JPH0967399A - Anti-mcp-1 human monoclonal antibody - Google Patents
Anti-mcp-1 human monoclonal antibodyInfo
- Publication number
- JPH0967399A JPH0967399A JP7221794A JP22179495A JPH0967399A JP H0967399 A JPH0967399 A JP H0967399A JP 7221794 A JP7221794 A JP 7221794A JP 22179495 A JP22179495 A JP 22179495A JP H0967399 A JPH0967399 A JP H0967399A
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- Japan
- Prior art keywords
- human
- mcp
- monoclonal antibody
- antibody
- monocyte
- Prior art date
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- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、ヒトモノクローナ
ル抗体に関するものである。詳しくは、本発明はヒト単
球走化活性化因子（以下ヒトＭＣＰ−１と称する）に対
して結合する能力を有するヒトモノクローナル抗体、ま
たは該ヒトモノクローナル抗体を含有する医薬組成物に
関するものである。TECHNICAL FIELD The present invention relates to a human monoclonal antibody. Specifically, the present invention relates to a human monoclonal antibody having the ability to bind to human monocyte chemotactic activator (hereinafter referred to as human MCP-1), or a pharmaceutical composition containing the human monoclonal antibody. .
【０００２】[0002]
【従来の技術】単球走化活性化因子（ＭＣＰ−１）は、
１９８９年にMatsushimaら及びYoshimuraらによりクロ
ーニングされた分子であり、単球やマクロファージに作
用してその走化性のこう進と活性化を促すサイトカイン
のひとつである（例えばOppenheimら: Annu. Rev. Immu
nol., 9, 617 (1991)）。炎症部位の拡大には、局所へ
の白血球の走化と浸潤が認められる。例えば特発性肺繊
維腫では、肺胞浸潤液中に単球やマクロファージの浸潤
が認められ、これが炎症部位の拡大に寄与していると考
えられている。この肺胞浸潤液中にはＭＣＰ−１の高発
現が認められ、特発性肺繊維腫のこう進にＭＣＰ−１が
影響するものと考えられている。子球体腎炎において
は、その病巣部位でＭＣＰ−１の高発現と単球やマクロ
ファージの浸潤が認められており、腎炎のこう進にＭＣ
Ｐ−１が関与していることが推定されている。また、動
脈硬化においては、酸化等の修飾を受けた低密度リポ蛋
白質がマクロファージに取り込まれ、やがて泡沫化して
硬化巣を形成することが知られているが、このマクロフ
ァージの走化にＭＣＰ−１が関与していると考えられて
いる。BACKGROUND OF THE INVENTION Monocyte chemotactic activator (MCP-1) is
It is a molecule cloned by Matsushima et al. And Yoshimura et al. In 1989, and is one of the cytokines that act on monocytes and macrophages to promote their chemotaxis and activation (eg Oppenheim et al .: Annu. Rev. Immu
nol., 9 , 617 (1991)). Localized leukocyte chemotaxis and infiltration are observed in the expansion of the inflammatory site. For example, in idiopathic pulmonary fibroma, infiltration of monocytes and macrophages is observed in the alveolar infiltrates, which is considered to contribute to the expansion of the inflammatory site. High expression of MCP-1 was observed in this alveolar infiltrate, and it is considered that MCP-1 affects the promotion of idiopathic pulmonary fibroma. In follicular nephritis, high expression of MCP-1 and infiltration of monocytes and macrophages were observed at the lesion site, and MC increased in the progression of nephritis.
It is estimated that P-1 is involved. Further, in arteriosclerosis, it is known that low density lipoprotein modified by oxidation and the like is taken up by macrophages and eventually foams to form sclerosing foci, but MCP-1 is involved in chemotaxis of these macrophages. Are believed to be involved.
