JP4583763B2 - 切断型24kDa塩基性線維芽細胞成長因子 - Google Patents
切断型24kDa塩基性線維芽細胞成長因子 Download PDFInfo
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Description
ジェン(分裂促進因子)の存在下においてさえ、遊走を内皮細胞では50%、乳癌MCF−7細胞では70%以上も抑制する。我々は、アミノ末端(アミノ末端55アミノ酸、「ATE」)に特異的な抗体または24kDa FGF−2の18kDa領域に対する抗体を用いて、ATEに対する遊走抑制と24kDa FGF−2の18kDaドメインに対する成長促進場所を突きとめた。したがって、24kDa FGF−2は、18kDa FGF−2とは別に細胞行動を影響し、18kDa FGF−2には存在しないATE領域にこの相違の原因があると結論した。
本発明は、細胞浸潤及び/または血管新生を抑制するに十分な投与量のオリゴペプチド、化学誘導体またはペプチド擬態を含む組成物を被験体に投与することによって、好ましくない制御不能なこの浸潤及び/または血管新生によって媒介される疾患とプロセスを治療するための方法並びに組成物を提供する。本発明は、特に腫瘍の成長、網膜症に起因する血管発生、あるいは血管形成に拠るその他の疾患の治療と抑制に有効である。血管新生前の転移腫瘍を有するヒトまたは被験体に対する本組成物の投与は、これらの腫瘍の成長または増大を防止する。
この組成物はタンパク質、変異体、または、ペプチド擬態または多量体ペプチド含む化学誘導体、並びに薬剤として許容される担体または賦形剤を含む。
、グリコシル化及びその他の置換基の付加(例えば、標識、血清半減期を増加する化合物(例えば、ポリエチレングリコール)等)を含む。
本発明者らは、塩基性線維芽細胞成長因子の24kDA、22.5kDA、並びに22kDa FGF−2型が培養細胞の遊走を抑制することを以前に測定した。
遊走及び成長アッセイ法 遊走アッセイ法では、MCF−7細胞または内皮細胞をトリプシンで収集、カウント、遠心分離してから、0.5mlのダルベッコ変法イーグル培地/0.5%ウシ血清アルブミン中で1 x 105 細胞になるように再懸濁した。細胞を、6.5mmトランスウェル(Costar)からなる2つのチェンバーを隔てる8.0μmポアサイズのポリカーボネート膜を含むボイデンチェンバーの上部ウェルに添加した。上部ウェルを、10ng/mlのIGF−2(Sigma)または10ng/mlのV
EGF(Sigma)を化学誘引物質として加えた、0.75mlのダルベッコ変法イーグル培地/0.5%ウシ血清アルブミンを含む下部チェンバー内に配置した。どちらのチェンバーも適切な濃度の24kDa FGF−2または切断型を含んだ。5%CO2中、37℃で4〜6時間インキュベートした後、上部膜表面にある非遊走細胞を綿棒で除去して、フィルタを横断してフィルタの下方表面上に拡がった拡がった細胞を固定してからDiff−Quik(Dade−Behring)で染色した。フィルタをスライドグラスにマウントして、4位相差顕微鏡写真/膜を100倍の倍率で撮影した。視野当たりの細胞数をコンタクトシートからカウントして、その結果を24kDa FGF−2を添加しなかった対照チェンバーと比較した。
マトリゲルプラグアッセイを用いる血管新生のインビボ評価 氷冷マトリゲル(500μL)(Collaborative Biomedical Products, Inc., Bedford, MA)にヘパリン(50μg/ml)、FGF−2(400ng/ml)、並びにATE+31を指示通りに混合した。マトリゲル混合物を、4〜8週齢のメス胸腺欠損Ncrヌードマウスの腹部正中線付近部位に、マウス1匹当たり3箇所、皮下注射した。注射部位は、各動物が陽性対照プラグ(FGF−2及びヘパリン)、陰性対照プラグ(ヘパリン+緩衝液)、並びに試験する処理を含むプラグ(FGF-2、ヘパリン、ATE +31).を受容するように選択した。全ての処理は3組で試験した。注射5日後に動物を頸椎脱臼で屠殺した。マウス皮膚は、腹部正中線に沿って剥離して、マトリゲルプラグを回収して、高分解能で直ちにスキャンした。次にプラグを水中に分散して、37℃で一晩インキュベートした。ヘモグロビン量は、Drabkin試薬(Sigma)を用いて測定した。
