled goat anti-hamster I.
It is gG. The results are shown in Figure 2. The left panel shows two-dimensionally the mouse thymus for CD4 and CD8 expression. The right panel shows the expression status of Fas antigen. As is clear from FIG. 2, among the thymocytes of Balb / c mice, 2
Double positive thymocyte subsets (CD4 + CD8 + ), single positive thymocyte subsets (CD4 + CD8 − and CD
4 - CD8 +) While expressing Fas antigen, thymocyte subsets double negative (CD4 - CD8 -) majority Fas of
It can be seen that the antigen is not expressed. On the other hand, MRL- lp
Thymocytes of r / lpr mice have a similar distribution of CD4 and CD8 antigens as Balb / c mice, but none of the thymocyte subsets express Fas antigen.
【0025】実験例2 Jo2抗体のマウス細胞および
個体に対する致死作用 (1)細胞溶解作用 96ウエルマイクロタイタープレート上でFas抗原を発
現するW4形質転換細胞(2x104)または非形質転
換細胞WR19L細胞(2x104)を、種々の濃度
(0.1−1000ng/ml)のJo2抗体と混合し、3
7℃で16時間インキュベーションした。次いでウエル
あたり0.5μCi[3H]チミジン(比活性:74GB
q/mmol)を加え、4時間インキュベーションした
後、収穫した。Jo2抗体による細胞溶解作用を、Jo
2抗体不在下での[3H]チミジンの取り込みに対する
割合(%)で示した。結果を図3に示す。図中、W4細
胞は(白丸)、WR19L細胞は(黒丸)で表されてい
る。図3から明らかに、Jo2抗体はFas抗原を発現す
る形質転換細胞W4を用量依存的に、16時間以内に溶
解するが、親細胞WR19Lには作用しない。これは、
Jo2抗体がFas抗原を発現する細胞を認識し、細胞溶
解作用を表すことを示すものである。 Experimental Example 2 Mouse cells of Jo2 antibody and
Lethal effect on individuals (1) Cytolytic effect W4-transformed cells (2x10 4 ) or non-transformed WR19L cells (2x10 4 ) expressing Fas antigen on 96-well microtiter plates were mixed at various concentrations (0.1). -1000 ng / ml) and mixed with Jo2 antibody
Incubated at 7 ° C for 16 hours. Then 0.5 μCi [3 H ] thymidine per well (specific activity: 74 GB
q / mmol) was added and the mixture was incubated for 4 hours and then harvested. The cytolytic action of the Jo2 antibody
2 Shown as a ratio (%) to [3 H ] thymidine incorporation in the absence of antibody. The results are shown in Fig. 3. In the figure, W4 cells are represented by (white circles) and WR19L cells are represented by (black circles). As is clear from FIG. 3, the Jo2 antibody lyses the Fas antigen-expressing transformed cell W4 within 16 hours in a dose-dependent manner, but does not act on the parent cell WR19L. this is,
It is shown that the Jo2 antibody recognizes cells expressing Fas antigen and exhibits cytolytic action.
【0026】(2)致死作用 マウスへのJo2抗体の腹腔内投与が及ぼす致死作用を
検討した。結果を図4に示す。即ち、10匹の3週令の
雌性Balb/cマウス(黒丸、白丸、黒三角)およびM
RL−lpr/lprマウス(黒四角)に、PBS200μl中
の、精製Jo2抗体100μg(黒丸、黒四角)または
10μg(白丸)、あるいは正常なハムスターIgG
(黒三角)を腹腔内投与し、以後の生死を観察した。図
4から明らかに、Jo2抗体100μgを投与されたB
alb/cマウスの90%(9/10)が投与後3時間以
内に死亡した。10μgを投与されたマウスにおいても
8時間以内に50%(5/10)が死亡した。他方、ハ
ムスターIgGを投与された対照群のマウスには死亡例
がない。なお、エンドトキシンの致死作用に対して極め
て高い耐性を有するC3H/HeJマウスもJo2抗体
によって死亡した(データ示さず)。このことは、Fas
抗原に対する抗体の致死作用がエンドトキシンによるも
のでないことを示唆している。(2) Lethal action The lethal action of intraperitoneal administration of Jo2 antibody to mice was examined. The results are shown in Fig. 4. That is, 10 3-week-old female Balb / c mice (black circles, white circles, black triangles) and M
RL- lpr / lpr mice (black squares) were treated with 100 μg of purified Jo2 antibody (black circles, black squares) or 10 μg (white circles) or normal hamster IgG in 200 μl of PBS.
