Factor VIII Chimeric and Hybrid Polypeptides and Methods of Use Thereof
Patent Information
- Application Number
- JP2023124309
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- Priority Date
- 2012-06-08
- Filing Date
- 2023-07-31
- Publication Date
- 2025-02-26
- Estimated Expiration
- 2032-07-06
AI Technical Summary
【0058】 本発明はまた、出血性障害であると診断されている対象が、長期間作用型FVIIIのポリペプチドの長い投与間隔の候補者であるかどうかを決定するための方法であって、当該対象における短時間作用型FVIIIの半減期を測定して、平均レベルのVWFを有する対象に投与される場合の短時間作用型FVIIIの半減期よりも少なくとも約1.2倍長い半減期が、この対象が長い投与間隔の候補者であることを示すことを含む、方法も提供する。一実施形態において、半減期は、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの半減期よりも少なくとも約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍長い。 特定の実施形態では、例えば以下が提供される: (項目1) 第VIII因子を、それを必要とするヒト対象に投与する方法であって、前記対象に、第VIII因子部分および第2の部分を含む治療用量のキメラポリペプチドを、前記第VIII因子部分からなる等価用量のポリペプチドに必要とされる投与間隔よりも少なくとも約1.5倍長い投与間隔で投与することを含み、前記第VIII因子部分は、重鎖を含む第1の鎖および軽鎖を含む第2の鎖の2つの鎖を有するプロセシングされた第VIII因子を含み、前記第1の鎖および前記第2の鎖は、金属結合により会合される、方法。 (項目2) 前記キメラポリペプチドの前記第VIII因子部分の少なくとも約50%、約60%、約70%、約75%、約80%、約85%、約90%、約95%、または約99%が、プロセシングされた第VIII因子である、項目1に記載の方法。 (項目3) 前記キメラポリペプチドの前記第VIII因子部分の約100%が、プロセシングされた第VIII因子である、項目2に記載の方法。 (項目4) 前記第VIII因子部分は、一本鎖である、一本鎖第VIII因子を含む、項目1または2のいずれか1項に記載の方法。 (項目5) 前記一本鎖第VIII因子は、無傷の細胞内プロセシング部位を含有する、項目4に記載の方法。 (項目6) 前記細胞内プロセシング部位は、完全長第VIII因子の残基1648のアルギニン(配列番号6のアミノ酸20~2351のアルギニン残基1667)に対応する、項目5に記載の方法。 (項目7) 前記キメラポリペプチドの前記第VIII因子部分の少なくとも約1%、約5%、約10%、約15%、約20%、または約25%が、一本鎖第VIII因子である、項目3~6のいずれか1項に記載の方法。 (項目8) 前記キメラポリペプチドの前記第VIII因子部分の約25%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約75%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目9) 前記キメラポリペプチドの前記第VIII因子部分の約20%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約80%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目10) 前記キメラポリペプチドの前記第VIII因子部分の約15%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約85%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目11) 前記キメラポリペプチドの前記第VIII因子部分の約10%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約90%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目12) 前記キメラポリペプチドの前記第VIII因子部分の約5%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約95%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目13) 前記キメラポリペプチドの前記第VIII因子部分の約1%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約99%が、プロセシングされた第VIII因子である、項目7に記載の方法。 (項目14) 前記キメラポリペプチドの前記第VIII因子部分の少なくとも約30%、約40%、約50%、約60%、約70%、約80%、約90%、約95%、または約99%が、一本鎖第VIII因子である、項目3~6のいずれか1項に記載の方法。 (項目15) 前記キメラポリペプチドの前記第VIII因子部分の約30%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約70%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目16) 前記キメラポリペプチドの前記第VIII因子部分の約40%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約60%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目17) 前記キメラポリペプチドの前記第VIII因子部分の約50%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約50%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目18) 