JP5149506B2 - 融合ポリペプチド、および抗血管腫瘍治療におけるその使用 - Google Patents
融合ポリペプチド、および抗血管腫瘍治療におけるその使用 Download PDFInfo
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- JP5149506B2 JP5149506B2 JP2006523616A JP2006523616A JP5149506B2 JP 5149506 B2 JP5149506 B2 JP 5149506B2 JP 2006523616 A JP2006523616 A JP 2006523616A JP 2006523616 A JP2006523616 A JP 2006523616A JP 5149506 B2 JP5149506 B2 JP 5149506B2
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Description
適切な新生血管新生は、進行性の腫瘍増殖に必須である(1(非特許文献1))。血管新生は、膨張性腫瘍増殖を維持するために特に必要とされる。なぜなら、充分な酸素供給のみが、腫瘍への栄養供給および腫瘍からの腫瘍分解産物の除去を確実にするからである。
この課題は、融合ポリペプチドを腫瘍血管内皮細胞に選択的にさせる3〜30個のアミノ酸のペプチド、および組織因子(TF)またはそのフラグメントを含む融合ポリペプチドによってここで解決され、この融合ポリペプチドは、この組織因子およびフラグメントが、この融合ポリペプチドが腫瘍血管内皮細胞に結合した際に血液凝固を活性化し得、これらのペプチドが、互いに直接結合しているかまたは15個までのアミノ酸を有するリンカーを介して互いに結合されていること特徴付けられる。このペプチドは、この融合ポリペプチドが腫瘍血管内皮細胞に選択的に結合することを可能にし、この融合ポリペプチドが腫瘍血管内皮細胞に結合したときに血液凝固を活性化し得るペプチドのC末端に結合されている。本発明はさらに、対応する融合ポリペプチドを含む薬学的組成物、および腫瘍の処置のためのその使用に関する。
先行技術で観察された課題は、以下のペプチドを含む融合ポリペプチドによっていまや克服された:
a)この融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチド;および
b)組織因子(TF)またはそのフラグメントであって、この組織因子およびこのフラグメントは、この融合ポリペプチドが腫瘍血管内皮細胞に結合したときに血液凝固を活性化し得ることによって特徴付けられる、組織因子またはそのフラグメント。ここで、このペプチドa)およびペプチドb)は、互いに直接結合しているかまたは15個までのアミノ酸を有するリンカーを介して互いに結合されており、この融合ポリペプチドは、この融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得るペプチドが、この融合ポリペプチドが腫瘍血管内皮細胞に結合した際に血液凝固を活性化し得るペプチドのC末端に結合されていることを特徴とする。本発明はさらに、対応する融合ポリペプチドを含む薬物、および腫瘍の処置のためのその使用に関する。
a)この融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチド;および
b)組織因子(TF)またはそのフラグメントであって、この組織因子およびこのフラグメントは、この融合ポリペプチドが腫瘍血管内皮細胞に結合したときに血液凝固を活性化し得ることによって特徴付けられる、組織因子またはそのフラグメント。ここで、このペプチドa)およびペプチドb)は、互いに直接結合しているかまたは15個までのアミノ酸を有するリンカーを介して互いに結合されている。本発明の特に好ましい実施形態によれば、この融合ポリペプチドは、以下を含む:
a)この融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチド;および
b)組織因子(TF)またはそのフラグメントであって、この組織因子およびこのフラグメントは、この融合ポリペプチドが腫瘍血管内皮細胞に結合したときに血液凝固を活性化し得ることによって特徴付けられる、組織因子またはそのフラグメント。ここで、このペプチドa)およびペプチドb)は、互いに結合している。
組織因子TFのN末端218アミノ酸(本明細書以下、tTFと省略する)をコードするcDNAを、ポリメラーゼ連鎖反応(PCR)によって、配列番号16および配列番号17(図27)に示すプライマーを用いて合成し、そして発現ベクターpET−30a(+)(Novagen)中にクローニングした。組換えプラスミドをE.coli(BL21)中に形質転換し、発現させ、そして精製した(Qiagen Plasmid Kit)。
