JP4966434B2 - 結合抗体の存在下における組換え血液凝固因子のpeg化 - Google Patents
結合抗体の存在下における組換え血液凝固因子のpeg化 Download PDFInfo
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Description
本発明は、例えば以下の項目を提供する。
(項目1)
血液凝固因子に、水溶性ポリマー(WSP)を結合する方法であって、
(a)前記血液凝固因子に血液凝固因子特異抗体を結合させる条件下で、前記抗体と共に前記血液凝固因子をインキュベートして、抗体−血液凝固因子複合体を形成させる工程と、
(b)前記抗体−血液凝固因子複合体に前記WSPを結合させる条件下で、前記WSPと共に前記抗体−血液凝固因子複合体をインキュベートする工程と、
(c)前記WSP結合血液凝固因子を、前記抗体から離す工程を含み、
前記血液凝固因子は、第IX因子(FIX)、第VIII因子(FVIII)、第VIIa因子(FVIIa)、フォンヴィレブランド因子(VWF)、第V因子(FV)、第X因子(FX)、第XI因子、第XII因子(FXII)、トロンビン(FII)、プロテインC、プロテインS、tPA、PAI−1、組織因子(TF)およびADAMTS13プロテアーゼからなる群から選択され、
前記WSPは、ポリエチレングリコール(PEG)、分枝PEG、ポリシアル酸(PSA)、炭水化物、多糖類、プルラン、キトサン、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、でんぷん、デキストラン、カルボキシメチルデキストラン、ポリアルキレンオキシド(PAO)、ポリアルキレングリコール(PAG)、ポリプロピレングリコール(PPG)ポリオキサゾリン、ポリアクリロイルモルホリン、ポリビニルアルコール(PVA)、ポリカルボキシレート、ポリビニルピロリドン、ポリホスファゼン、ポリオキサゾリン、ポリエチレン−co−マレイン酸無水物、ポリスチレン−co−マレイン酸無水物、ポリ(1−ヒドロキシメチルエチレンヒドロキシメチルホルマール)(PHF)、および/または2−メタクリロイルオキシ−2’−エチルトリメチルアンモニウムホスフェート(MPC)からなる群から選択される、方法。
(項目2)
前記血液凝固因子がFVIIIである、項目1に記載の方法。
(項目3)
前記FVIIIが完全長FVIIIである、項目2に記載の方法。
(項目4)
前記WSPが約2,000から約150,000Daの分子量を有する、項目1に記載の方法。
(項目5)
前記WSPが直鎖状構造または分枝構造を有する、項目4に記載の方法。
(項目6)
前記WSPがPEGである、項目5に記載の方法。
(項目7)
前記WSPがPSAである、項目5に記載の方法。
(項目8)
前記抗体が樹脂に固定化されている、項目1に記載の方法。
(項目9)
(a)血液凝固因子、および
(b)前記血液凝固因子に結合している少なくとも1種の水溶性ポリマー(WSP)分子
を含むタンパク質性構築体であって、
前記少なくとも1種のWSPは、前記血液凝固因子に特異的に結合する抗体の存在下で、前記血液凝固因子に結合し、
前記血液凝固因子は、第IX因子(FIX)、第VIII因子(FVIII)、第VIIa因子(FVIIa)、フォンヴィレブランド因子(VWF)、第V因子(FV)、第X因子(FX)、第XI因子、第XII因子(FXII)、トロンビン(FII)、プロテインC、プロテインS、tPA、PAI−1、組織因子(TF)およびADAMTS13プロテアーゼからなる群から選択され、
前記WSPは、ポリエチレングリコール(PEG)、分枝PEG、ポリシアル酸(PSA)、炭水化物、多糖類、プルラン、キトサン、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、でんぷん、デキストラン、カルボキシメチルデキストラン、ポリアルキレンオキシド(PAO)、ポリアルキレングリコール(PAG)、ポリプロピレングリコール(PPG)ポリオキサゾリン、ポリアクリロイルモルホリン、ポリビニルアルコール(PVA)、ポリカルボキシレート、ポリビニルピロリドン、ポリホスファゼン、ポリオキサゾリン、ポリエチレン−co−マレイン酸無水物、ポリスチレン−co−マレイン酸無水物、ポリ(1−ヒドロキシメチルエチレンヒドロキシメチルホルマール)(PHF)、および/または2−メタクリロイルオキシ−2’−エチルトリメチルアンモニウムホスフェート(MPC)からなる群から選択される、構築体。
