JP5473960B2 - Egfレセプターシグナル伝達の調節のためのtaceまたはアンフィレグリンの阻害 - Google Patents
Egfレセプターシグナル伝達の調節のためのtaceまたはアンフィレグリンの阻害 Download PDFInfo
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- JP5473960B2 JP5473960B2 JP2011025668A JP2011025668A JP5473960B2 JP 5473960 B2 JP5473960 B2 JP 5473960B2 JP 2011025668 A JP2011025668 A JP 2011025668A JP 2011025668 A JP2011025668 A JP 2011025668A JP 5473960 B2 JP5473960 B2 JP 5473960B2
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Description
1.1 細胞培養、プラスミドおよびレトロウイルストランスフェクション
すべての細胞系(American Type Culture Collection, Manassas, VA)を、取り扱い説明書にしたがって、規定通りに増殖させた。HEK−293細胞のトランスフェクションを、従来知られているように、リン酸カルシウム共沈により実施した(1)。抗−アンフィレグリン(AR)、抗−HB−EGF中和抗体(R&D Systems, Minneapolis, MN、PTX、ヘパリン(Sigma, St.Louis, MO)マリマステート(BB2516、Sugen Inc., South San Francisco, CA)、バチマステート(BB94, British Biotech, Oxford, UK)を血清枯渇細胞に、それぞれの増殖因子添加前に添加した。
細胞を溶解し、かつタンパク質を従来の方法で免疫沈降した(13)。ウエスタンブロットを、標準的な方法で実施した。ヒトEGFR(108.1)およびSHC(1)ならびにGST−Grb2融合タンパク質(5)に対する抗体を、予め特徴付けした。ホスホチロシンを、4G10モノクローナル抗体(UBI, Lake Placid, NY)を用いて検出した。ポリクローナル抗−ホスホ−p44/p42(Thr202/Tyr204)MAPK抗体および抗−ホスホ−Akt(Ser473)抗体を、New England Biolabs(Beverly, MA)から入手した。ポリクローナル抗−Akt1/2および抗−ERK2抗体は、Santa Cruz Biotechnology(Santa Curz, CA)、抗−TACE−抗体はChemicon(Harrow, UK)からのものである。
ACS分析を従来の方法で実施した(1)。細胞を、HB−EGF、AR(R&D Systems)またはTGFa(Oncogene, Boston, MA)に対するエクトドメイン−特異的抗体を用いて染色した。PBSで洗浄した後に、細胞をFITC−結合二次抗体を用いてインキュベートし、かつBacton Dickinson FACScalibur フローサイトメーター上で分析した。
内在遺伝子のターゲッティングのための21−ヌクレオチドsiRNAデュープレックス(Dharmacon Research, Lafayette, CO, USA)のトランスフェクションは、標的化された内在性遺伝子を、オリゴフェクタミン(Invitrogen)および4.2μg siRNAデュープレックス/6ウェルプレートを用いて、従来方法にしたがっておこなった(30)。トランスフェクトしたSCC−9細胞を、血清−枯渇にし、かつトランスフェクションの4日後にアッセイをおこなった。サイレンシングにおける標的遺伝子中の最も高い効率は、重要な遺伝子の種々の領域を標的化するsiRNAデュープレックスの混合物を用いて得られた。使用されたsiRNAの配列は以下のとおりであった。
3H−チミジン取り込みアッセイ(5)のために、SCC−15細胞を、12ウェルプレート中に3x104細胞/ウェルで播種した。48時間に亘っての血清枯渇後に、公知方法のように細胞をプレインキュベートし、かつ刺激した。18時間後に細胞を、3H−チミジン(1μCi/ml)で、4時間に亘ってパルスラベルし、かつチミジン取り込みを、三塩化酢酸沈降によって測定し、その後に、液体シンチレーションカウンターを用いて測定した。