【０００３】この様に、ＭＣＰ−１は単球及びマクロフ
ァージに作用して、炎症局所への走化性のこう進と活性
化を促す役割を担っており、いくつかの炎症反応の進展
に関わっていると考えられている。従って、ＭＣＰ−１
の作用を抑制するような物質がこれら炎症反応の予防あ
るいは治療に役立てられると考えられ、ＭＣＰ−１に対
するモノクローナル抗体はそのひとつの候補と考えられ
る。ＭＣＰ−１に対して結合するマウスモノクローナル
抗体は、例えばYoshimuraら（Yoshimuraら: J. Immuno
l., 147, 2229 (1991)）あるいは特開平６−２０５６９
０号公報に記載されている。すなわち、抗原としてヒト
ＭＣＰ−１をマウスに免疫した後、そのマウスから得ら
れた脾臓細胞とミエローマ細胞とを細胞融合してハイブ
リドーマを作製し、該ハイブリドーマから抗ＭＣＰ−１
マウスモノクローナル抗体を得ている。しかし、これら
の報告では、ヒト以外の動物由来の抗ＭＣＰ−１モノク
ローナル抗体は記載されているものの、ヒト由来の抗Ｍ
ＣＰ−１モノクローナル抗体に関しては何等記載されて
いない。As described above, MCP-1 acts on monocytes and macrophages to promote the promotion and activation of chemotaxis to local inflammation, and is involved in the development of some inflammatory reactions. It is believed that Therefore, MCP-1
It is considered that a substance that suppresses the action of is useful for prevention or treatment of these inflammatory reactions, and a monoclonal antibody against MCP-1 is considered as one of the candidates. Mouse monoclonal antibodies that bind to MCP-1 are described in, for example, Yoshimura et al. (Yoshimura et al .: J. Immuno).
L., 147 , 2229 (1991)) or JP-A-6-20569.
No. 0 publication. That is, after immunizing a mouse with human MCP-1 as an antigen, spleen cells and myeloma cells obtained from the mouse are cell-fused to prepare a hybridoma, and anti-MCP-1 is prepared from the hybridoma.
We have mouse monoclonal antibodies. However, although these reports describe anti-MCP-1 monoclonal antibodies derived from animals other than humans, human-derived anti-MCP-1 monoclonal antibodies have been described.
No mention is made of the CP-1 monoclonal antibody.
【０００４】ヒト以外の動物由来の抗体は、ヒトに対し
て投与した場合異物として認識され、その結果種々の抗
体反応が起こり、投与した動物抗体に対する抗体が形成
される。この抗体反応は、投与した抗体自体の反応性を
減少させると同時に、ヒト血中での半減期を著しく短縮
させる。例えばマウスモノクローナル抗体をヒトに投与
すると、抗マウス抗体反応（ＨＡＭＡ）が生じる。ＨＡ
ＭＡを減ずる方策として、マウスモノクローナル抗体の
抗原結合部位とあるヒト抗体の定常部位を組み換えて、
キメラ抗体あるいはヒト化抗体を作製する試みがなされ
ている。しかし、この方法においてもやはりマウス抗体
に由来する成分がヒト体内で抗原として認識されるた
め、ＨＡＭＡの問題は完全に回避できない。このため、
完全なヒトモノクローナル抗体が有用であろうと期待さ
れる。Antibodies derived from animals other than humans are recognized as foreign substances when administered to humans, and as a result, various antibody reactions occur to form antibodies against the administered animal antibodies. This antibody reaction reduces the reactivity of the administered antibody itself and, at the same time, significantly shortens the half-life in human blood. For example, when a mouse monoclonal antibody is administered to humans, an anti-mouse antibody reaction (HAMA) occurs. HA
As a measure to reduce MA, recombine the antigen binding site of mouse monoclonal antibody and the constant site of a human antibody,
Attempts have been made to produce chimeric or humanized antibodies. However, even in this method, since the component derived from the mouse antibody is recognized as an antigen in the human body, the problem of HAMA cannot be completely avoided. For this reason,
It is expected that fully human monoclonal antibodies will be useful.
【０００５】一方、ヒトの血清中にＭＣＰ−１と結合す
るポリクローナル抗体が存在することが、Sylvesterら
（Sylvesterら: J. Immunol., 151, 3292 (1993)）によ
り報告されている。しかし、この中においてもヒトポリ
クローナル抗体に関しては記載されているものの、ヒト
モノクローナル抗体については何等記載されていない。On the other hand, the presence of a polyclonal antibody which binds to MCP-1 in human serum has been reported by Sylvester et al. (Sylvester et al .: J. Immunol., 151 , 3292 (1993)). However, even though there is a description about a human polyclonal antibody in this, there is no description about a human monoclonal antibody.
【０００６】[0006]
【発明が解決しようとする課題】本発明の目的は、抗Ｍ
ＣＰ−１ヒトモノクローナル抗体を提供することであ
る。詳しくは、単球やマクロファージの走化性のこう進
と活性化を促すＭＣＰ−１に結合してＭＣＰ−１の働き
を抑制する、ヒトモノクローナル抗体を提供することで
ある。また本発明の他の目的は、炎症反応に関与するＭ
ＣＰ−１に結合することにより、単球やマクロファージ
の関与する炎症を伴う疾患に対する治療、予防剤として
期待される薬剤を提供することである。The object of the present invention is to provide anti-M
To provide a CP-1 human monoclonal antibody. Specifically, it is to provide a human monoclonal antibody that inhibits the action of MCP-1 by binding to MCP-1 that promotes chemotaxis promotion and activation of monocytes and macrophages. Another object of the present invention is to involve M involved in inflammatory response.