(Black triangle) was intraperitoneally administered, and the subsequent life and death was observed. As is clear from FIG. 4, B administered with 100 μg of Jo2 antibody
90% (9/10) of alb / c mice died within 3 hours after administration. 50% (5/10) also died within 8 hours in the mice administered with 10 μg. On the other hand, there are no deaths in the control group of mice that received hamster IgG. C3H / HeJ mice, which have extremely high resistance to the lethal action of endotoxin, also died due to the Jo2 antibody (data not shown). This is Fas
It suggests that the lethal action of the antibody against the antigen is not due to endotoxin.
【0027】 (3)血清中の生化学的パラメーターの変化 PBS200μl中の精製Jo2抗体100μgを3週
令の雌性Balb/cマウスに腹腔内投与し、様々な時期
に血液試料を採取した。血清の生物学的パラメーターを
標準的な自動臨床分析装置(日立、7150型)で検査
した。検査した生物学的パラメーターは以下の通りであ
る。GOT:グルタミン酸オキザロ酢酸トランスアミナ
ーゼ;GPT:グルタミン酸ピルビン酸トランスアミナ
ーゼ;ALP:アルカリホスホターゼ;AMY:アミラ
ーゼ;CPK:クレアチンホスホキナーゼ;HBD:α
−ヒドロキシ酪酸デヒドロゲナーゼ;BUN:血中尿素
窒素;T−BIL:総ビリルビン。結果を表1に示す。(3) Changes in Biochemical Parameters in Serum 100 μg of purified Jo2 antibody in 200 μl of PBS was intraperitoneally administered to 3-week-old female Balb / c mice, and blood samples were collected at various times. Serum biological parameters were examined on a standard automated clinical analyzer (Hitachi, Model 7150). The biological parameters tested are as follows. GOT: Glutamate oxaloacetate transaminase; GPT: Glutamate pyruvate transaminase; ALP: Alkaline phosphotase; AMY: Amylase; CPK: Creatine phosphokinase; HBD: α
-Hydroxybutyrate dehydrogenase; BUN: blood urea nitrogen; T-BIL: total bilirubin. The results are shown in Table 1.
【0028】[0028]
【表1】 表1から明らかに、血清中のGOT、GPT、HBDお
よびT−BILは抗体の注射から3時間後に劇的に増加
し、基準値の200、1000、50および10倍に達
したが、ALP、AMYおよびBUNの増加は僅かであ
った。ヘマトクリット値、血球数およびNa+、K+およ
びCl-等のイオン濃度には有意な変化を認めなかっ
た。ハムスターのIgGを投与されたマウスでは同様の
変化を認めなかった。これらの結果は、マウスに投与さ
れたFas抗体が、肝臓および心臓の細胞を特異的に損傷
することを示唆している。[Table 1] It is clear from Table 1 that GOT, GPT, HBD and T-BIL in serum increased dramatically 3 hours after injection of the antibody, reaching 200, 1000, 50 and 10 times the reference value, but ALP, The increase in AMY and BUN was slight. No significant changes were observed in hematocrit value, blood cell count and ion concentrations of Na + , K + and Cl − . Similar changes were not observed in mice administered hamster IgG. These results suggest that Fas antibody administered to mice specifically damages liver and heart cells.