HNSCC細胞中での、GPCR−誘導トランス活性化シグナルは、広汎性メタロプロテアーゼインヒビター、たとえばバチマステート(BB94)(13)およびマリマステート(BB2516;図1A)に対して敏感である。EGFRシグナルトランス活性化のリガンド依存性機序との一貫性に関しては、我々は、モノクローナル抗−EGFR抗体ICR−3Rが、この場合、EGF−様の増殖因子が、レセプターの細胞外領域と結合するのを防ぎ(14)、SCC−9細胞中で、GPCR−およびEGF−誘導EGFRチロシンリン酸化を排除することが見出された(図1A)。これとは対照的に、ICR−3Rは、ペルバナデートによって、多くの細胞間タンパク質のチロシンリン酸化含量を増加させる、潜在的チロシンホスファターゼインヒビター(15)によって誘導された応答と干渉することはなかった。従来の報告では、GPCR−誘導EGFRチロシンリン酸化が、HB−EGF(1−3)のタンパク質分解を必要とすることが証明され、これによって、HB−EGFまたは他のEGF−様増殖因子が、ERGFRトランス活性化経路中で、頭部および頸部癌細胞中に含まれるのではないかということが示唆された。cDNA顕微鏡分析よって、HB−EGF、TGFαおよびAR mRNAsの、SCC−4、SCC−9、SCC−15およびSCC−25細胞中での発現が見出された(データは示されていない)。さらに、これらリガンドの発現および細胞表面の局在は、フローサイトメトリーによって、エクトドメイン特異的抗体を用いて確認した(図1B、SCC−9に関する典型的データ)。驚くべきことに、頭部および頸部癌細胞のLPA(10μM)またはホルボールエステルTPA(1mM)での処理は、この場合、これらは、発散(shedding)の一般的誘導物質として作用し、内因性proARの細胞表面含量を減少させる(図1C)。しかしながら、これらの細胞性内容物において、さらにLPAは、proTGFαまたはproHB−EGFのタンパク質分解を誘発しないが、その一方でTPAでの刺激は、双方のEGF−様増殖因子前駆体(データは示していない)とのエクトドメイン分断を生じる。これらの図は、LPA刺激が、HNSCC中のproARの発散を誘発する。さらに、バチマステート(10μM)は完全に、proARのLPA−誘発エクトドメイン分解を回避し、これよって、proAR発散のためのメタロプロテアーゼ活性要求性が確認された。Gi/Oファミリーの優性な百日咳毒(PTX)−敏感性Gタンパク質が、LPA−誘導EGFRチロシンリン酸化の介在物質であり(図1A)、PTX(100ng/mL)が部分的に、SCC−9細胞の細胞表面上で、proARシェーディングを阻害する(図1C)。
試験結果の増加した量は、EGFRが、異なるGPCRシグナルの中心的インテグレーターとしての機能し、それによって下流の経路に集められるといった概念を支持するものである(4,6,12)。存在するデータは、ヒト癌細胞での望ましくないEGFRトランス活性化機序を支持し、かつGPCRシグナル中のTACEに関する新規生物学的機能を同定する。これらの結果は、TACEのGPCR−誘導活性が、遊離ARの量の増加に寄与しうるといった生物学的因果関係を有することを証明したものである。EGFRのHB−EGF−依存型トランス活性化が、肺上皮細胞(3)およびCOS−7細胞(27)中のADAM10によってか、あるいは、心筋細胞(2)中のADAM12によって介在されるといった他の機序が記載されている。しかしながら、膜貫通proARが、GPCR刺激に応じて分解され、さらにARが、GPCRアゴニストにより、真の癌細胞特性を介在するための機能的等価物であることが初めて示された。TACE−依存性AR放出は、GPCR−誘導EGFR刺激、ERK/MAPK経路の活性、Akt/PKBのリン酸化、細胞増殖および遊走の誘発に対して不可欠であることを示した。
2.1モノクローナル抗体の製造
モノクローナル抗体(Mabs)は、ヒトTACE(ADAM17)のメタロプロテアーゼドメインに対して製造された。組換えタンパク質は、BALB/cマウスの免疫化に関して使用され(J.H. Peters, H. Baumgarten and M. Schulze, Monoclonale Antikoerper-Herstellung und Charakterisierung, Springer- Verlag, 1985, Berlin Heidelberg New York Tokio)、モノクローナル抗体の精製は、T−GelTM Absorbentを用いておこなった(Pierce, Rockford, IL, USA)。