前記キメラポリペプチドの前記第VIII因子部分の約60%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約40%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目19) 前記キメラポリペプチドの前記第VIII因子部分の約70%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約30%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目20) 前記キメラポリペプチドの前記第VIII因子部分の約80%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約20%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目21) 前記キメラポリペプチドの前記第VIII因子部分の約85%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約15%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目22) 前記キメラポリペプチドの前記第VIII因子部分の約90%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約10%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目23) 前記キメラポリペプチドの前記第VIII因子部分の約95%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約5%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目24) 前記キメラポリペプチドの前記第VIII因子部分の約99%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約1%が、プロセシングされた第VIII因子である、項目14に記載の方法。 (項目25) 前記キメラポリペプチドの前記第VIII因子部分の約100%が、一本鎖第VIII因子である、項目14に記載の方法。 (項目26) 一本鎖第VIII因子を含む前記キメラポリペプチドは、第VIII因子活性が発色アッセイによってインビトロで測定される場合、2つのFc部分と、前記2つのFc部分のうちの1つと融合するプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵するレベルで、前記第VIII因子活性を有する、項目4~25のいずれか1項に記載の方法。 (項目27) 一本鎖第VIII因子を含む前記キメラポリペプチドは、2つのFc部分と、前記2つのFc部分のうちの1つと融合するプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵するインビボでの第VIII因子活性を有する、項目4~26のいずれか1項に記載の方法。 (項目28) 一本鎖第VIII因子を含む前記キメラポリペプチドは、2つのFc部分と、前記2つのFc部分のうちの1つと融合するプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵する第Xa因子の発生率を有する、項目4~27のいずれか1項に記載の方法。 (項目29) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵するレベルで、活性化プロテインCによって不活性化される、項目4~28のいずれか1項に記載の方法。 (項目30) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵する第IXa因子の相互作用率を有する、項目4~29のいずれか1項に記載の方法。 (項目31) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵するレベルでフォンウィルブランド因子に結合する、項目4~30のいずれか1項に記載の方法。 (項目32) 前記キメラポリペプチドは、 前記第VIII因子部分からなるポリペプチドの能力に匹敵するリン脂質ベシクルと相互作用する能力と、 前記第VIII因子部分からなるポリペプチドの能力に匹敵する第X因子を活性化するXアーゼ複合体を形成する能力と、 前記第VIII因子部分からなるポリペプチドの能力に匹敵する5分以内にαトロンビンによって活性化される能力と 前記第VIII因子部分からなるポリペプチドの能力に匹敵する第IXa因子と相互作用する能力と、からなる群から選択される1つ以上の特性を有する、項目1~31のいずれか1項に記載の方法。 (項目33) 前記キメラポリペプチドは、 Kmが第X因子の濃度の一関数として測定される、前記第VIII因子部分からなるポリペプチドのKmの約1、約1.5、または約2の標準偏差以内のKmで第X因子を活性化するXアーゼ複合体を形成することと、 Vmaxが第X因子の濃度の一関数として測定される、前記第VIII因子部分からなるポリペプチドのVmaxの約1、約1.5、または約2の標準偏差以内のVmaxで第X因子を活性化するXアーゼ複合体を形成することと、 Kdが第IXa因子の濃度の一関数として測定される、前記第VIII因子部分からなるポリペプチドのKdの約1、約1.5、または約2の標準偏差以内のKdで第X因子を活性化するXアーゼ複合体を形成することと、 Vmaxが第IXa因子の濃度の一関数として測定される、前記第VIII因子部分からなるポリペプチドのVmaxの約1、約1.5、または約2の標準偏差以内のVmaxで第X因子を活性化するXアーゼ複合体を形成することと、からなる群から選択される1つ以上の特性を有する、項目32に記載の方法。 (項目34) 前記用量は、1IU/kg当たり1.38IU/dLより大きい平均増分回収率(K値)(活性;観察値)を有する、項目1~33のいずれかに記載の方法。 (項目35) 前記用量は、1IU/kg当たり少なくとも約1.5、少なくとも約1.85、または少なくとも約2.46IU/dLの平均増分回収率(K値)(活性;観察値)を有する、項目34に記載の方法。 (項目36) 前記キメラポリペプチドは、前記患者集団または前記対象において、以下からなる群から選択される、1つ以上の薬物動態パラメーターを示す、項目1~35のいずれかに記載の方法: 25IU/kgの前記キメラポリペプチドが投与された前記対象におけるCmax_OBSが、一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる25IU/kgのポリペプチドが投与された対象におけるCmax_OBSに匹敵すること、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約60.5IU/dLの前記対象におけるCmax_OBS、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約53.1~69IU/dLの前記対象におけるCmax_OBS、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約119IU/dLの前記対象におけるCmax_OBS、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約103~136IU/dLの前記対象におけるCmax_OBS、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約76.5IU/dLの前記対象におけるCmax_OBS、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約64.9~90.1IU/dLの前記対象におけるCmax_OBS、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約182IU/dLの前記対象におけるCmax_OBS、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約146~227IU/dLの前記対象におけるCmax_OBS、 約2.33±1.08mL/時間/kg以下の前記患者集団における平均クリアランス(CL)(活性)、 約1.8~2.69mL/時間/kgの前記患者集団における平均クリアランス(CL)(活性)、 前記第VIII因子部分からなるポリペプチドのクリアランスの約65%である、前記患者集団における平均クリアランス(CL)(活性)、 約1.22~5.19mL/時間/kgの前記対象における平均クリアランス(CL)(活性)、 前記対象におけるクリアランス(CL)(活性)が、一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる同じ量のポリペプチドが投与された対象におけるクリアランスよりも0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.60、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、または0.70倍低いこと、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約1.68mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約1.31~2.15mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約2.32mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約1.64~3.29mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約1.49mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約1.16~1.92mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約1.52mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約1.05~2.20mL/時間/kgの前記対象におけるクリアランス(CL)(活性)、 少なくとも約26.3±8.33時間の前記患者集団における平均平均滞留時間(MRT)(活性)、 約25.9~26.5時間の前記患者集団における平均MRT(活性)、 前記第VIII因子部分からなるポリペプチドの平均MRTよりも約1.5倍長い前記患者集団における平均MRT(活性)、 約14~41.3時間の前記対象における平均滞留時間(MRT)(活性)、 前記対象におけるMRTが、一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる同じ量のポリペプチドが投与された対象におけるMRTよりも少なくとも1.50、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.60、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.70、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.80、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、または1.90倍高いこと、 一段階(aPTT)アッセイによって測定される場合、約27時間の前記対象におけるMRT(活性)、 一段階(aPTT)アッセイによって測定される、約20.6~35.3時間の前記対象におけるMRT(活性)、 二段階(発色)アッセイによって測定される、約23.9~28.5時間の前記対象におけるMRT(活性)、 二段階(発色)アッセイ二段階(発色)アッセイによって測定される、約19.8~28.9時間の前記対象におけるMRT(活性)、 二段階(発色)アッセイ二段階(発色)アッセイによって測定される、約20.5~39.6時間の前記対象におけるMRT(活性)、 約18.3±5.79時間の前記患者集団における平均t1/2ベータ(活性)、 約18~18.4時間である前記患者集団における平均t1/2ベータ(活性)、 前記第VIII因子部分からなるポリペプチドの平均t1/2ベータよりも約1.5倍長い前記患者集団における平均t1/2ベータ(活性)、 約11~26.4時間の前記対象における平均t1/2ベータ(活性)、 