It is intended to provide a drug expected to be a therapeutic or preventive agent for a disease associated with inflammation involving monocytes and macrophages by binding to CP-1.
【０００７】[0007]
【課題を解決するための手段】本発明者らは、上記課題
を解決するために鋭意検討を行った結果、ヒトリンパ球
よりヒトＭＣＰ−１に結合するヒトモノクローナル抗体
を取得できることを見いだし、本発明を解決するに至っ
た。すなわち本発明は、ヒトＭＣＰ−１に結合するヒト
モノクローナル抗体を提供するものであり、単球やマク
ロファージの関与する炎症反応を伴う疾患の治療、予防
剤として期待される新規な薬剤を提供するものである。Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a human monoclonal antibody that binds to human MCP-1 can be obtained from human lymphocytes, and the present invention Came to solve. That is, the present invention provides a human monoclonal antibody that binds to human MCP-1, and provides a novel drug expected as a therapeutic or prophylactic agent for diseases associated with an inflammatory reaction involving monocytes and macrophages. Is.
【０００８】[0008]
【発明の実施の形態】本発明のモノクローナル抗体は、
ヒトリンパ球に由来するヒトモノクローナル抗体であ
る。本発明のヒトモノクローナル抗体のグロブリンタイ
プは、ＭＣＰ−１との結合性を有する限り特に制限され
ないが、例えばＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ、Ｉｇ
Ｄ等が挙げられる。本発明のヒトモノクローナル抗体
は、ヒトモノクローナル抗体を産生する細胞株から得ら
れるものの他に、酵素消化や遺伝子組み換え等により作
製したフラグメント、他の蛋白質あるいは因子との融合
体蛋白質であって、ヒトＭＣＰ−１との結合を有する限
り本発明の範疇である。また、ヒトＭＣＰ−１との結合
性を損なわない限り、遺伝子組み換えによる改変も可能
である。BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention
It is a human monoclonal antibody derived from human lymphocytes. The globulin type of the human monoclonal antibody of the present invention is not particularly limited as long as it has a binding property to MCP-1, but for example, IgG, IgM, IgA, IgE, Ig
D etc. are mentioned. The human monoclonal antibody of the present invention includes, in addition to those obtained from cell lines producing human monoclonal antibodies, fragments produced by enzymatic digestion, gene recombination, etc., and fusion proteins with other proteins or factors. As long as it has a bond with -1, it is within the scope of the present invention. Further, modification by gene recombination is also possible as long as the binding property to human MCP-1 is not impaired.
【０００９】本発明のヒトモノクローナル抗体を産生す
る細胞株は、抗ＭＣＰ−１ヒトモノクローナル抗体を産
生する限り特に限定はないが、抗ヒトＭＣＰ−１抗体を
産生するヒトリンパ球をエプスタイン・バー・ウイルス
（ＥＢＶ）で形質転換して得ることができ、更にこの形
質転換細胞とヒトミエローマ細胞とを細胞融合したハイ
ブリドーマとして得ることができる。また、抗ヒトＭＣ
Ｐ−１抗体を産生するヒトリンパ球とヒトミエローマ細
胞とを細胞融合したハイブリドーマとして得ることもで
きる。さらに、ヒトリンパ球から抗体遺伝子を取り出し
て、これを適当なベクターに組み込むことにより産生細
胞を作製しても良い。このような抗ＭＣＰ−１ヒトモノ
クローナル抗体産生細胞株の例としては、例えばハイブ
リドーマ株ＭＡ０１（ＦＥＲＭ Ｐ−１５０５２）があ
げられる。The cell line producing the human monoclonal antibody of the present invention is not particularly limited as long as it produces the anti-MCP-1 human monoclonal antibody, but human lymphocytes producing the anti-human MCP-1 antibody are Epstein-Barr virus. It can be obtained by transforming with (EBV), and further can be obtained as a hybridoma in which this transformed cell and human myeloma cell are fused. Also, anti-human MC
It can also be obtained as a hybridoma in which human lymphocytes producing the P-1 antibody and human myeloma cells are cell-fused. Further, a production cell may be prepared by taking out an antibody gene from human lymphocytes and incorporating it into an appropriate vector. An example of such an anti-MCP-1 human monoclonal antibody-producing cell line is, for example, hybridoma strain MA01 (FERM P-15052).