ATE+31の腫瘍成長に対する影響のインビボ評価 2x106 個のMatLyLu前立腺癌細胞を400nM ATE+31が存在する場合または存在しない場合に0.5mlのマトリゲル中に移植して、このゲルを4〜8週齢のメス胸腺欠損Ncrヌードマウスの腹部正中線付近部位の皮下に配置した。7日後にこのゲルを回収して、写真撮影並びに計量した。各動物に細胞もタンパク質も含まない、細胞のみ、そして細胞とタンパク質を含むマトリゲルを注射して、それぞれの相対重量を同一個体で比較した。
本発明の好ましい動物被験体は哺乳類である。本発明は、ヒト被験者の治療に特に有効である。用語「治療する」とは、被験体に、細胞遊走に対する抑制活性を有し、腫瘍発生及び血管新生の抑制を誘導する任意の切断型24kDa FGF−2を含む医薬品組成物を投与することを意味する、
本発明の医薬品組成物は、その切断型(複数でもよい)を薬剤として許容される賦形剤または担体と併用して、所期の目的を達成するための任意の手段によって投与することができる。投与量及びレジメンは、これらの特定の疾患のいずれかを治療する臨床分野の当業者によって容易に決定され得る。好ましい量は以下に説明する。
は吸入ルートを用いることができる。あるいは、または同時に、経口ルートで投与してもよい。投与量は、レシピエントの年齢、健康状態、及び体重、併用療法の種類、適応される場合は、処置の頻度、並びに所望する効果の本質に依存する。
ない。
用いて競合結合実験が行われた(図4)。標識24kDa FGF−2間の競合は、標識タンパク質の結合を用量依存性で低下させ、100倍過剰では 結合を81±3%低下させた。この濃度では、18kDa FGF−2もまた、24kDa FGF−2の結合を同様に減少した(79±5%)。しかしながら、ATE+31は、1000倍過剰濃度でも、125 I−24kDa FGF−2との結合に有意な影響はなく、24kDa FGF−2のアミノ末端部分内には主要なFGFR1結合部位が見当たらないことが示唆された。ATE+31がFGFR1制御によるシグナル伝達経路を活性化する能力を有するか否かを決定するため、リン酸化特異抗体を用いて、ERK1/2活性化に対するその影響を分析した(図5)。MCF−7細胞及び内皮細胞のどちらにおいても、24kDa FGF−2は、濃度4×10-11 Mでは、ERK1/2のリン酸化を促進し、この応答は3T3細胞でも生じる。しかし、同一モル濃度で、ATE+31は、これらの細胞においてERK1/2リン酸化には影響しなかった。濃度を4×10-10 Mまで増加してもERKリン酸化量に影響はなかった。
ATE+31が存在する場合は対照値の1.5倍のみであった。したがって、高濃度のATE+31による血管発生の純増加は、18kDa FGF−2のみを含むプラグによる増加の22%のみであった。
ネットワークを示した。血管密度の相違の他に、成長する血管の統合性がまたペプチドの存在によって影響された。高拡大率では、処理領域が血管の断片(矢印)を含むことを示す。これらの血管を通る血流のビデオ分析は、血流速度の減速を明らかにし、これは低分化な血管の形成に起因する影響であると考えられる。これらの相違は、15日目にはさらに拡大した。15日目までには、未処理動物の血管神経叢は極めて高密度となり、スフェロイド内のほとんどの空間を充満するが、処理動物はこの領域にほとんど血管が見られない。15日目に行った皮膚の組織学的評価は、ATE+31による広範な腫瘍成長抑制を示す。濃青色領域で表示される腫瘍を比較すると、その大きさが劇的に異なることが示される。15日後の処理腫瘍スフェロイドの平均サイズは、未処理の7.8%のみであった(1.3mm2 対16.8mm2 、 n=3)。
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Bikfalvi,A. 1994. Studies on FGF−2: nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin.(FGF−2における、核転座及び高分子量型の機能並びにヘパリン不在におけるレセプター結合に関する研究)Mol.Reprod.Dev. 39:102−105.