tTF−GRGDSP(配列番号3;図14;本明細書以下、tTF−RGDと省略する;PCRプライマー配列番号18および配列番号19(図28)を用いた);
tTF−GNGRAHA(配列番号4;図15;本明細書以下、tTF−NGRと省略する;PCRプライマー配列番号20および配列番号21(図29)を用いた);
tTF−GALNGRSHAG(配列番号5;図16;PCRプライマー配列番号28および配列番号29(図33)を用いた)。
tTF−GCNGRCG(配列番号6;図17;本明細書以下、tTF−シクロNGR1と省略する;PCRプライマー配列番号22および配列番号23(図30)を用いた);
tTF−GCNGRCVSGCAGRC(配列番号7;図18;本明細書以下、tTF−シクロNGR2と省略する;PCRプライマー配列番号24および配列番号25(図31)を用いた);
tTF−GCVLNGRMEC(配列番号8;図19;本明細書以下、tTF−シクロNGR3と省略する;PCRプライマー配列番号26および配列番号27(図32)を用いた)。
第VIIa因子を介した第X因子から第Xa因子への活性化における補因子活性についてのこれらの融合タンパク質の機能活性を、ミカエリス−メンテン分析によりインビトロで実証した。tTFおよびtTF融合ポリペプチドが、リン脂質の存在下でFVIIaを介したFXの特異的タンパク質分解活性化を強化する能力を、Ruf(45)によって記載された方法のわずかに改変したバージョンにおいて決定した。これに関しては、各々20μlの以下の試薬を、マイクロタイタープレート中ピペッティングした:(a)TBS−BSA中の50nM組換えFVIIa(Novo−Nordisk);(b)TBS−BSA中の0.16nM〜1.6μMのtTF/tTF融合ポリペプチド;(c)25mM CaCl2および500μMのリン脂質小胞(ホスファチジルコリン/ホスファチジルセリン、70/30、M/M;Sigma)。室温にて10分間のインキュベーション後、20μlの天然基質FX(Enzyme Research Laboratories)を5μMの濃度で添加した。次いで、サンプルをピペットによって1分間隔で採取し、そして100mM EDTA溶液の添加によって反応を停止させた。形成されたFXaの量を、405nmでの吸光度の変化によって決定することにより、Microplate Reader中での色素形成性基質Spectrozyme FXaの添加によって測定し、そしてミカエリス−メンテン反応速度についてのパラメーターを、Rufによって記載された方法により分析した。結果は、tTFおよびtTF融合ポリペプチドが両方とも、これらの条件下で機能的に活性であることを示す(図3)。融合ポリペプチドについて見出されたミカエリス定数(Km)は、0.12〜1.2nMの範囲内にあり(図3)、従って、tTFについて公開されているよりも低い範囲であった、それゆえ、機能活性は、ペプチドとtTFとの融合によって影響を受けないとみなされ得る。
αvβ3インテグリンに対するtTF−RGDおよびtTF−NGRの結合を、精製αvβ3をマイクロタイタープレートに固定することにより、ELISA(酵素結合免疫吸着アッセイ)において実証した(図4を参照のこと)。αvβ3に対するtTF−RGDの結合の特異性は、配列GRGDSPを有する合成ペプチド(Gibco社製)がこの試験系においてtTF−RGDのαvβ3に対する結合を完全に阻害するという事実によって強調された(図5を参照のこと)。
tTF−RGD融合タンパク質およびtTF−NGR融合タンパク質を、無胸腺ヌードマウスにおけるヒト腫瘍異種移植片に対するそれらの効果および副作用に関して評価した。本発明者らの実験室で確立したモデルをこのために用いた(33、34)。細胞株CCL185(ヒト肺腺癌)およびM−21(ヒト黒色腫)を、雄性BALB/cヌードマウス(9〜12週齢)の脇腹に皮下注射した。約50〜100mm3(CCL185)または400〜600mm3(M−21)の腫瘍体積が得られたら、マウスをランダムに4つの群に割り当てた。群1は、生理学的生理食塩水溶液(NaCl)のみを受け、群2はtTFを、群3はtTF−RGDを、そして群4はtTF−NGRを受けた(各々の場合、1.5〜2.0mg/kg体重(BW)のタンパク質)。(特定の細胞株の増殖速度に依存して)1〜3日間の間隔で動物の尾静脈に注射を行った。融合タンパク質のかなりの治療活性が観察された。tTF−RGD融合タンパク質またはtTF−NGR融合タンパク質で処置したマウスの腫瘍は、それらの増殖が有意に阻害されたか、または部分的後退の場合は、tTFまたはNaClと比較してサイズが低下した(図7および図8を参照のこと)。
tTF−RGD融合タンパク質の抗腫瘍活性をまた、線維肉腫(HT1080)を有するBALB/cヌードマウスにおいて調べた。これらの腫瘍は、迅速に増殖し、充分に新生血管形成されている。2つの実験の結果を表2および図34に提示する。tTF−RGDの2回目の注射後、コントロール群と比較して有意なHT1080腫瘍増殖阻害が観察された。この効果は、7日目の実験の最後まで持続した(緩衝液コントロール(生理学的食塩水溶液)と比較して、tTF−RGDについてP=0.021、tTFと比較してtTF−RGDについてP=0.005)。先の実験と同様に、このモデルにおいて、腫瘍体積の部分的後退が観察された。