一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる同じ量のポリペプチドが投与された対象におけるt1/2ベータ(活性)よりも少なくとも1.50、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.60、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.70、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.80、1.81、1.82、1.83、1.84、1.85、1.86、または1.87倍高い前記対象におけるt1/2ベータ(活性)、 一段階(aPTT)アッセイによって測定される、約18.8時間の前記対象におけるt1/2ベータ(活性)、 一段階(aPTT)アッセイによって測定される、約14.3~24.5時間の前記対象におけるt1/2ベータ(活性)、 二段階(発色)アッセイによって測定される、約16.7時間の前記対象におけるt1/2ベータ(活性)、 二段階(発色)アッセイによって測定される、約13.8~20.1時間の前記対象におけるt1/2ベータ(活性)、 二段階(発色)アッセイによって測定される、約19.8時間の前記対象におけるt1/2ベータ(活性)、 二段階(発色)アッセイによって測定される、約14.3~27.5時間の前記対象におけるt1/2ベータ(活性)、 1IU/kg当たり約2.01±0.44IU/dLの前記患者集団における平均増分回収率(K値)(活性;観察値)、 1IU/kg当たり約1.85~2.46IU/dLの前記患者集団における平均増分回収率(K値)(活性;観察値)、 前記第VIII因子部分からなるポリペプチドの平均増分回収率の約90%である前記患者集団における平均増分回収率(K値)(活性;観察値)、 1IU/kg当たり約1.38~2.88IU/dLの前記対象における増分回収率(K値)(活性;観察値)、 一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなるポリペプチドが投与された対象における増分回収率に匹敵する前記対象における増分回収率、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約2.44IU/dLの前記対象における増分回収率、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約2.12~2.81IU/dLの前記対象における増分回収率、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1.83IU/dLの前記対象における増分回収率、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1.59~2.10IU/dLの前記対象における増分回収率、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約3.09IU/dLの前記対象における増分回収率、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約2.80IU/dLの前記対象における増分回収率、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約2.61~3.66IU/dLの前記対象における増分回収率、および 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約2.24~3.50IU/dLの前記対象における増分回収率、 約55.1±12.3mL/kgの前記患者集団における平均Vss(活性)、 約45.3~56.1mL/kgの前記患者集団における平均Vss(活性)、 約37.7~79.4mL/kgの前記対象における平均Vss(活性)、 一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる同じ量のポリペプチドが投与された対象におけるVss(活性)に匹敵する前記対象におけるVss(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約45.5mL/kgの前記対象におけるVss(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約39.3~52.5mL/kgの前記対象におけるVss(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約62.8mL/kgの前記対象におけるVss(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、約55.2~71.5mL/kgの前記対象におけるVss(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約35.9mL/kgの前記対象におけるVss(活性)、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約30.4~42.3mL/kgの前記対象におけるVss(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約43.4mL/kgの前記対象におけるVss(活性)、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、約38.2~49.2mL/kgの前記対象におけるVss(活性)、 1IU/kg当たり約49.9±18.2IU*h/dLの前記患者集団における平均AUC/用量(活性)、 1IU/kg当たり約44.8~57.6IU*h/dLの前記患者集団における平均AUC/用量(活性)、および 1IU/kg当たり約19.2~81.7IU*h/dLの前記対象におけるAUC/用量、 一段階(aPTT)アッセイまたは二段階(発色)アッセイによって測定される場合、前記完全長成熟第VIII因子からなる同じ量のポリペプチドが投与された前記対象におけるAUCINFよりも少なくとも1.45 