JP4456165B2 - ヒト第vii凝固因子変異型 - Google Patents
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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Description
非特許文献2(Iwanaga et al., Thromb. Haemost. (supplement August 1999), 466, abstract 1474 )は残基316〜320を欠失させたか又は残基311〜322をトリプシンからの対応の残基により置換した第VIIa因子変異型に関する。
本明細書中に記載の第VII因子変異型を組み換え核酸技術の手段により製造する。一般的に、クローンした野生型第VII因子核酸配列を修飾し所望のタンパク質をコードさせる。次に、この修飾配列を発現ベクター内に挿入し、次にこの発現ベクターを宿主細胞内に形質転換又はトランスフェクトする。高度な真核細胞、特に培養哺乳動物細胞が宿主細胞として好ましい。ヒト第VII因子についての完全なヌクレオチド及びアミノ酸配列が知られている(V.S.4,784,950を参照のこと、ここに組み換えヒト第VII因子のクローニング及び発現が記載されている)。ウシ第VII因子配列が Takeya et al. J. Biol. Chem. 263 : 14868-14872 (1988)に記載されている。
本発明は、本発明による好ましい第VII因子変異型を選択するための安定性アッセイをも提供する。これらのアッセイは簡単なインビトロ予備試験として実施されうる。
本発明による第VII因子変異型は、いくつかの原因、例えば凝固因子欠損(例えばA型若しくはB型血友病又は第XI若しくはVII 凝固因子の欠損)又は凝固因子阻害剤を有する出血障害の制御に使用されるか、あるいはそれらは正常に機能する血液凝固カスケード(凝固因子欠損又は凝固因子のいずれかに対する阻害剤がない)を有する対象に起こる過剰な出血の制御のために使用される。前記出血は不完全な血小板機能、血小板減少症又はフォン・ウィルブランド症により引き起こされる。それらは、高められた線溶活性がさまざまな刺激により誘発された対象において見られる。
〔L305V/M306D/D309S〕−FVII、〔L305V〕−FVII、〔L305I〕−FVII、〔L305T〕−FVII、及び〔F374P〕−FVIIをコードするDNA構築物を、着目の挿入物を伴う高次コイルの2本鎖DNAベクター及び所望の変異を含む2の合成プライマーを用いた特定部位突然変異誘発により製造した。以下のプライマーを用いた。
原則的に以前に記載されたとおり(Thim et. al. (1988) Biochemistry 27, 7785-7793 ; Persson and Nielson (1996) FEBS Lett. 385, 241-243 )、BHK細胞を形質移入して前記変異型〔L305V/M306D/D309S〕−FVIIの発現を得た。前記第VII因子変異型を以下のとおり精製した: 5mM EDTA、0.1% Triton X−100、及び10mM Trisの添加、8.0へのpH調整、並びに水の添加による10〜11mS/cmへの導電率 の調整の後、培養上清をQ Sepharose Fast Flow(Pharmacia Biotech)の25mlカラムに添加した。タンパク質の溶出を10mM Tris、50mM NaCl、0.1% Triton X−100、pH8.0から10mM Tris、1M NaCl、5mM CaCl2 、0.1% Triton X−100、pH7.5への勾配により果した。〔L305V/M306D/D309S〕−FVIIを含む画分をプールし、10mM CaCl2 を添加し、そしてCNBr活性化Sepharose 4B(Pharmacia Biotech)に結合したモノクローナル抗体F1A2(Novo Nordisk, Bagsvaerd, Denmark)を含む25mlカラムに適用した。前記カラムを10mM CaCl2 、100mM NaCl、及び0.02% Triton X−100含有 50mM Hepes、pH7.5により平衡化した。平衡化バッファー及び2M NaCl含有平衡化バッファーを用いて洗浄した後、結合物質をCaCl2 の代わりに10mM EDTAを含む平衡化バッファーを用いて溶出した。使用又は保存の前に、EDTAを上回る過剰なCaCl2 を添加するか、又は〔L305V/M306D/D309S〕−FVIIをCa2+含有バッファーに移した。各ステップの収量を第VII因子ELISA計測法により追跡し、そして前記精製タンパク質をSDS−PAGEにより分析した。