Claims (15)
- 融合ポリペプチドであって、
a)該融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチド;および
b)組織因子(TF)のフラグメントであって、該フラグメントは、配列番号2に示される配列からなり、そして、該フラグメントは、該融合ポリペプチドが腫瘍血管内皮細胞に結合した際に血液凝固を活性化し得ることによって特徴付けられる、フラグメント
を含み、
ここで、該ペプチドa)および該ペプチドb)は、互いに直接結合しているかまたは15個までのアミノ酸を有するリンカーを介して互いに結合されており、該融合ポリペプチドは、該融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得るペプチドが、該融合ポリペプチドが腫瘍血管内皮細胞に結合した際に血液凝固を活性化し得るペプチドのC末端に結合されていることを特徴とし、
そして、該融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチドが、アミノ酸配列RGDまたはNGRを含むことを特徴とする、融合ポリペプチド。 - 前記ペプチドa)および前記ペプチドb)、ならびに15個までのアミノ酸を有するリンカーからなる、請求項1に記載の融合ポリペプチド。
- 前記ペプチドa)と前記ペプチドb)とが互いに直接結合している、請求項1に記載の融合ポリペプチド。
- 前記融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得る、3〜30アミノ酸のペプチドが、直鎖状または環状の構造を有することによって特徴付けられる、請求項1〜3のいずれか1項に記載の融合ポリペプチド。
- 前記融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得るペプチドが、GRGDSPおよびGNGRAHAを含む群より選択されることによって特徴付けられる、請求項1に記載の融合ポリペプチド。
- 前記融合ポリペプチドを腫瘍血管内皮細胞へと選択的に結合させ得るペプチドが、GCNGRCG、GCNGRCVSGCAGRC、GCVLNGRMECおよびGALNGRSHAGを含む群より選択されることによって特徴付けられる、請求項1に記載の融合ポリペプチド。
- 配列番号3〜8に示される配列のうちの1つを有することによって特徴付けられる、請求項1〜6のいずれか1項に記載の融合ポリペプチド。
- 請求項1〜7のいずれか1項に記載の融合ポリペプチドをコードする、核酸。
- 配列番号10〜15に示す配列のうちの1つを有することによって特徴付けられる、請求項8に記載の核酸。
- 請求項8または請求項9に記載の核酸を含む、ベクター。
- 請求項8もしくは請求項9に記載の核酸または請求項10に記載のベクターを含む、細胞。
- 薬学的組成物であって、請求項1〜請求項7のいずれか1項に記載の融合ポリペプチド、請求項8もしくは請求項9に記載の核酸、請求項10に記載のベクター、または請求項11に記載の細胞を含む、薬学的組成物。
- 薬学的に受容可能なキャリア、賦形剤または佐剤をさらに含む、請求項12に記載の薬学的組成物。
- 新生物疾患の処置のための、請求項12または請求項13に記載の組成物。
- 前記新生物疾患が、気管支癌ならびに胸郭および縦隔の他の腫瘍、胸部癌および他の婦人科腫瘍、結腸直腸癌、膵臓癌および胃腸管の他の腫瘍、悪性黒色腫および皮膚の他の腫瘍、頭部および頚部の領域の腫瘍、前立腺癌および他の尿生殖器腫瘍、肉腫、内分泌活性腫瘍、白血病および骨髄形成異常症候群ならびにホジキンリンパ腫および非ホジキンリンパ腫を含む群より選択されることによって特徴付けられる、請求項14に記載の組成物。
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DE10338733A DE10338733A1 (de) | 2003-08-22 | 2003-08-22 | Fusionspolypeptide und deren Verwendung für die antivaskuläre Tumortherapie |
DE10338733.1 | 2003-08-22 | ||
PCT/EP2004/009364 WO2005021593A1 (de) | 2003-08-22 | 2004-08-20 | Fusionspolypeptide und deren verwendung für die antivaskuläre tumortherapie |
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