【0029】実験例3 マウス肝臓および胸腺細胞にお
けるJo2誘導アポプトーシス (1)Jo2抗体のマウス肝細胞に対する作用 3週令のBalb/cマウスにJo2抗体100μgまた
は対照としてハムスターIgGを投与した。2時間後、
死亡したマウスの肝臓を摘出し、厚さ5μmの切片を
得、10%ホルムアルデヒドで固定化し、ヘマトキシリ
ンおよびエオシンで染色して組織の変化を調べた。結果
を図5に示す。図中、パネルAおよびBは対照(ハムス
ターIgG)、パネルCおよびDはJo2抗体100μ
gを投与されて2時間以内に死亡したマウスの肝臓組織
であり、パネルAおよびCは10倍、パネルBおよびD
は40倍の写真である。図から明らかに、Jo2抗体を
投与されたマウスの肝臓には多くの病巣出血と壊死が認
められる。この領域には正常な肝細胞は少ししか残って
おらず、大多数の損傷した細胞の特徴は細胞質濃縮と核
濃縮(ピクノーゼ)であり、これら細胞の死がアポトー
シスによることを示唆している。肝細胞に誘導されたア
ポトーシスは、ヒトの激症肝炎の動物モデルとなると考
えられる。このような出血性病巣および細胞壊死領域は
ハムスターのIgGを投与された対照群マウスの肝臓切
片には認められなかった。 Experimental Example 3 In mouse liver and thymocytes
Jo2 Induced Apoptosis (1) Effect of Jo2 Antibody on Mouse Hepatocytes Three-week-old Balb / c mice were injected with 100 μg of Jo2 antibody or hamster IgG as a control. Two hours later,
The livers of dead mice were excised, 5 μm-thick sections were obtained, fixed with 10% formaldehyde, and stained with hematoxylin and eosin to examine tissue changes. Results are shown in FIG. In the figure, panels A and B are controls (hamster IgG), panels C and D are Jo2 antibody 100μ.
g is liver tissue of a mouse that died within 2 hours after administration of g, panels A and C are 10 times, panels B and D
Is a 40x photo. As is clear from the figure, many focal bleeding and necrosis are observed in the liver of the mouse to which the Jo2 antibody was administered. Few normal hepatocytes remain in this region, and the majority of damaged cells are characterized by cytoplasmic and nuclear enrichment (picnose), suggesting that death of these cells is due to apoptosis. Hepatocyte-induced apoptosis is considered to be an animal model for human severe hepatitis. Such hemorrhagic lesions and cell necrosis areas were not found in the liver slices of control mice that were administered with hamster IgG.
【0030】 (2)Jo2抗体のマウス胸腺細胞に対する作用 Balb/cマウスにJo2Fas抗体を注射し、少なくと
も5時間生存したマウスから、胸腺DNAを調製して
0.5μg/mlの臭化エチジウムを含有するTBEバッ
ファー中の1%アガロースゲルによる電気泳動にかけ、
分析した。結果を図6に示す。DNAは、投与前(レー
ン1)、0.5時間後(レーン2)、1時間後(レーン
3)、2時間後(レーン4)、3時間後(レーン5)ま
たは5時間後(レーン6)に調製した。対照として、ハ
ムスターIgGの投与から5時間後のマウスから調製し
た胸腺DNAを用いた(レーン7)。胸腺組織は、肝臓
組織と異なり、明確な異常を呈していなかったが(デー
タ示さず)、その染色体DNAは明らかにアポトーシス
に特徴的な分解を示した。即ち、3時間後に得た胸腺D
NAははしご状に分離するアポトーシスに典型的なフラ
グメンテーション(切断)を示した。DNAの切断は後
になる程増加し、抗体投与から5時間後には、全染色体
DNAの80%以上が分解された。対照マウスでは染色
体DNAの分解を認めなかった。これらの結果は、Fas
抗原を発現する胸腺細胞がインビボで、抗Fas抗体によ
り誘導されるアポトーシスの影響を受け易いことを示唆
している。(2) Effect of Jo2 antibody on mouse thymocytes Balb / c mice were injected with Jo2Fas antibody, and thymus DNA was prepared from mice that survived for at least 5 hours to contain 0.5 μg / ml ethidium bromide. Electrophoresed on a 1% agarose gel in TBE buffer
analyzed. Results are shown in FIG. DNA was administered before (lane 1), after 0.5 hour (lane 2), after 1 hour (lane 3), after 2 hours (lane 4), after 3 hours (lane 5) or after 5 hours (lane 6). ) Was prepared. As a control, thymus DNA prepared from mice 5 hours after the administration of hamster IgG was used (lane 7). Thymic tissue, unlike liver tissue, did not exhibit distinct abnormalities (data not shown), but its chromosomal DNA clearly showed degradation characteristic of apoptosis. That is, thymus D obtained after 3 hours
NA showed a fragmentation typical of apoptosis that separated in a ladder. The amount of DNA cleavage increased later, and 80% or more of the total chromosomal DNA was degraded 5 hours after the antibody administration. No chromosomal DNA degradation was observed in control mice. These results are
It is suggested that thymocytes expressing the antigen are susceptible to apoptosis induced by anti-Fas antibody in vivo.