13. Piotrowicz,R.S., Martin,J.L., Dillmann, W.H., and Levin,E.G. 1997. The 27−kDa heat shock protein facilitates basic fibroblast growth factor release from endothelial cells.(27−kDa熱ショックタンパク質は、内皮細胞からの塩基性線維芽細胞成長因子の放出を促進する)J:Bio/.Chem. 272:7042−7047.
Claims (19)
- 配列番号5の核酸分子からなる単離核酸分子。
- 配列番号5の核酸分子を含み、抗血管新生、腫瘍抑制、及び抗遊走活性を有するが細胞増殖を促進しない、切断型24kDa塩基性繊維芽細胞成長因子(FGF−2)をコードする単離核酸分子。
- 前記核酸分子が検出可能な基に共有結合している、請求項1または2に記載の単離核酸分子。
- 前記検出可能基が放射性標識及び蛍光基から成る群から選択される、請求項3に記載の単離核酸分子。
- 前記核酸分子が1つ以上の発現制御エレメントに作動可能に連結されている、請求項1または2に記載の単離核酸分子。
- 前記核酸配列が1つ以上の欠失を含む、請求項1または2に記載の核酸分子。
- 請求項1または2に記載の核酸を含むベクター。
- 請求項1または2に記載のベクターを含み、それによって前記細胞が前記核酸を発現できる、組み換え型宿主細胞。
- 前記宿主が、原核及び真核宿主から成る群から選択される、請求項8に記載の宿主細胞。
- 配列番号2のポリペプチドからなる単離ポリペプチド。
- 配列番号2のポリペプチドを含み、抗血管新生、腫瘍抑制、及び抗遊走活性を有するが細胞増殖を促進しない、切断型24kDa塩基性繊維芽細胞成長因子(FGF−2)であ
る単離ポリペプチド。 - 請求項10または11に記載のポリペプチドを薬剤として有効量含む医薬品組成物。
- 前記医薬品組成物がさらに薬剤として許容される担体を1つ以上含む、請求項12に記載の医薬品組成物。
- 少なくとも二つの成分から成る融合タンパク質であって、前記融合タンパク質の一つの成分が請求項10または11に記載のポリペプチドである抗血管新生、腫瘍抑制、及び抗遊走活性を有するが細胞増殖を促進しない融合タンパク質。
- 一つの単量体が請求項10または11に記載のポリペプチドである、二量体。
- 少なくとも一つの単量体が請求項10または11に記載のポリペプチドである、多量体。
- 抗血管新生、腫瘍抑制、及び抗遊走活性を有するが細胞増殖を促進しない切断型24kDa塩基性繊維芽細胞成長因子(FGF−2)のポリペプチド産生の方法であって、前記方法が、
a)好適な栄養条件で、配列番号5の核酸分子で形質転換または形質移入された宿主細胞を生育すること、及び
b)前記核酸分子の発現によるポリペプチド産物を単離すること、を含む方法。 - 前記ポリペプチドが固形支持体またはキャリア上に固定化されている請求項10または11に記載のポリペプチド。
- 配列番号2のポリペプチドとは1つまたは2つのアミノ酸の保存的置換の分だけ異なるアミノ酸配列を有する単離ポリペプチドであって、抗血管新生、腫瘍抑制、及び抗遊走活性を有するが細胞増殖を促進しない、切断型24kDa塩基性繊維芽細胞成長因子(FGF−2)であるポリペプチド。
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PCT/US2003/010719 WO2003087318A2 (en) | 2002-04-08 | 2003-04-07 | Truncated 24 kda basic fibroblast growth factor |
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US7803772B2 (en) | 2010-09-28 |
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US7629146B2 (en) | 2009-12-08 |
US7432243B2 (en) | 2008-10-07 |
EP1492559A2 (en) | 2005-01-05 |
NZ536040A (en) | 2008-10-31 |
JP2005528895A (ja) | 2005-09-29 |
WO2003087318A2 (en) | 2003-10-23 |
US20100144632A1 (en) | 2010-06-10 |
AU2003223506A1 (en) | 2003-10-27 |
CA2480423C (en) | 2012-01-03 |
WO2003087318A3 (en) | 2004-02-19 |
AU2003223506B2 (en) | 2008-10-23 |
EP1492559A4 (en) | 2005-07-27 |
US20080051356A1 (en) | 2008-02-28 |
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