【００１０】本発明のヒトモノクローナル抗体を産生す
る細胞を得るための、抗ヒトＭＣＰ−１抗体を産生する
ヒトリンパ球は、ヒト末梢血やリンパ節等から取得でき
る。ヒト生体内に存在し、ヒトＭＣＰ−１と結合できる
抗体を作り得るリンパ球であれば本発明に用いることが
できる。通常、マウスやウサギ等でモノクローナル抗体
やポリクローナル抗体を取得する際に行われるＭＣＰ−
１の免疫は、必要がない。Human lymphocytes producing anti-human MCP-1 antibody for obtaining cells producing the human monoclonal antibody of the present invention can be obtained from human peripheral blood or lymph nodes. Any lymphocyte existing in the human body and capable of producing an antibody capable of binding to human MCP-1 can be used in the present invention. MCP- which is usually performed when obtaining monoclonal or polyclonal antibodies in mice, rabbits, etc.
Immune 1 is not necessary.
【００１１】本発明のヒトモノクローナル抗体の調製
は、上記の抗ＭＣＰ−１ヒトモノクローナル抗体を産生
する細胞株を各種公知の方法を用いて培養・精製すれば
よく、細胞の増殖及びＭＣＰ−１ヒトモノクローナル抗
体の産生を阻害しないものであれば特に制限はない。The human monoclonal antibody of the present invention can be prepared by culturing and purifying the above-mentioned cell line producing the anti-MCP-1 human monoclonal antibody by various known methods, for the purpose of cell growth and MCP-1 human. There is no particular limitation as long as it does not inhibit the production of the monoclonal antibody.
【００１２】本発明の抗ＭＣＰ−１ヒトモノクローナル
抗体を含有する薬剤は、単球及びマクロファージの関与
する炎症反応の関わる疾患、例えば特発性肺繊維腫、子
球体腎炎、動脈硬化等の予防や治療に有用となることが
期待される。The drug containing the anti-MCP-1 human monoclonal antibody of the present invention is used for the prevention and treatment of diseases associated with inflammatory reaction involving monocytes and macrophages, such as idiopathic pulmonary fibroma, follicular nephritis, arteriosclerosis and the like. It is expected to be useful for.
【００１３】[0013]
【実施例】以下実施例を示して本発明を説明するが、本
発明はこれに限られるものではない。 実施例１ 抗ＭＣＰ−１ヒトモノクローナル抗体の作製 ａ）ＥＢＶ形質転換 健常人より末梢血２０ｍｌを採取し、フィコール分離液
（大日本製薬）により血球分離を行い、ヒト単核球画分
を得た。これをＲＰＭＩ−１６４０培地（日水製薬）で
２度洗浄し、ヒト単核球３×１０7 個を調製した。遠心
分離（１，６００ｒｐｍ、６分間）によりペレットを
得、あらかじめ３７℃に保温したＢ９５−８細胞の培養
上清（Ｂ９５−８細胞を、２０％牛胎児血清（ＦＣＳ）
を含むＲＰＭＩ−１６４０培地で培養したときの培養上
清）３０ｍｌを加えて再懸濁した。３７℃の炭酸ガスイ
ンキュベーターに移し、９０分間保温してＥＢＶを感染
させた。遠心分離（１，６００ｒｐｍ、６分間）により
ペレットを得、該ペレットを２０％ＦＣＳを含むＲＰＭ
Ｉ−１６４０培地６０ｍｌに懸濁し、懸濁液を９６穴プ
レート（Corning）６枚に分注した。３７℃の炭酸ガス
インキュベーターに移し、３〜５日毎に半量ずつ培地交
換を行いながら培養した。EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited thereto. Example 1 Preparation of anti-MCP-1 human monoclonal antibody a) EBV transformation 20 ml of peripheral blood was collected from a healthy person and blood cells were separated with a Ficoll separation solution (Dainippon Pharmaceutical Co., Ltd.) to obtain a human mononuclear cell fraction. . This was washed twice with RPMI-1640 medium (Nissui Pharmaceutical) to prepare 3 × 10 7 human mononuclear cells. A pellet was obtained by centrifugation (1,600 rpm, 6 minutes), and the culture supernatant of B95-8 cells (B95-8 cells preliminarily kept at 37 ° C. was added to 20% fetal calf serum (FCS)).
30 ml of a culture supernatant obtained by culturing in RPMI-1640 medium containing the above was added and resuspended. It was transferred to a carbon dioxide gas incubator at 37 ° C. and kept warm for 90 minutes to infect EBV. A pellet was obtained by centrifugation (1,600 rpm, 6 minutes), and the pellet was added to RPM containing 20% FCS.