原則的に以前に記載されたとおり(Thim et. al. (1988) Biochemistry 27, 7785-7793 ; Persson and Nielson (1996) FEBS Lett. 385, 241-243 )、BHK細胞を形質移入して前記変異型〔L305V〕−FVIIの発現を得た。前記第VII因子変異型を以下のとおり精製した: 5mM EDTA、0.1% Triton X−100、及び10mM Trisの添加、8.0へのpH調整、並びに水の添加による10〜11mS/cmへの導電率の調整の後、培養上清をQ Sepharose Fast Flow(Pharmacia Biotech )の25mlカラムに添加した。タンパク質の溶出を10mM Tris、50mM NaCl、0.1% Triton X−100、pH8.0から10mM Tris、1M NaCl、5mM CaCl2 、0.1% Triton X−100、pH7.5への勾配により果した。〔L305V〕−FVIIを含む画分をプールし、10mM CaCl2 を添加し、そしてCNBr活性化Sepharose 4B(Pharmacia Biotech )に結合したモノクローナル抗体F1A2(Novo Nordisk, Bagsvaerd, Denmark)を含む25mlカラムに適用した。前記カラムを10mM CaCl2 、100mM NaCl、及び0.02% Triton X−100含有 50mM Hepes、pH7.5により平衡化した。平衡化バッファー及び2M NaCl含有平衡化バッファーを用いて洗浄した後、結合物質をCaCl2 の代わりに10mM EDTAを含む平衡化バッファーを用いて溶出した。使用又は保存の前に、EDTAを上回る過剰なCaCl2 を添加するか、又は〔L305V〕−FVIIをCa2+含有バッファーに移した。各ステップの収量を第VII因子ELISA計測法により追跡し、そして前記精製タンパク質をSDS−PAGEにより分析した。
原則的に以前に記載されたとおり(Thim et. al. (1988) Biochemistry 27, 7785-7793 ; Persson and Nielson (1996) FEBS Lett. 385, 241-243 )、BHK細胞を形質移入して前記変異型〔F374P〕−FVIIの発現を得た。前記第VII因子変異型を以下のとおり精製した: 5mM EDTA、0.1% Triton X−100、及び10mM Trisの添加、8.0へのpH調整、並びに水の添加による10〜11mS/cmへの導電率の調整の後、培養上清をQ Sepharose Fast Flow(Pharmacia Biotech )の25mlカラムに添加した。タンパク質の溶出を10mM Tris、50mM NaCl、0.1% Triton X−100、pH8.0から10mM Tris、1M NaCl、5mM CaCl2 、0.1% Triton X−100、pH7.5への勾配により果した。〔F374P〕−FVIIを含む画分をプールし、10mM CaCl2 を添加し、そしてCNBr活性化Sepharose 4B(Pharmacia Biotech )に結合したモノクローナル抗体F1A2(Novo Nordisk, Bagsvaerd, Denmark)を含む25mlカラムに適用した。前記カラムを10mM CaCl2 、100mM NaCl、及び0.02% Triton X−100含有 50mM Hepes、pH7.5により平衡化した。平衡化バッファー及び2M NaCl含有平衡化バッファーを用いて洗浄した後、結合物質をCaCl2 の代わりに10mM EDTAを含む平衡化バッファーを用いて溶出した。使用又は保存の前に、EDTAを上回る過剰なCaCl2 を添加するか、又は〔F374P〕−FVIIをCa2+含有バッファーに移した。各ステップの収量を第VII因子ELISA計測法により追跡し、そして前記精製タンパク質をSDS−PAGEにより分析した。