TACEのメタロプロテアーゼ−ドメインを認識する8個のモノクローナル抗体を、ELISAによって同定された。これらの抗体は、ヘマグルチニンエピトープ(TACE−HA)でタグされた、TACEをコードする真核細胞性発現プラスミドで一時的にトランスフェクトされたHEK−293細胞のライセートを免疫沈降するために使用した(31)。モノクローナル抗体432−2、400−1、343−3および432−7は、特にTACEの成熟体を免疫沈降するものであるのに対し、α−HA抗体は、優先的にTACEの前駆体を免疫沈降した(図5)。
Claims (3)
- アンフィレグリン特異的インヒビターとしてのアンフィレグリンに対する中和抗体または少なくとも1個のアンフィレグリンに対する結合部位を含む前記中和抗体フラグメントを含有する、Gタンパク質−結合レセプターの刺激により誘発されるEGFRチロシンリン酸化によって引き起こされるか、あるいはこれに関連する疾病の治療のための医薬組成物であって、その際、疾病が扁平上皮細胞癌である医薬組成物。
- 更に、TACE/ADAM17の特異的なインヒビターとしてのTACEに対するアンチセンスまたはsiRNAが含有される、請求項1に記載の医薬組成物。
- 付加的に、製薬学的に許容可能なキャリア、希釈剤および/またはアジュバンドを含む、請求項1又は2に記載の医薬組成物。
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WO2007016597A2 (en) * | 2005-07-29 | 2007-02-08 | The Regents Of The University Of California | Targeting tnf-alpha converting enzyme (tace)-dependent growth factor shedding in cancer therapy |
JP2007135581A (ja) * | 2005-10-20 | 2007-06-07 | Japan Science & Technology Agency | 自己免疫性血小板減少性紫斑病(itp)患者の血液細胞特異的遺伝子群 |
US20100111965A1 (en) * | 2006-10-11 | 2010-05-06 | Fusion Antibodies Limited | Combination therapy |
WO2008156707A1 (en) * | 2007-06-15 | 2008-12-24 | University Of Florida Research Foundation | Therapeutic compounds |
US20090309617A1 (en) * | 2007-08-24 | 2009-12-17 | Ye Fang | Biosensor antibody functional mapping |
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WO2010137012A1 (en) | 2009-05-25 | 2010-12-02 | Ramot At Tel-Aviv University Ltd. | Peptide therapy for amphiregulin mediated diseases |
AU2012213240A1 (en) * | 2011-02-01 | 2013-08-15 | Cancer Research Technology Limited | Anti-TACE antibody molecules and their uses |
CA2887069A1 (en) * | 2012-10-05 | 2014-04-10 | Bioneer Corporation | Amphiregulin-specific double-helical oligo-rna, double-helical oligo-rna structure comprising double-helical oligo-rna, and composition for preventing or treating respiratory diseases containing same |
JPWO2014157229A1 (ja) * | 2013-03-28 | 2017-02-16 | 国立大学法人東北大学 | Taceペプチドエピトープ、抗ヒトtaceタンパク質抗体及び当該抗体を産生するハイブリドーマ |
CN106459974A (zh) | 2014-04-04 | 2017-02-22 | 柏业公司 | 新颖的双链寡rna和包含它的用于预防或治疗纤维化或呼吸系统疾病的药物组合物 |
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