JP6404349B2 - サクサチリン−Fc融合タンパク質及びその用途 - Google Patents
サクサチリン−Fc融合タンパク質及びその用途 Download PDFInfo
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- JP6404349B2 JP6404349B2 JP2016531527A JP2016531527A JP6404349B2 JP 6404349 B2 JP6404349 B2 JP 6404349B2 JP 2016531527 A JP2016531527 A JP 2016531527A JP 2016531527 A JP2016531527 A JP 2016531527A JP 6404349 B2 JP6404349 B2 JP 6404349B2
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- saccharin
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Description
(a)本発明は、増加した半減期を有するサクサチリン誘導体及びその用途に関するものである。
(b)本発明のサクサチリン誘導体は、親タンパク質であるサクサチリンと類似した血栓溶解能を有するだけではなく、顕著に増加したタンパク質の半減期を有し、それを用いてFeCl3−誘導された頸動脈動物モデルで血管内に既に形成された血栓を長期間の間に非常に効率的に溶解させる。
(c)したがって、本発明のサクサチリン誘導体を有効成分として含む組成物は、貫通後、再狭窄も起こさず、微小血管まで効果的に開放させて、血管の狭窄又は閉塞疾患(例えば、脳血管疾患、心血管疾患、動脈硬化性血管疾患、冠状動脈疾患、末梢血管疾患など)の治療に非常に効果的である。
組換えサクサチリンタンパク質発現ベクターの製作、発現及び精製
Fc−サクサチリン及びサクサチリン−Fcタンパク質発現ベクターの製作
組換えサクサチリンタンパク質を発現させるために、本発明者らは、下記の表1に記載のプライマー対(サクサチリン−F及びサクサチリン−R)を用いてPCRを実施した。得られたPCR産物を制限酵素sfiI(thermo scientific)で切断して、pYK603ベクター及びpYK602ベクター(A&Rセラピューティクス)にT4 DNAリガーゼ(thermo scientific)を用いてクローニングして、それぞれFc−サクサチリン及びサクサチリン−Fcタンパク質発現用組換えベクターを製造した(図1)。
前述したFc−サクサチリン又はサクサチリン−Fcを含む組換えベクターをそれぞれ292E細胞にPEI(polyethylenimine;Polysciences)を用いてトランスフェクション(transfection)させた。トランスフェクション後、細胞培養2日又は5日目に上澄み液(supernatant)を収去して、SDS−PAGEを実施した後、NC膜(membrane;Millipore)に移し、2次抗体である抗−HuFc−HRP(1:2000希釈;thermo scientific)と常温で1時間反応させた。前記NC膜をPBS/Tで3回にわたって洗浄した後、ECL溶液でフィルムを現像してウェスタンブロッティングを実施した(図2)。
前述したように、トランスフェクションされた293E細胞の培養で3、5又は7日目の培養上澄み液を0.22μmトップフィルター(top−filter;Millipore)を使って濾過した。該濾過された上澄み液を500μlタンパク質Aビーズ(GE healthcare)がパッキングされている5mlカラムにローディングしてビーズと結合させた。ポンプ(peri−start pump;Bio−rad)を使って0.9ml/minの流速で4℃で一晩結合させた後、100ml以上のPBSで洗浄し、0.1Mグリシン(Glycine)−HClを用いて6つの画分(fraction)に溶出させた。前記溶出液は、1M Tris(pH9.0)溶液で中和(neutralization)させた。以後、タンパク質の量を定量し、2〜3つの画分に含まれたタンパク質をアミコンウルトラ遠心分離フィルター(amicon ultra centrifugal filter;Millipore)を使って濃縮させた。PBSで10回以上緩衝液を交換させた。
ゾレチル(zoletil 50;VirBac)とロムプン(rompun;Bayer)とを3:1の比率で混合した後、生理食塩水で10倍希釈させた麻酔溶液を製造した。雄ICR(Institute of Cancer Research)マウス((株)オリンエントバイオ、大韓民国)の体重を測定して、g当たり10μlの前記麻酔溶液を腹腔内に注入した。麻酔されたマウスを固定させて開腹した後、腹大靜脈で血液を採取してクエン酸ナトリウムチューブ(sodium citrate tube、#363048;BD Science)に入れ、よく混ぜた。前記採取された血液を15mlチューブに移して、1,000rpmで15分間遠心分離させて血小板(platelet)を含む血漿層(Platelet−Rich Plasma、PRP)を収得した。また、下側の血球層を再び3,000rpmで10〜15分間遠心分離させて上澄み液(血漿)を得た。前記上澄み液(600μl)を10,000rpmで2分間遠心分離させて血球細胞などが除去されたPPP(Platelet−Poor Plasma)を収得し、残った血漿は、以前工程で分離したPRPと混合して、引き続き実験に用いた。血小板凝集アッセイは、凝集測定器(Aggregometer Model 700;Chrono−log)を用いて実施した。