原則的に以前に記載されたとおり(Thim et. al. (1988) Biochemistry 27, 7785-7793 ; Persson and Nielson (1996) FEBS Lett. 385, 241-243 )、BHK細胞を形質移入して前記変異型〔L305I〕−FVII又は〔L305T〕−FVIIの発現を得た。前記第VII因子変異型を以下のとおり精製した: 5mM EDTA、0.1% Triton X−100、及び10mM Trisの添加、8.0へのpH調整、並びに水の添加による10〜11mS/cmへの導電率の調整の後、培養上清をQ Sepharose Fast Flow(Pharmacia Biotech )の25mlカラムに添加した。タンパク質の溶出を10mM Tris、50mM NaCl、0.1% Triton X−100、pH8.0から10mM Tris、1M NaCl、5mM CaCl2 、0.1% Triton X−100、pH7.5への勾配により果した。〔L305I〕−FVII又は〔L305T〕−FVIIを含む画分をプールし、10mM CaCl2 を添加し、そしてCNBr活性化Sepharose 4B(Pharmacia Biotech )に結合したモノクローナル抗体F1A2(Novo Nordisk, Bagsvaerd, Denmark)を含む25mlカラムに適用した。前記カラムを10mM CaCl2 、100mM NaCl、及び0.02% Triton X−100含有 50mM Hepes、pH7.5により平衡化した。平衡化バッファー及び2M NaCl含有平衡化バッファーを用いて洗浄した後、結合物質をCaCl2 の代わりに10mM EDTAを含む平衡化バッファーを用いて溶出した。使用又は保存の前に、EDTAを上回る過剰なCaCl2 を添加するか、又は〔L305I〕−FVII又は〔L305T〕−FVIIをCa2+含有バッファーに移した。各ステップの収量を第VII因子ELISA計測法により追跡し、そして前記精製タンパク質をSDS−PAGEにより分析した。
天然(野生型)第VIIa因子と第VII因子変異型(共に以下「第VIIa因子」と呼ぶ)をそれらの特異的活性を直接比較するために同時にアッセイした。前記アッセイをマイクロタイター・プレート(MaxiSorp, Nunc, Denmark )中で行った。終濃度1mMの色素原性基質D−Ile−Pro−Arg−p−ニトロアニリド(S-2288, Chromogenix, Sweden )を、0.1M NaCl、5mM CaCl2 、及び1mg/mlウシ血清アルブミン含有50mM Hepes、pH7.4中の第VIIa因子(終濃度100nM)に添加した。405nmでの吸光度をSpectra Max(商標)340プレート・リーダー(Molecular Devices, USA)により連続的に計測した。酵素を含まないブランク・ウェルにおける吸光度を差し引いた後、20分間インキュベーションの間に発生した吸光度を第 VIIa 因子の変異型と野生型の活性の間の比を計算するために使用した: 比=(A405nm 第VIIa因子変異型)/(A405nm 第VIIa因子野生型)。
天然(野生型)第VIIa因子と第VII因子変異型(共に以下「第VIIa因子」と呼ぶ)をそれらの特異的活性を直接比較するために同時にアッセイした。前記アッセイをマイクロタイター・プレート(MaxiSorp, Nunc, Denmark )中で行った。0.1M NaCl、5mM CaCl2 、及び1mg/mlウシ血清アルブミン含有50mM Hepes、pH7.4 100μL中、第VIIa因子(10nM)と第 X因子(0.8μM)を15分間インキュベートした。次に第X因子の開裂を0.1M NaCl、20mM EDTA、及び1mg/mlウシ血清アルブミン含有50mM Hepes、pH7.4 50μLの添加により停止させた。産生された第Xa因子の量を、終濃度0.5mMの色素原性基質Z−D−Arg−Gly−Arg−p−ニトロアニリド(S -2765, Chromogenix, Sweden )の添加により計測した。405nmでの吸光度をSpectra Max(商標)340プレート・リーダー(Molecular Devices, USA)により連続的に計測した。FVIIa を含まないブランク・ウェルにおける吸光度を差し引いた後、10分間に発生した吸光度を第VIIa因子の変異型と野生型の活性の間の比を計算するために使用した: 比=(A405nm 第VIIa因子変異型)/(A405nm 第VIIa因子野生型)。
Claims (13)