【0031】[0031]
【発明の効果】本発明のハイブリドーマを培養すること
により得られるマウス細胞表面抗原に特異的なハムスタ
ーモノクローナル抗体Jo2は、抗Fas抗体の臨床適用
を安全かつ効果的に行うための研究開発に大きく貢献す
るのみならず、肝臓、胸腺、心臓等の細胞のアポトーシ
スの研究、並びに激症肝炎等の疾患の動物モデルの開発
に有用である。EFFECT OF THE INVENTION The hamster monoclonal antibody Jo2 specific for the mouse cell surface antigen obtained by culturing the hybridoma of the present invention greatly contributes to research and development for safe and effective clinical application of anti-Fas antibody. In addition, it is useful for studying apoptosis of cells such as liver, thymus, heart, etc., and for developing animal models of diseases such as severe hepatitis.
【図1】マウス胸腺から得た細胞におけるFas抗原の発
現を表すフローサイトメトリーの結果を示すグラフ。FIG. 1 is a graph showing the results of flow cytometry showing the expression of Fas antigen in cells obtained from mouse thymus.
【図2】マウスの胸腺由来の細胞サブセット[CD4+
CD8+(DP)、CD4+CD8-(CD4SP)、C
D4-CD8+(CD8SP)およびCD4-CD8-(D
N)]におけるFas抗原の発現を表す3色フローサイト
メトリーの結果を示すグラフ。FIG. 2. Cell subset derived from mouse thymus [CD4 +
CD8 + (DP), CD4 + CD8 - (CD4SP), C
D4 - CD8 + (CD8SP) and CD4 - CD8 - (D
N)] is a graph showing the results of three-color flow cytometry showing the expression of Fas antigen.
【図3】Fas抗原を発現するW4形質転換細胞または非
形質転換細胞WR19L細胞に対するJo2抗体の細胞
溶解作用を、Jo2抗体不在下での[3H]チミジンの
取り込みに対する割合(%)で示したグラフ。FIG. 3 shows the cytolytic effect of Jo2 antibody on W4 transformed cells expressing Fas antigen or non-transformed WR19L cells, as a ratio (%) to [3 H ] thymidine incorporation in the absence of Jo2 antibody. Graph.
【図4】Jo2抗体を腹腔内投与されたマウスの生存率
を示すグラフ。FIG. 4 is a graph showing the survival rate of mice intraperitoneally administered with the Jo2 antibody.
【図5】Jo2抗体を投与されて死亡したマウスの肝臓
組織の変化を示す写真の模写図。FIG. 5 is a copy of a photograph showing changes in liver tissue of a mouse that died after administration of a Jo2 antibody.