The suspension was suspended in 60 ml of I-1640 medium, and the suspension was dispensed into six 96-well plates (Corning). The cells were transferred to a carbon dioxide gas incubator at 37 ° C., and cultured while exchanging the medium by half every 3 to 5 days.
【００１４】ｂ）抗体産生の確認 上記ａ）の培養から２０日後にコロニー形成の確認され
たウエルについて、培養上清中の抗体活性をヒトＭＣＰ
−１を抗原として用いたドット・イムノバインディング
アッセイ（ＤＩＢＡ）法により測定した。リコンビナン
ト・ヒトＭＣＰ−１（PeproTech）を５０μｇ／ｍｌ濃
度に水に溶解した。これをグリッド付のニトロセルロー
ス膜（トーヨー）に０．２μｌずつスポットした後風乾
し、固定化抗原とした。固定化抗原を、ブロック液（１
０％ＦＣＳを含む生理的塩濃度のトリス塩酸緩衝液（Ｔ
ＢＳ））でブロッキングした。サンプルとなる培養液５
０μｌを９６穴Ｕ底プレートに取り、これにブロッキン
グした固定化抗原を入れ、室温で２時間振とうした。Ｔ
ＢＳでウエルを洗浄後、パーオキシダーゼ標識抗ヒトイ
ムノグロブリン抗体（DAKO）をブロック液で５００倍に
希釈してウエルあたり５０μｌずつ添加し、室温で２時
間振とうした。ウエルをＴＢＳで洗浄後、コニカイムノ
ステイン（コニカ）により染色し、発色を肉眼で確認し
た。B) Confirmation of antibody production Regarding the wells in which colony formation was confirmed 20 days after the culture in the above a), the antibody activity in the culture supernatant was measured by human MCP
It was measured by the dot immunobinding assay (DIBA) method using -1 as an antigen. Recombinant human MCP-1 (PeproTech) was dissolved in water to a concentration of 50 μg / ml. This was spotted on a nitrocellulose membrane (Toyo) with a grid in an amount of 0.2 μl each and then air-dried to obtain an immobilized antigen. Block the immobilized antigen with blocking solution (1
Tris-hydrochloric acid buffer solution (T containing 0% FCS at physiological salt concentration)
BS)). Sample culture fluid 5
0 μl was transferred to a 96-well U-bottom plate, the blocked immobilized antigen was added thereto, and the mixture was shaken at room temperature for 2 hours. T
After washing the wells with BS, peroxidase-labeled anti-human immunoglobulin antibody (DAKO) was diluted 500 times with the block solution, 50 μl was added to each well, and the mixture was shaken at room temperature for 2 hours. After the wells were washed with TBS, they were stained with Konica Immunostain (Konica), and the color development was visually confirmed.
【００１５】ｃ）細胞融合 上記ｂ）において抗体活性の確認されたウエルの細胞
を、６穴プレートまで拡大培養した。２×１０6 個の細
胞と、あらかじめ２０％ＦＣＳを含むＩＭＤＭ培地で培
養したＳＨＭ−Ｄ３３（ＡＴＣＣより入手）１×１０6
個を遠心チューブに集め、遠心分離（１，６００ｒｐ
ｍ、６分間）によりペレットを得た。このペレットを細
胞融合用培地（０．２５Ｍマンニトール、０．１ｍＭ塩
化カルシウム、０．１ｍＭ塩化マグネシウム、０．２ｍ
Ｍトリス塩酸緩衝液（ｐＨ７．２））で２度洗浄して再
びペレットを得た後、ペレットを細胞融合用培地０．４
ｍｌに再懸濁し、電気融合法（融合装置：島津ＳＳＨ−
１、融合チャンバー：島津ＦＴＣ−０２）により細胞融
合を行った。融合条件は、交流高周波電界：１ＭＨｚ、
４０Ｖ、１０秒、直流パルス電界：２．３ｋＶ／ｃｍ、
４０μｓ、１０秒。融合後の細胞は、ＨＡＴ（三光純
薬）と１μＭウアバイン、２０％ＦＣＳ、５μｇ／ｍｌ
ヒト・トランスフェリン、８μｇ／ｍｌウシ・インスリ
ン、５０ｍＭ２−メルカプトエタノールを含むＩＭＤＭ
培地に懸濁し、９６穴プレートに播種した。３７℃炭酸
ガスインキュベーターに移し、３〜５日毎に半量ずつ培
地交換を行いながら培養した。１４〜２０日後、コロニ
ー形成の確認されたウエルについて、培養上清中の抗体
活性を上記ｂ）に記載のＤＩＢＡ法で確認した。活性の
確認できた細胞については限界希釈法によるクローニン
グを３度行い、抗ＭＣＰ−１ヒトモノクローナル抗体産
生ハイブリドーマ株を得た。ハイブリドーマ株のうち１
つをＭＡ０１として取得した。このハイブリドーマ株Ｍ
Ａ０１は、茨城県つくば市東１丁目１番３号の通商産業
省工業技術院生命工学工業技術研究所に寄託番号ＦＥＲ
Ｍ Ｐ−１５０５２として寄託されている。C) Cell fusion The cells in the well whose antibody activity was confirmed in b) above were expanded and cultured up to a 6-well plate. 2 × 10 6 cells and SHM-D33 (obtained from ATCC) 1 × 10 6 cultured in IMDM medium containing 20% FCS in advance
Collect them in a centrifuge tube and centrifuge (1,600 rp
m, 6 minutes) to obtain pellets. This pellet was used as a medium for cell fusion (0.25 M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium chloride, 0.2 m
M Tris-HCl buffer (pH 7.2)) was washed twice to obtain a pellet again, and the pellet was used as a cell fusion medium 0.4.