(項目10)
前記血液凝固因子がFVIIIである、項目9に記載のタンパク質性構築体。
(項目11)
前記FVIIIが完全長FVIIIである、項目10に記載のタンパク質性構築体。
(項目12)
前記WSPが約2,000から約150,000Daの分子量を有する、項目9に記載のタンパク質性構築体。
(項目13)
前記WSPが直鎖状構造または分枝構造を有する、項目12に記載のタンパク質性構築体。
(項目14)
前記WSPがPEGである、項目13に記載のタンパク質性構築体。
(項目15)
前記WSPがPSAである、項目13に記載のタンパク質性構築体。
(項目16)
前記抗体が樹脂に固定化されている、項目9に記載のタンパク質性構築体。
ポリペプチド
本明細書に記載する場合、第IX因子(FIX)、第VIII因子(FVIII)、第VIIa因子(FVIIa)、フォンヴィレブランド因子(VWF)、第V因子(FV)、第X因子(FX)、第XI因子、第XII因子(FXII)、トロンビン(FII)、プロテインC、プロテインS、tPA、PAI−1、組織因子(TF)およびADAMTS13プロテアーゼを含むが、それだけに限らない「血液凝固タンパク質」または「血液凝固因子」が、本発明によって意図される。本明細書で使用する場合、「血液凝固タンパク質」または「血液凝固因子」という用語は、このような特定の天然の血液凝固タンパク質に関係する生物活性を示す、第IX因子(FIX)、第VIII因子(FVIII)、第VIIa因子(FVIIa)、フォンヴィレブランド因子(VWF)、第V因子(FV)、第X因子(FX)、第XII因子(FXII)、トロンビン(FII)、プロテインC、プロテインS、tPA、PAI−1、組織因子(TF)およびADAMTS13プロテアーゼ、または他の血液凝固因子のいずれかを指す。
一態様では、提供される血液凝固因子(例えばFVIII、FVIIまたはFIX)、または他の血液凝固因子誘導体の分子は、限定なしに含む水溶性ポリマーに結合している。臨床的に許容される適切な水溶性ポリマーには、以下に限定されないが、PEG、ポリエチレングリコールプロピオンアルデヒド、エチレングリコール/プロピレングリコールのコポリマー、ポリオキサゾリン、ポリアクリロイルモルホリン、モノメトキシポリエチレングリコール、カルボキシメチルセルロース、ポリアセタール類、ポリビニルアルコール(PVA)、ポリビニルピロリドン、ポリ−1,3−ジオキソラン、ポリ−1,3,6−トリオキサン、エチレン/マレイン酸無水物のコポリマー、ポリ(β−アミノ酸)(ホモポリマーまたはランダムコポリマーのいずれか)、ポリ(n−ビニルピロリドン)ポリエチレングリコール、プロプロピレングリコールのホモポリマー類(PPG)、および他のポリアルキレンオキシド類、ポリプロピレンオキシド/エチレンオキシドのコポリマー類、ポリオキシエチル化ポリオール類(POG)(例えばグリセロール)および他のポリオキシエチル化ポリオール類、ポリオキシエチル化ソルビトール、またはポリオキシエチル化グルコース、コロン酸または他の炭水化物ポリマー類、フィコールまたはデキストラン、ならびにそれらの混合物が含まれる。
本発明の組換え血液凝固因子(例えばrFVIII)をコードする核酸は、例えば以下に限定されないが、遺伝子、mRNA前駆体(pre−mRNA)、mRNA、多型変異体、対立遺伝子、合成および天然に存在する変異体を含む。rFVIIIという用語によって包含されるタンパク質は、以下に限定されないが、例えば、本明細書中の上記のタンパク質およびポリペプチド、上記の核酸によってコードされるタンパク質、種間ホモログ、および他のポリペプチドを含む。
血液凝固因子(例えばFVIII、FVII、FIX)、または他の血液凝固因子の産生は、以下に挙げる当技術分野において公知の任意の方法を含む。(i)遺伝子工学によって組換えDNAを産生する、(ii)以下に限定されないが、例えば、トランスフェクション、電気穿孔法、またはマイクロインジェクションによって、組換えDNAを原核細胞または真核細胞内に導入する、(iii)前記形質転換させた細胞を培養する、(iv)FVIIIを、例えば構成的に、または誘導時に発現させる、および(v)例えば培地から、または形質転換された細胞を採取することにより、前記FVIIIを単離することによって、(vi)精製したrFVIIIを得る。