PPP(500μl)を添加した最初のキュベット(cuvettes、#312;Chrono−log)とPRP(480μl)とサクサチリン(10μl)又は組換えサクサチリン(Fc−サクサチリン又はサクサチリン−Fc;10μl)を添加した二番目のキュベット(cuvette)とを凝集測定器にさし、基準線(baseline)をセッティングした後、前記二番目のキュベットに10μlのADP(最終濃度、20μM;#384;Chrono−log)を入れてピペッティング(pipetting)で混合させ、凝集程度を測定した。この際、ADPのみを入れた時の凝集程度を100%に算定し、これについての相対的な数値で各試料の凝集程度を計算した。すなわち、100%で各試料の凝集率(%)を差し引いた値が抑制率(%)で計算される。血小板凝集に対する天然サクサチリンのIC50濃度は、150nMであり、本発明のFc−サクサチリン及びサクサチリン−FcのIC50濃度は、それぞれ196nM及び362nMであったが(図4)、これは、本発明の組換えサクサチリンがサクサチリンと比較されるほどの優れた血小板凝集活性を表わすということを示す。
サクサチリン、Fc−サクサチリン及びサクサチリン−Fc(in PBS、pH7.4)を含む反応チューブにNHS−ローダミン(N−Hydroxysuccinimide−Rhodamine;#46406、10mg/ml;Thermo Scientific Pierce,米国)を添加し、常温で1時間反応させた。PD10脱塩カラム(salting column)(#17−0851−01;GE Healthcare,米国)を用いて反応していない(non−reacted)NHS−ローダミンを除去した。ローダミン−コンジュゲーションされた(conjugated)タンパク質の濃度をBCAタンパク質アッセイキット(#23225;Thermo Scientific Pierce,米国)で測定した後、実験に利用するまで4℃で暗条件で保管した。
96ウェルプレートに各インテグリン(αIIbβ3、αvβ3、αMβ2、及びαLβ2)を100ng/ウェルずつ添加し、4℃で一晩中コーティングした。コーティング後、PBS−T(0.5% Tween 20)で3回洗浄した。1% BSA(in PBS−T)溶液で常温で2時間ブロッキング(blocking)した。ブロッキング後、PBS−Tで3回洗浄した。サクサチリン−Fc又はFc−サクサチリンを100nMから1/4ずつ連続希釈して8つのサンプル溶液を製造した。
32〜34gの8週齢の雄ICRマウスを用いた。実験室動物の管理及び利用は、国際実験動物管理認証協会(AAALAC)の指針に適当な基準に延世大学校医科大学動物実験倫理委員会(IACUC)の検討と承認とを受けたプロトコルによって実施した。手術過程を説明すれば、動物は、70% N2O及び30% O2で構成された混合物(大韓特殊ガス、大韓民国)内に5%イソフルラン(日盛新薬、大韓民国)の吸入を通じて麻酔させた。麻酔は、2%イソフルランで保持させた。手術過程の間に、動物の体温は、直腸用プローブで持続的にモニタリングし、恒温被覆調節ユニット(Homeothermic blanket control unit)及びヒーティングパッド(Harvard Apparatus,Holliston,MA)を用いて37.0±0.2℃に保持した。サクサチリン及びサクサチリン−Fcのインビボ血栓溶解活性をテストするために、FeCl3(Sigma−Aldrich,米国)−誘導された頸動脈血栓モデルを用いた。頸部正中切開(midline cervical incision)を実施して、左側総頸動脈を手術顕微鏡(SEILER,米国)下で気をつけて解剖した。超音波ドップラー流速プローブ(ultrasonic Doppler flow probe;Transonic MA0.7PSB)を総頸動脈(CCA)の中央部位に位置させた。頸動脈血流量は、超音波TS420血流量計(Transonic Instruments,Ithaca,NY)及びiWorx IX−304Tデータ獲得システム(iWorx Systems,Inc.,Dover,NH)を用いて測定した。対照群としてCCAの基本血流量を5分間測定した。対照群の基本血流量の決定後、プローブを除去した。露出されたCCA中央地点の外側表面(adventitial surface)に50% FeCl3で飽和された濾過紙(700×500μM;ADVANTEC,日本)を5分間接触させることによって、化学的ストレスによる酸化的血管損傷が誘導された。濾過紙を除去した後、CCAを生理食塩水で洗浄し、その血流量を測定した。血流量の減少を通じて血栓形成及び動脈閉塞を決定し、完全閉塞は、血流が10分間不在した場合と定義された。
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Claims (18)
- 配列番号2のアミノ酸配列からなるサクサチリンと免疫グロブリンのFc領域がコンジュゲーションされたことを特徴とするサクサチリン誘導体。
- 前記免疫グロブリンのFc領域は、前記サクサチリンのN末端又はC末端にコンジュゲーションされている請求項1に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、N末端にリーダー配列がさらにコンジュゲーションされている請求項1に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、天然のサクサチリンよりも4〜6.5倍の増加した半減期を有する請求項1に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、血栓内に存在するインテグリン(integrin)に結合して血栓を分解する請求項1に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、血栓を構成する血小板表面のGP IIb−IIIaに結合して血栓を分解する請求項5に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、好中球に存在するインテグリンαMβ2に対して1×10−8〜1×10−10MのKd値の結合力を有する請求項1に記載のサクサチリン誘導体。