Resuspend in ml, electrofusion method (fusion device: Shimadzu SSH-
1. Fusion chamber: Cell fusion was performed by Shimadzu FTC-02). Fusion condition is AC high frequency electric field: 1MHz,
40 V, 10 seconds, DC pulse electric field: 2.3 kV / cm,
40 μs, 10 seconds. The fused cells were HAT (Sanko Junyaku) and 1 μM ouabain, 20% FCS, 5 μg / ml.
IMDM containing human transferrin, 8 μg / ml bovine insulin, 50 mM 2-mercaptoethanol
The cells were suspended in a medium and seeded in a 96-well plate. The cells were transferred to a 37 ° C. carbon dioxide incubator and cultured while replacing the medium by half every 3 to 5 days. After 14 to 20 days, antibody activity in the culture supernatant of the well in which colony formation was confirmed was confirmed by the DIBA method described in b) above. The cells whose activity was confirmed were cloned three times by the limiting dilution method to obtain an anti-MCP-1 human monoclonal antibody-producing hybridoma strain. 1 of the hybridoma strains
One was obtained as MA01. This hybridoma strain M
A01 was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, 1-3-1 Higashi, Tsukuba, Ibaraki, Japan.
Deposited as MP-15052.
【００１６】ｄ）抗体の精製 ハイブリドーマ株ＭＡ０１を無血清培地ＳＦＭ−１０１
（日水製薬）で培養し、培養上清３５０ｍｌを取得し
た。この培養上清に対し５０％飽和硫酸アンモニウム分
画を行った後、生理的塩濃度のリン酸緩衝液（ＰＢＳ）
に再溶解した。これを０．２μｍのフィルターで濾過
後、Ｓｕｐｅｒｄｅｘ２００カラム（ファルマシア）を
用いて精製した。得られた抗体画分は、限外濾過膜（３
０ｋ分画、フジフィルター）により濃縮し、無菌的にフ
ィルター濾過（０．２μｍ）後小分けし、使用時まで−
８０℃に凍結保存した。D) Purification of antibody Hybridoma strain MA01 was added to serum-free medium SFM-101.
(Nissui Pharmaceutical), and 350 ml of the culture supernatant was obtained. The culture supernatant was subjected to 50% saturated ammonium sulfate fractionation, and then phosphate buffer (PBS) having a physiological salt concentration.
Redissolved in. This was filtered through a 0.2 μm filter and then purified using Superdex 200 column (Pharmacia). The obtained antibody fraction was passed through an ultrafiltration membrane (3
Concentrate with 0k fraction, Fuji filter), filter aseptically (0.2 μm), and divide into small portions.
It was stored frozen at 80 ° C.