ある態様では、血液凝固因子(例えば、FVIII、FVII、FIX)、または他の血液凝固因子の分子は、任意の種々の化学的方法によって水溶性ポリマーに結合している(Roberts JMら、Advan Drug Delivery Rev、2002年、54巻、459〜76頁)。例えば、一実施形態では、FVIIIは、N−ヒドロキシスクシンイミド(NHS)エステルを用いて、タンパク質の遊離アミノ基にPEGを結合することによって修飾される。別の実施形態では、水溶性ポリマー(例えばPEG)は、マレイミドの化学を利用して遊離SH基に結合される、あるいは予め酸化を行った後、FVIIIの炭水化物部位にPEGヒドラジドまたはPEGアミンがカップリングされる。
本明細書で使用する場合、「シアル酸部分」は、水溶液または水性懸濁液に可溶なシアル酸のモノマーまたはポリマー(「多糖類」)を含み、医薬として有効な量のPSA−FVIII結合体を哺乳動物に投与した場合、副作用などの負の影響がほとんどない、または全くないものである。ポリマーは、一態様では、1から4単位を有するものとして特徴付けられる。ある態様では、異なるシアル酸単位が、鎖に組み込まれている。
本明細書で使用する場合、「抗体」という用語は、標的タンパク質またはその断片に特異的な、モノクローナルおよびポリクローナル抗体、一本鎖抗体、キメラ抗体、ニ官能性/ニ特異性抗体、ヒト化抗体、ヒト抗体、ならびに相補性決定領域(CDR)移植抗体を指す。「抗体」という用語は、さらに、in vivoにおける治療用抗体の遺伝子移入をも含む。Fab、Fab’、F(ab’)2、scFv、およびFvを含む抗体断片もまた、本発明によって提供される。抗体は、一部の好ましい実施形態において、モノクローナル抗体、ヒト化抗体、霊長類化抗体、一本鎖抗体、またはキメラ抗体であってよい。
本発明のタンパク質性構築体を含む組成物を、ヒトまたは試験動物に投与するために、一態様では、その組成物は1つまたは複数の医薬として許容される担体を含む。「医薬として」または「薬理学的に許容される」という用語は、安定しており、凝集産物および開裂産物などのタンパク質分解を抑制し、さらに、以下に記載されるように、当技術分野で周知の経路を用いて投与される場合、アレルギー反応または他の有害反応をさらに生じない分子構成体(molecular entity)および組成物を指す。「医薬として許容される担体」は、上で開示した薬剤を含め、任意およびすべての臨床的に有用な溶媒、分散媒体、被覆剤、抗菌剤および抗真菌剤、等張剤および吸収遅延剤などを含む。
rFVIII中のリジン残基のPEG化
A.FVIIIの固定化
樹脂上に固定化された抗FVIII mAbを完全に飽和させる条件下で、平衡化した抗FVIII免疫親和性樹脂と共に、rFVIIIサンプルをインキュベートした(>3mgFVIII/g樹脂)。一実施形態では、抗FVIII mAbは、rFVIIIの活性と関連のあるrFVIII分子上のエピトープに結合して、このエピトープが水溶性ポリマーに結合するのを阻止する。回転装置内で、2〜8℃でインキュベートを一晩実施した。結合FVIIIを有する樹脂を、ガラスフリットを用いてろ過により集め、緩衝液AQ2(WFI中、20mM Hepes、20mM CaCl2、0.5M NaCl、0.1%(v/v)ポリソルベート80、室温でpH6.8±0.2)で2度洗浄した。
固定化FVIIIのPEG化は、NEKTAR社(San Carlos、カリフォルニア)によって提供されているPEG化試薬を用いて、パイロットスケールのPEG化FVIII製造に適用される手順に従って実施した。試薬は、安定なFVIII−PEG結合体をもたらす化学的性質を有する20kDaのPEGとした。PEG化速度は、FVIIIタンパク質に対し過剰モルのPEG化試薬を用いて調節した。反応後、過剰の未反応PEG試薬は、グリシン溶液を添加することによって不活性化させた。