- 前記サクサチリン誘導体は、好中球に存在するインテグリンαLβ2に対して1×10−8〜1×10−10MのKd値の結合力を有する請求項1に記載のサクサチリン誘導体。
- 請求項1から8のいずれかに記載のサクサチリン誘導体をコードすることを特徴とするヌクレオチド。
- (a)請求項9に記載のヌクレオチド;及び(b)前記ヌクレオチドに作動的に連結されたプロモーターを含むことを特徴とする組換えベクター。
- 請求項10に記載の組換えベクターで形質転換されたことを特徴とする細胞。
- (a)請求項1から8のいずれかに記載のサクサチリン誘導体の薬剤学的有効量;及び(b)薬剤学的に許容される担体を含むことを特徴とする血栓溶解用組成物。
- 請求項12に記載の組成物を含むことを特徴とする血管の狭窄又は閉塞疾患の予防又は治療用の薬剤学的組成物。
- 前記血管は、大動脈、頸動脈、鎖骨下動脈、腹腔動脈、腸間膜動脈、腎動脈、腸骨動脈、小動脈、毛細血管、又は小静脈である請求項13に記載の薬剤学的組成物。
- 前記血管の狭窄又は閉塞疾患は、脳卒中、脳梗塞、脳血栓症、脳塞栓症、小窩性脳梗塞、急性冠動脈症候群、狭心症、大動脈狭窄症、心筋梗塞症、片頭痛、脚ブロック、脳虚血、急性虚血性心血管疾患、血栓性静脈炎、静脈血栓塞栓症、深部静脈血栓症、肺塞栓症、末梢血管疾患、血管性頭痛、アテローム性動脈硬化症、血管痙攣、再狭窄症、器具血管形成術以後再狭窄症、及び血管炎による血管閉塞症からなる群から選択される請求項13に記載の薬剤学的組成物。
- 前記薬剤学的組成物は、血管内に直接注入される方式で投与される請求項13に記載の薬剤学的組成物。
- 前記薬剤学的組成物は、血栓によって血管が閉塞した患者に投与される請求項13に記載の薬剤学的組成物。
- 前記閉塞は、部分的閉塞又は完全閉塞である請求項17に記載の薬剤学的組成物。
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US5879657A (en) | 1993-03-30 | 1999-03-09 | The Dupont Merck Pharmaceutical Company | Radiolabeled platelet GPIIb/IIIa receptor antagonists as imaging agents for the diagnosis of thromboembolic disorders |
EP1259595B1 (en) * | 2000-02-25 | 2007-04-04 | Immunex Corporation | Integrin antagonists |
CN1184313C (zh) * | 2000-07-26 | 2005-01-12 | 郑光会 | 衍生自黑眉蝮蛇的新型蛋白以及制备该蛋白的方法 |
WO2004078137A2 (en) * | 2003-03-04 | 2004-09-16 | Greenville Hospital System | Antitumor agents comprising a targeting portion and an immune response triggering portion |
FR2900734B1 (fr) | 2006-05-05 | 2009-03-06 | Diagnostica Stago Soc Par Acti | Detection des maladies veineuses thromboemboliques par le dosage des d-dimeres et de la fibrine soluble |
US7943728B2 (en) * | 2006-12-26 | 2011-05-17 | National Cheng Kung University | Disintegrin variants and their use in treating osteoporosis-induced bone loss and angiogenesis-related diseases |
US8183201B2 (en) | 2006-12-26 | 2012-05-22 | National Cheng Kung University | Methods of treating αvβ3 integrin-associated diseases by administering polypeptides selective for αvβ3 integrin |
KR20090036908A (ko) * | 2007-10-10 | 2009-04-15 | 연세대학교 산학협력단 | 뱀독 유래의 디스인테그린인 삭사틸린을 포함하는동맥경화증 예방 또는 치료용 조성물 |
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JP2016527265A (ja) | 2016-09-08 |
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WO2015016616A1 (ko) | 2015-02-05 |
EP3029071A1 (en) | 2016-06-08 |
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