【００１７】実施例２ サンドイッチＥＬＩＳＡ法によ
る液相でのヒトＭＣＰ−１抗原との結合性の確認 抗ＭＣＰ−１ウサギポリクローナル抗体（PeproTech）
を０．１Ｍ炭酸緩衝液（ｐＨ９．６）で４μｇ／ｍｌに
希釈し、希釈液を９６穴プレート（Greiner）にウエル
あたり５０μｌずつ分注した。プレートを４℃で一晩放
置し、抗体固定化プレートとした。このプレートを洗浄
液（０．０２％アジ化ナトリウム、０．０５％Ｔｗｅｅ
ｎ２０を含む１０ｍＭトリス塩酸緩衝液（ｐＨ８．
０））で洗浄後、ブロック液（０．５％ウシ血清アルブ
ミンを含むＰＢＳ）でブロッキングした。一方、別の９
６穴Ｕ底プレート内で、ハイブリドーマ株ＭＡ０１より
精製した抗ＭＣＰ−１ヒトモノクローナル抗体２０及び
５μｇ／ｍｌとヒトＭＣＰ−１抗原（PeproTech）０〜
１００ｎｇ／ｍｌを混合し、室温で２時間振とうした。
反応物は、ブロッキングの終了した抗体固定化プレート
に移し、室温で２時間振とうした。洗浄液で洗浄後、ブ
ロック液で５００倍に希釈したアルカリホスファターゼ
標識抗ヒトＩｇＭ抗体（BIO SOURCE）をウエルあたり１
００μｌずつ分注し、室温で２時間振とうした。洗浄液
で洗浄後、基質溶液（０．６％ｐ−ニトロフェニルリン
酸、０．５ｍＭ塩化マグネシウムを含む９．６％ジエタ
ノールアミン緩衝液（ｐＨ９．６））５０μｌを加え、
室温で３０分振とうした。反応液に３Ｎ水酸化ナトリウ
ム５０μｌを加えて反応を停止した後、４０５ｎｍにお
ける吸光度をイムノリーダー（日本インターメッド）に
より測定した。その結果、図１（図１）に示す通り、ハ
イブリドーマ株ＭＡ０１より調製した抗体はヒトＭＣＰ
−１抗原量に依存して反応が認められた。同様の反応
を、反応特異性の異なるヒトＩｇＭ抗体（Rockland）を
抗ＭＣＰ−１ヒトモノクローナル抗体の代わりに用いた
場合、及びヒトモノクローナル抗体を用いなかった場合
（Ｃｏｎｔｒｏｌ）は、ヒトＭＣＰ−１と全く反応が認
められなかった。このことより、ハイブリドーマ株ＭＡ
０１の産生する抗ＭＣＰ−１ヒトモノクローナル抗体
は、ヒトＭＣＰ−１を液相で認識し、結合できることが
確認できた。Example 2 Confirmation of Binding to Human MCP-1 Antigen in Liquid Phase by Sandwich ELISA Anti-MCP-1 Rabbit Polyclonal Antibody (PeproTech)
Was diluted to 4 μg / ml with 0.1 M carbonate buffer (pH 9.6), and the diluted solution was dispensed into a 96-well plate (Greiner) at 50 μl per well. The plate was left overnight at 4 ° C. to prepare an antibody-immobilized plate. Wash the plate with wash solution (0.02% sodium azide, 0.05% Tween).
10 mM Tris-HCl buffer (pH 8.
After washing with 0)), blocking was performed with a blocking solution (PBS containing 0.5% bovine serum albumin). On the other hand, another 9
Anti-MCP-1 human monoclonal antibody 20 and 5 μg / ml purified from hybridoma strain MA01 and human MCP-1 antigen (PeproTech) 0 to 6 in a 6-well U-bottom plate
100 ng / ml was mixed and shaken at room temperature for 2 hours.
The reaction product was transferred to a blocking antibody-immobilized plate and shaken at room temperature for 2 hours. After washing with wash solution, add 1-fold per well of alkaline phosphatase-labeled anti-human IgM antibody (BIO SOURCE) diluted with blocking solution 500 times.
It was dispensed by 00 μl each and shaken at room temperature for 2 hours. After washing with a washing solution, 50 μl of a substrate solution (9.6% diethanolamine buffer solution (pH 9.6) containing 0.6% p-nitrophenyl phosphate and 0.5 mM magnesium chloride) was added,
Shake for 30 minutes at room temperature. After 50 μl of 3N sodium hydroxide was added to the reaction solution to stop the reaction, the absorbance at 405 nm was measured by an immunoreader (Nippon Intermed). As a result, as shown in FIG. 1 (FIG. 1), the antibody prepared from the hybridoma strain MA01 was found to be human MCP.
A reaction was observed depending on the amount of the -1 antigen. Similar reactions were carried out with human MCP-1 when human IgM antibody (Rockland) with different reaction specificity was used instead of anti-MCP-1 human monoclonal antibody and when no human monoclonal antibody was used (Control). No reaction was observed. From this, the hybridoma strain MA
It was confirmed that the anti-MCP-1 human monoclonal antibody produced by 01 can recognize and bind human MCP-1 in the liquid phase.