PEG化FVIII結合体を有する抗FVIII樹脂をカラムに充填し、緩衝液AQ2およびAM3(WFI中、20mM MES、20mM CaCl2、0.5M NaCl、0.1%(v/v)ポリソルベート80、室温でpH5.9±0,2)で洗浄した。緩衝液AH2(WFI中、20mM Hepes、20mM CaCl2、250mM NaCl、0.1%(v/v)ポリソルベート80、室温でpH6.8±0.2)、次いで、50%のエチレングリコールを含有する緩衝液AE1(WFI中、50%(v/v)エチレングリコール、20mM L−ヒスチジン、20mM CaCl2、250mM NaCl、0.01%(v/v)ポリソルベート80、室温でpH6.8±0.2)を、カラムにポンプ注入することによって、FVIIIの脱着を行った。PEG化FVIIIを画分で集め、UV280シグナルに従ってプールした。
全タンパク質量は、実験的に求めた換算係数1,315(1mg/mlタンパク質=1,315のUV280吸収)を用いて、UV280の吸収によって決定した。この係数は、ブラッドフォード(Bradford)法(DF1013/024)に準拠して全タンパク質の測定から導いた。
FVIIIのA2領域に局在しているエピトープに結合しているモノクローナル抗体F8.1とrFVIIIの複合体上の直鎖状20Kポリエチレングリコール分子を用いて、FVIIIのPEG化実験を実施した。
in vivoにおける、PEG化rFVIIIの生化学的特徴付け
PEG化rFVIIIの生化学的特徴付けの結果を、以下の表1および表2にまとめる。
血液凝固因子中のリジン残基のPEG化
本発明において、上の実施例1による血液凝固タンパク質のPEG化が計画される。すなわち、以下:第IX因子(FIX)、第VIIa因子(FVIIa)、フォンヴィレブランド因子(VWF)、第V因子(FV)、第X因子(FX)、第XI因子、第XII因子(FXII)、トロンビン(FII)、プロテインC、プロテインS、tPA、PAI−1、組織因子(TF)およびADAMTS13プロテアーゼのうちのいずれか1つを用いて、実施例1を繰り返す。特定された各血液凝固因子に対して使用される抗体は、前記血液凝固因子の活性と関連のある血液凝固因子上のエピトープに対する結合親和性を有することができるが、それを必要とするわけではない。
Claims (7)
- 第VIII因子(FVIII)に水溶性ポリマー(WSP)を結合する方法であって、
(a)前記FVIIIにFVIII特異抗体を結合させる条件下で、前記抗体と共に前記FVIIIをインキュベートして、抗体−FVIII複合体を形成させる工程と、
(b)前記抗体−FVIII複合体に前記WSPを結合させる条件下で、前記WSPと共に前記抗体−FVIII複合体をインキュベートする工程と、
(c)前記WSP結合FVIIIを、前記抗体から離す工程を含み、
前記WSPは、ポリエチレングリコール(PEG)、分枝PEG、ポリシアル酸(PSA)、炭水化物、多糖類、プルラン、キトサン、ヒアルロン酸、コンドロイチン硫酸、デルマタン硫酸、でんぷん、デキストラン、カルボキシメチルデキストラン、ポリアルキレンオキシド(PAO)、ポリアルキレングリコール(PAG)、ポリプロピレングリコール(PPG)ポリオキサゾリン、ポリアクリロイルモルホリン、ポリビニルアルコール(PVA)、ポリカルボキシレート、ポリビニルピロリドン、ポリホスファゼン、ポリオキサゾリン、ポリエチレン−co−マレイン酸無水物、ポリスチレン−co−マレイン酸無水物、ポリ(1−ヒドロキシメチルエチレンヒドロキシメチルホルマール)(PHF)、および/または2−メタクリロイルオキシ−2’−エチルトリメチルアンモニウムホスフェート(MPC)からなる群から選択される、方法。 - 前記FVIIIが完全長FVIIIである、請求項1に記載の方法。
- 前記WSPが約2,000から約150,000Daの分子量を有する、請求項1に記載の方法。
- 前記WSPが直鎖状構造または分枝構造を有する、請求項3に記載の方法。
- 前記WSPがPEGである、請求項4に記載の方法。
- 前記WSPがPSAである、請求項4に記載の方法。
- 前記抗体が樹脂に固定化されている、請求項1に記載の方法。
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