【図6】Jo2抗体を投与されたマウスの胸腺細胞の染
色体DNAの変化を示すアガロースゲルによる電気泳動
の泳動パターンの模写図。FIG. 6 is a reproduction of electrophoretic patterns on an agarose gel showing changes in chromosomal DNA of thymocytes of mice administered with the Jo2 antibody.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 C12R 1:91) ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification code Office reference number FI technical display location C12R 1:91)
Claims (2)
抗原を発現するW4形質転換細胞で免疫された哺乳類の
脾臓細胞とを融合させて得られるハイブリドーマにより
産生されるモノクローナル抗体であって、 (1)マウス細胞表面抗原を発現する形質転換細胞に対
して用量依存性の細胞溶解作用を示すが、マウス細胞表
面抗原を発現しない細胞には細胞溶解作用を示さず、 (2)マウス胸腺細胞のサブセットの内CD4+CD
8+、CD4+CD8-およびCD4-CD8+を免疫化学
的に認識するがCD4-CD8-を認識せず、 (3)マウスに腹腔内投与した時、正常なマウスの胸腺
および肝臓の細胞の細胞表面抗原を免疫化学的に認識
し、アポトーシスに基づく肝細胞の死をもたらし、数時
間以内にマウスを死に至らしめるが、細胞表面抗原を発
現しないMRL−lpr/lprマウスに腹腔内投与した時に
は、そのような細胞死および個体死をもたらさないこと
を特徴とする抗体。1. A monoclonal antibody produced by a hybridoma, which is obtained by fusing mouse myeloma cells with mammalian spleen cells immunized with W4-transformed cells expressing a mouse cell surface antigen, which is (1) mouse It shows a dose-dependent cytolytic effect on transformed cells that express cell surface antigens, but does not show cytolytic effect on cells that do not express mouse cell surface antigens. (2) Among the subsets of mouse thymocytes CD4 + CD
Immunochemically recognizes 8 + , CD4 + CD8 − and CD4 − CD8 + but does not recognize CD4 − CD8 − , and (3) when administered intraperitoneally to mice, the cells of thymus and liver of normal mice were detected. It immunochemically recognizes cell surface antigens, causes apoptosis-based hepatocyte death, and causes death of mice within a few hours, but when intraperitoneally administered to MRL- lpr / lpr mice that do not express cell surface antigens. An antibody characterized by not causing such cell death and individual death.
れているハイブリドーマが産生するモノクローナル抗体
である請求項1のモノクローナル抗体。2. The monoclonal antibody according to claim 1, which is a monoclonal antibody produced by the hybridoma deposited under FERM P-13635.
Priority Applications (1)
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JP12390593A JP3447322B2 (en) | 1993-05-26 | 1993-05-26 | Hamster monoclonal antibody against mouse cell surface antigen |
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JP12390593A JP3447322B2 (en) | 1993-05-26 | 1993-05-26 | Hamster monoclonal antibody against mouse cell surface antigen |
Publications (2)
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JPH06327491A true JPH06327491A (en) | 1994-11-29 |
JP3447322B2 JP3447322B2 (en) | 2003-09-16 |
Family
ID=14872249
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP12390593A Expired - Lifetime JP3447322B2 (en) | 1993-05-26 | 1993-05-26 | Hamster monoclonal antibody against mouse cell surface antigen |
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JP (1) | JP3447322B2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997015326A1 (en) * | 1995-10-27 | 1997-05-01 | Sumitomo Electric Industries, Ltd. | Remedy for hepatitides |
WO2010098471A1 (en) | 2009-02-27 | 2010-09-02 | 株式会社バイオマトリックス研究所 | Immunization method using cancer cell |
WO2012026615A1 (en) | 2010-08-25 | 2012-03-01 | 株式会社バイオマトリックス研究所 | Method for producing antibodies using cancer cells |
-
1993
- 1993-05-26 JP JP12390593A patent/JP3447322B2/en not_active Expired - Lifetime
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997015326A1 (en) * | 1995-10-27 | 1997-05-01 | Sumitomo Electric Industries, Ltd. | Remedy for hepatitides |
US6068841A (en) * | 1995-10-27 | 2000-05-30 | Sumitomo Electric Industries, Ltd. | Antibodies to Fas-L for treatment of hepatitis |
WO2010098471A1 (en) | 2009-02-27 | 2010-09-02 | 株式会社バイオマトリックス研究所 | Immunization method using cancer cell |
WO2012026615A1 (en) | 2010-08-25 | 2012-03-01 | 株式会社バイオマトリックス研究所 | Method for producing antibodies using cancer cells |
US8598408B2 (en) | 2010-08-25 | 2013-12-03 | Ordermade Medical Research Inc. | Method of producing an antibody using a cancer cell |
Also Published As
Publication number | Publication date |
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JP3447322B2 (en) | 2003-09-16 |
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