- 配列番号1の153〜406位のプロテアーゼ・ドメイン内において、少なくとも、374位のPhe残基がProにより置き換えられているヒト第VII凝固因子変異型であって、組織因子の不在下でのインビトロ加水分解アッセイによる活性が、配列番号1で表される野生型ヒト第VIIa凝固因子の活性に対して1.25倍以上である、ヒト第VII凝固因子変異型。
- 前記のプロテアーゼ・ドメイン内における374位以外の位置で、最大20のアミノ酸残基が置き換えられている、請求項1に記載のヒト第VII凝固因子変異型。
- 274位及び/又は300〜304位及び/又は306〜312位の中の少なくとも1の残基が置き換えられている、請求項1又は2に記載のヒト第VII凝固因子変異型。
- 少なくとも274位の残基が置き換えられている、請求項1〜3のいずれか1項に記載のヒト第VII凝固因子変異型。
- 300〜304位の中の少なくとも1の残基が置き換えられている、請求項1〜4のいずれか1項に記載のヒト第VII凝固因子変異型。
- 306〜312位の中の少なくとも1の残基が置き換えられている、請求項1〜5のいずれか1項に記載のヒト第VII凝固因子変異型。
- 274位のAla残基が、Met、Leu、Lys若しくはArgにより置き換えられ;及び/又は、304位のArg残基が、Tyr、Phe、Leu若しくはMetにより置き換えられ;及び/又は、306位のMet残基が、Asp若しくはAsnにより置き換えられ;及び/又は、309位のAsp残基が、Ser若しくはThrにより置き換えられている、請求項3に記載のヒト第VII凝固因子変異型。
- F374P−FVIIaである、請求項1に記載のヒト第VII凝固因子変異型。
- 請求項1〜8のいずれか1項に記載の第VII因子変異型をコードするヌクレオチド配列を含む核酸構築物。
- 請求項9に記載の核酸構築物を含む組み換えベクター。
- 請求項9に記載の核酸構築物又は請求項10に記載のベクターを含む組み換え宿主細胞。
- 請求項1〜8のいずれか1項に記載のヒト第VII凝固因子変異型を製造する方法であって、前記核酸構築物の発現が可能な条件下、適当な育成培地中で請求項11に記載の細胞を培養し、得られたポリペプチドを培地から回収することを含む方法。
- 請求項1〜8のいずれか1項に記載のヒト第VII凝固因子変異型と、医薬として許容される担体とを含む医薬組成物。
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TWI538916B (zh) | 2008-04-11 | 2016-06-21 | 介控生化科技公司 | 經修飾的因子vii多肽和其用途 |
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FR2947181B1 (fr) * | 2009-06-26 | 2012-05-04 | Lfb Biotechnologies | Composition de facteur vii |
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JP2013533871A (ja) | 2010-06-30 | 2013-08-29 | ノヴォ ノルディスク アー/エス | 組織因子経路インヒビターに特異的に結合することが可能な抗体 |
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US5580560A (en) * | 1989-11-13 | 1996-12-03 | Novo Nordisk A/S | Modified factor VII/VIIa |
US5788965A (en) * | 1991-02-28 | 1998-08-04 | Novo Nordisk A/S | Modified factor VII |
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EP1282693B1 (en) | 2010-10-20 |
WO2001083725A1 (en) | 2001-11-08 |
EP1282693A1 (en) | 2003-02-12 |
JP2009060902A (ja) | 2009-03-26 |
ATE485371T1 (de) | 2010-11-15 |
AU2001254624A1 (en) | 2001-11-12 |
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