【００１８】[0018]
【発明の効果】本発明により、ヒトＭＣＰ−１に対して
結合するヒトモノクローナル抗体が提供される。これ
は、単球やマクロファージの関与する炎症反応に深く関
わっていると考えられているヒトＭＣＰ−１に結合する
ことができるため、この様な炎症反応を伴う疾患に対す
る予防、治療効果が期待できる。さらに、これまで報告
されているＭＣＰ−１の特異的阻害剤である抗ＭＣＰ−
１マウスモノクローナル抗体と比べ、ヒトに投与する際
に抗原性の問題がないヒトモノクローナル抗体が示され
た。この様な特性から、本発明により提供される抗ＭＣ
Ｐ−１ヒトモノクローナル抗体を含有する薬剤は、ヒト
に対する有用な薬剤となることが期待される。INDUSTRIAL APPLICABILITY The present invention provides a human monoclonal antibody that binds to human MCP-1. Since it can bind to human MCP-1 which is thought to be deeply involved in the inflammatory reaction involving monocytes and macrophages, it can be expected to have preventive and therapeutic effects on diseases associated with such inflammatory reaction. . Furthermore, anti-MCP- which is a specific inhibitor of MCP-1 reported so far.
Human monoclonal antibodies were shown to have no antigenicity problems when administered to humans as compared to 1 mouse monoclonal antibody. From such characteristics, the anti-MC provided by the present invention
A drug containing the P-1 human monoclonal antibody is expected to be a useful drug for humans.
【図１】抗ＭＣＰ−１ヒトモノクローナル抗体の液相に
おけるヒトＭＣＰ−１抗原に対する結合性を示す図であ
る。図中、ＭＡ０１はハイブリドーマ株ＭＡ０１より精
製した抗ＭＣＰ−１ヒトモノクローナル抗体を、ＩｇＭ
はヒトＩｇＭ抗体（Rockland）であることを示してい
る。FIG. 1 is a diagram showing the binding properties of anti-MCP-1 human monoclonal antibody to human MCP-1 antigen in a liquid phase. In the figure, MA01 is an anti-MCP-1 human monoclonal antibody purified from the hybridoma strain MA01, which is IgM.
Indicates a human IgM antibody (Rockland).
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 //(Ｃ１２Ｐ 21/08 Ｃ１２Ｒ 1:91） (72)発明者 里澤 昇 千葉県茂原市東郷1144番地 三井東圧化学 株式会社内 (72)発明者 渡邊 綾子 千葉県茂原市東郷1900番地の１ 三井東圧 化学株式会社内 (72)発明者 横松 知子 千葉県茂原市東郷1144番地 三井東圧化学 株式会社内─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 6 Identification number Internal reference number FI technical display location // (C12P 21/08 C12R 1:91) (72) Inventor Noboru Satozawa Togo, Mobara-shi, Chiba 1144 Mitsui Toatsu Chemical Co., Ltd. (72) Inventor Ayako Watanabe 1900 Togo, Mobara-shi, Chiba Prefecture 1900 Mitsui Toatsu Chemical Co., Ltd. (72) Inventor Tomoko Yokomatsu 1144 Togo, Mobara City Chiba Prefecture Mitsui Toatsu Chemical Within the corporation
Claims (5)
１）に結合するヒトモノクローナル抗体。1. A human monocyte chemotactic activator (human MCP-
A human monoclonal antibody that binds to 1).
を産生する細胞株。2. A cell line producing the human monoclonal antibody according to claim 1.
ブリドーマ株ＦＥＲＭ Ｐ−１５０５２。3. A hybridoma strain FERM P-15052 which produces a human monoclonal antibody.
５２の産生するヒトモノクローナル抗体。4. A hybridoma strain FERM P-150.
A human monoclonal antibody produced by 52.
ナル抗体を含有することを特徴とする医薬組成物。5. A pharmaceutical composition comprising the human monoclonal antibody according to claim 1 or 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7221794A JPH0967399A (en) | 1995-08-30 | 1995-08-30 | Anti-mcp-1 human monoclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7221794A JPH0967399A (en) | 1995-08-30 | 1995-08-30 | Anti-mcp-1 human monoclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0967399A true JPH0967399A (en) | 1997-03-11 |
Family
ID=16772309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7221794A Pending JPH0967399A (en) | 1995-08-30 | 1995-08-30 | Anti-mcp-1 human monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0967399A (en) |
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|---|---|---|---|---|
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| WO2004024921A1 (en) | 2002-09-12 | 2004-03-25 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Human antihuman mcp-1 antibody and antibody fragment thereof |
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1995
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