1.46、1.47、1.48、1.49、1.50、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.60、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.70、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.80、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.90倍高い前記対象におけるAUCINF、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1440±316時間*IU/dLの前記対象におけるAUCINF、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1160~1880時間*IU/dLの前記対象におけるAUCINF、 約25IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1480時間*IU/dLの前記対象におけるAUCINF、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約2910±1320時間*IU/dLの前記対象におけるAUCINF、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約1980~3970時間*IU/dLの前記対象におけるAUCINF、 約65IU/kgの前記キメラポリペプチドが投与される場合、一段階(aPTT)アッセイによって測定される、1IU/kg当たり約2800時間*IU/dLの前記対象におけるAUCINF、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約1660時間*IU/dLの前記対象におけるAUCINF、 約25IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約1300~2120時間*IU/dLの前記対象におけるAUCINF、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約4280時間*IU/dLの前記対象におけるAUCINF、 約65IU/kgの前記キメラポリペプチドが投与される場合、二段階(発色)アッセイによって測定される、1IU/kg当たり約2960~6190時間*IU/dLの前記対象におけるAUCINF。 (項目37) 前記治療用量は、約10~約150、100~110、110~120、120~130、130~140、140~150、110、115、120、125、130、135、140、145、または150IU/kgである、項目1~36のいずれかに記載の方法。 (項目38) 前記投与間隔は、1.5~5、1.5、2、3、4、もしくは5日、またはそれ以上である、項目1~37のいずれかに記載の方法。 (項目39) 第VIII因子活性を有するキメラポリペプチドを含む組成物であって、前記ポリペプチドの少なくとも約30%、約40%、約50%、約60%、約70%、約80%、約90%、約95%、約96%、約97%、約98%、約99%、または約100%が、一本鎖第VIII因子である第VIII因子部分、および第2の部分を含み、前記一本鎖第VIII因子は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と少なくとも90%または95%同一である、組成物。 (項目40) 前記一本鎖第VIII因子は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と同一である、項目39に記載の組成物。 (項目41) 第VIII因子活性を有するキメラポリペプチドを含む組成物であって、前記ポリペプチドの少なくとも約30%、約40%、約50%、約60%、約70%、約80%、約90%、約95%、約96%、約97%、約98%、約99%、または約100%が、一本鎖第VIII因子である第VIII因子部分、および第2の部分を含み、前記一本鎖第VIII因子は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と少なくとも90%または95%同一である、組成物。 (項目42) 前記一本鎖第VIII因子は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と同一である、項目41に記載の組成物。 (項目43) 前記第2の部分は、Fc領域、アルブミン、PAS配列、トランスフェリンもしくはCTP(4つのO-グリカンを有するhCGの28アミノ酸C末端ペプチド(CTP))、ポリエチレングリコール(PEG)、ヒドロキシエチルデンプン(HES)、アルブミン結合ポリペプチド、アルブミン結合小分子、またはそれらのいずれかの組み合わせである、項目39~42のいずれかに記載の組成物。 (項目44) 前記第2の部分は、Fc領域である、項目43に記載の組成物。 (項目45) 前記Fcは、表2に示される前記Fcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と少なくとも90%または95%同一である、項目44に記載の組成物。 (項目46) 前記Fcは、表2に示される前記Fcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と同一である、項目45に記載の組成物。 (項目47) 前記ポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目39に記載の組成物。 (項目48) 前記ポリペプチドは、シグナル配列を含まない表2A(i)に示される前記第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と同一であるか、またはシグナル配列を含む表2A(i)に示される前記第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目39に記載の組成物。 (項目49) 前記ポリペプチドは、第2のポリペプチドを含むハイブリッドの形態であり、前記第2のポリペプチドは、Fcから本質的になる、項目39~48のいずれかに記載の組成物。(項目50) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目49に記載の組成物。 (項目51) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示される前記アミノ酸配列(配列番号4のアミノ酸21~247)と同一であるか、またはシグナル配列を含む表2A(ii)に示される前記アミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目50に記載の組成物。 (項目52) 前記ポリペプチドは、前記第VIII因子からなるポリペプチドに対して少なくとも1.5~6倍長い、1.5~5倍長い、1.5~4倍長い、1.5~3倍長い、または1.5~2倍長い半減期を有する、項目39~51のいずれかに記載の組成物。 (項目53) 前記第VIII因子部分からなるポリペプチドの能力に匹敵するリン脂質ベシクルと相互作用する能力と、 前記第VIII因子部分からなるポリペプチドの能力に匹敵する第X因子を活性化するXアーゼ複合体を形成する能力と、 前記第VIII因子部分からなるポリペプチドの能力に匹敵する5分以内にαトロンビンによって活性化される能力と 前記第VIII因子部分からなるポリペプチドの能力に匹敵する第IXa因子と相互作用する能力と、からなる群から選択される、1、約1.5、または約2つ以上の特性を有する、項目39~52のいずれかに記載の組成物。 (項目54) 前記ポリペプチドの前記第VIII因子部分の約70%、約60%、約50%、約40%、約30%、約20%、約10%、約5%、約4%、約3%、約2%、または約1%が、プロセシングされた第VIII因子である、項目39~53のいずれかに記載の組成物。 (項目55) 一本鎖第VIII因子を含む前記キメラポリペプチドは、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵する第VIII因子活性を有し、前記プロセシングされた第VIII因子は、前記第VIII因子が発色アッセイによってインビトロで測定される場合、前記2つのFc部分のうちの1つのFcと融合している、項目39~54のいずれか1項に記載の組成物。 (項目56) 一本鎖第VIII因子を含む前記キメラポリペプチドは、プロセシングされた第VIII因子に匹敵する第Xa因子の発生率を有する、項目39~55のいずれか1項に記載の組成物。 (項目57) 一本鎖第VIII因子を含む前記キメラポリペプチドは、2つのFc部分と、前記2つのFc部分のうちの1つと融合するプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵する第Xa因子の発生率を有する、項目39~56のいずれか1項に記載の組成物。 (項目58) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵する第Xa因子の相互作用率を有する、項目39~57のいずれか1項 に記載の組成物。 (項目59) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵するレベルで、活性化プロテインCによって不活性化される、項目39~58のいずれか1項に記載の組成物。 (項目60) 前記キメラポリペプチド中の前記一本鎖第VIII因子は、2つのFc部分とプロセシングされた第VIII因子とからなるキメラポリペプチド中のプロセシングされた第VIII因子に匹敵するレベルで、フォンウィルブランド因子に結合する、項目39~59のいずれか1項に記載の組成物。 (項目61) 一本鎖第VIII因子を含む前記キメラポリペプチドは、2つのFc部分と、前記2つのFc部分のうちの1つと融合するプロセシングされた第VIII因子とからなるキメラポリペプチドに匹敵するインビボでの第VIII因子活性を有する、項目39~60のいずれか1項に記載の組成物。 (項目62) 前記キメラポリペプチドの前記第VIII因子部分の約30%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約70%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目63) 前記キメラポリペプチドの前記第VIII因子部分の約40%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約60%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目64) 前記キメラポリペプチドの前記第VIII因子部分の約50%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約50%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目65) 前記キメラポリペプチドの前記第VIII因子部分の約60%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約40%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目66) 前記キメラポリペプチドの前記第VIII因子部分の約70%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約30%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目67) 前記キメラポリペプチドの前記第VIII因子部分の約80%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約20%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目68) 前記キメラポリペプチドの前記第VIII因子部分の約90%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約10%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目69) 前記キメラポリペプチドの前記第VIII因子部分の約95%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約5%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目70) 前記キメラポリペプチドの前記第VIII因子部分の約99%が、一本鎖第VIII因子であり、前記キメラポリペプチドの前記第VIII因子部分の約5%が、プロセシングされた第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目71) 前記キメラポリペプチドの前記第VIII因子部分の約100%が、一本鎖第VIII因子である、項目39~61のいずれか1項に記載の組成物。 (項目72) 賦形剤をさらに含む薬学的組成物である、項目39~71のいずれか1項に記載の組成物。 (項目73) 治療上有効量の項目39~72のいずれか1項に記載の組成物を投与することを含む、出血性状態を治療する方法。 (項目74) 前記治療は、予防的処置である、項目73に記載の方法。 (項目75) 前記治療は、オンデマンド治療である、項目73に記載の方法。 (項目76) 前記出血性状態は、出血凝固障害、関節血症、筋肉出血、口の出血、出血、筋肉への出血、口腔出血、外傷、頭部外傷(trauma capitis)、胃腸出血、頭蓋内出血、腹腔内出血、胸腔内出血、骨折、中枢神経系の出血、咽頭後隙の出血、後腹膜腔内出血、および腸腰筋鞘(illiopsoas sheath)内の出血からなる群から選択される、項目73~75のいずれか1項に記載の方法。 (項目77) 前記出血凝固障害は、血友病である、項目76に記載の方法。 (項目78) 前記出血凝固障害は、血友病Aである、項目77に記載の方法。 (項目79) 前記投与は、1%を超えるレベルで、前記患者集団の少なくとも90%において、第VIII因子のトラフレベルを維持する、項目1~38および73~78のいずれか1項に記載の方法。 (項目80) 前記投与は、3%を超えるレベルで、前記集団の少なくとも60%において、第VIII因子のトラフレベルを維持する、項目1~38および73~78のいずれか1項に記載の方法。 (項目81) 1%を超えるレベルで少なくとも1日、2日、または3日間、患者集団において、第VIII因子を維持する方法であって、前記集団に、治療上有効量のrFVIIIFcを投与することを含み、前記治療上有効量は、少なくとも25IU/kgのrFVIIIFcであり、前記集団の少なくとも90%が、少なくとも1%以上の前記第VIII因子のトラフレベルを維持する、方法。 (項目82) 3%を超えるレベルで少なくとも1日、2日、または3日間、患者集団において、第VIII因子を維持する方法であって、前記集団に、治療上有効量のrFVIIIFcを投与することを含み、前記治療上有効量は、少なくとも65IU/kgのrFVIIIFcであり、前記集団の少なくとも90%が、少なくとも3%以上の前記第VIII因子のトラフレベルを維持する、方法。 (項目83) 前記一本鎖第VIII因子は、アルギニン1645に対応するアミノ酸位置での置換または突然変異を含む、項目4~38および73~82のいずれか1項に記載の方法。 (項目84) 前記一本鎖第VIII因子は、アルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目4~38および73~83のいずれか1項に記載の方法。 (項目85) 前記一本鎖第VIII因子は、完全長第VIII因子中のアルギニン1645およびアルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目4~38および73~84のいずれか1項に記載の方法。 (項目86) アルギニン1645に対応するアミノ酸位置で置換されたアミノ酸は、アルギニン1648に対応するアミノ酸位置で置換されたアミノ酸とは異なるアミノ酸である、項目85に記載の方法。 (項目87) 前記置換または突然変異は、アルギニン以外のアミノ酸である、項目83~86のいずれか1項に記載の方法。 (項目88) 前記一本鎖FVIIIは、無傷の細胞内プロセシング部位を含む、項目39~72のいずれか1項に記載の組成物。 (項目89) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1645に対応するアミノ酸位置での置換または突然変異を含む、項目39~72のいずれか1項に記載の組成物。 (項目90) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目39~72のいずれか1項に記載の組成物。 (項目91) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1645およびアルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目39~72のいずれか1項に記載の組成物。 (項目92) アルギニン1645に対応するアミノ酸位置で置換されたアミノ酸は、アルギニン1648に対応するアミノ酸位置で置換されたアミノ酸とは異なるアミノ酸である、項目91に記載の組成物。 (項目93) 前記置換または突然変異は、アルギニン以外のアミノ酸である、項目89~92のいずれか1項に記載の組成物。 (項目94) 前記一本鎖第VIII因子は、無傷の細胞内プロセシング部位を含む、項目73~82のいずれか1項に記載の方法。 (項目95) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1645に対応するアミノ酸位置での置換または突然変異を含む、項目73~82のいずれか1項に記載の方法。 (項目96) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目73~82のいずれか1項に記載の方法。 (項目97) 前記一本鎖第VIII因子は、完全長成熟第VIII因子中のアルギニン1645およびアルギニン1648に対応するアミノ酸位置での置換または突然変異を含む、項目73~82のいずれか1項に記載の方法。 (項目98) アルギニン1645に対応するアミノ酸位置で置換されたアミノ酸は、完全長第VIII因子中のアルギニン1648に対応するアミノ酸位置で置換されたアミノ酸とは異なるアミノ酸である、項目97に記載の方法。 (項目99) 前記置換または突然変異は、アルギニン以外のアミノ酸である、項目95~98のいずれか1項に記載の方法。 (項目100) アルギニン以外のアミノ酸残基は、イソロイシン、ロイシン、リシン、メチオニン、フェニルアラニン、トレオニン、トリプトファン、バリン、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン酸、グルタミン、グリシン、プロリン、セレノシステイン、セリン、チロシン、ヒスチジン、オルニチン、ピロリシン、またはタウリンからなる群から選択される、項目87または99に記載の方法。 (項目101) アルギニン以外のアミノ酸残基は、イソロイシン、ロイシン、リシン、メチオニン、フェニルアラニン、トレオニン、トリプトファン、バリン、アラニン、アスパラギン、アスパラギン酸、システイン、グルタミン酸、グルタミン、グリシン、プロリン、セレノシステイン、セリン、チロシン、ヒスチジン、オルニチン、ピロリシン、またはタウリンからなる群から選択される、項目93に記載の組成物。 (項目102) 前記対象は、小児対象である、項目1~38、73~87、または94~100のいずれか1項に記載の方法。 (項目103) 対象に、出血エピソードを予防、減少、または治療する方法であって、前記対象に、有効量の長時間作用型第VIII因子(FVIII)タンパク質を投与することを含み、前記対象は、血漿中に高レベルのフォンウィルブランド因子(VWF)を発現する、方法。(項目104) 前記対象は、血漿中に高レベルのVWFを発現するとして特定されている、項目103に記載の方法。 (項目105) 対象において、出血エピソードを予防、減少、または治療する方法であって、 (a)高レベルのVWFを有する対象を、前記対象の血漿中のVWFレベルを測定することによって特定することであって、少なくとも約100IU/dLのVWFレベルが、高レベルのVWFを有するものとして前記対象を特定する、対象を特定することと、 (b)前記対象に、有効量の長時間作用型FVIIIタンパク質を投与することと、を含む、方法。 (項目106) 前記対象は、ヒトである、項目103~105のいずれかに記載の方法。 (項目107) 前記対象は、小児対象である、項目103~106のいずれかに記載の方法。 (項目108) 前記対象は、血友病Aを有する、項目103~107のいずれかに記載の方法。 (項目109) 前記高レベルのVWFは、少なくとも約100IU/dLである、項目103~108のいずれかに記載の方法。 (項目110) 前記高レベルのVWFは、約100IU/dL~約200IU/dLである、項目103~109のいずれかに記載の方法。 (項目111) 前記高レベルのVWFは、約110IU/dL、約120IU/dL、約130IU/dL、約140IU/dL、約150IU/dL、約160IU/dL、約170IU/dL、約180IU/dL、約190IU/dL、または約200IU/dLである、項目110に記載の方法。 (項目112) 前記対象は、血清型A、B、またはABを有する、項目103~111のいずれかに記載の方法。 (項目113) 前記長期間作用型FVIIIタンパク質は、前記対象において、20時間~約40時間の半減期を有する、項目103~112のいずれかに記載の方法。 (項目114) 前記長期間作用型FVIIIタンパク質は、約21時間、22時間、23時間、24時間、25時間、26時間、27時間、28時間、29時間、30時間、31時間、31時間、32時間、33時間、34時間、35時間、36時間、37時間、38時間、39時間、または40時間の半減期を有する、項目113に記載の方法。 (項目115) 前記長期間作用型FVIIIタンパク質は、約20~約27時間の半減期を有する、項目114に記載の方法。 (項目116) 前記長期間作用型FVIIIタンパク質は、平均レベルのVWFを有する個人に投与される場合の前記前記長期間作用型FVIIIタンパク質の半減期よりも少なくとも約1.2倍高い半減期を有する、項目103~115のいずれかに記載の方法。 (項目117) 前記長期間作用型FVIIIタンパク質は、平均レベルのVWFを有する個人に投与される場合の前記前記長期間作用型FVIIIタンパク質の半減期よりも少なくとも約約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍高い半減期を有する、項目116に記載の方法。 (項目118) 前記有効量は、少なくとも約20IU/kg、少なくとも約25IU/kg、少なくとも約30IU/kg、少なくとも約35IU/kg、少なくとも約40IU/kg、少なくとも約45IU/kg、少なくとも約50IU/kg、少なくとも約55IU/kg、少なくとも約60IU/kg、少なくとも約65IU/kg、少なくとも約70IU/kg、少なくとも約75IU/kg、少なくとも約80IU/kg、少なくとも約85IU/kg、または少なくとも約90IU/kgである、項目103~117のいずれか1項に記載の方法。 (項目119) 前記有効量は、少なくとも約65IU/kg~少なくとも約90IU/kgである、項目103~118のいずれかに記載の方法。 (項目120) 前記有効量は、80IU/kgである、項目119に記載の方法。 (項目121) 前記長期間作用型FVIIIタンパク質は、72時間ごとまたはそれ以上で投与される、項目103~120のいずれかに記載の方法。 (項目122) 前記長期間作用型FVIIIタンパク質は、1週間またはそれ以上で約1回投与される、項目121に記載の方法。 (項目123) 前記有効量の前記長期間作用型FVIIIタンパク質は、10日ごとに約1回、2週間ごとに約1回、15日ごとに約1回、20日ごとに約1回、3週間ごとに約1回、25日ごとに約1回、4週間ごとに約1回、または1ヶ月ごとに約1回投与される、項目121に記載の方法。 (項目124) 前記長期間作用型FVIIIは、80IU/kgの投与量で72時間ごとに1回投与される、項目121に記載の方法。 (項目125) 前記対象は、小児対象である、項目124に記載の方法。 (項目126) 前記投与は、出血エピソードの5~20%超、5~15%超、5~10%超、10~20%超、または10~15%超を解消する、項目103~125のいずれかに記載の方法。 (項目127) 前記対象において、血漿第VIII因子:Cのトラフレベルは、1~3または3~5IU/dLを超えて維持される、項目103~126のいずれかに記載の方法。 (項目128) 前記投与は、前記対象において、出血エピソードを予防する、項目103~127のいずれかに記載の方法。 (項目129) 前記出血エピソードは、突発的である、項目128に記載の方法。 (項目130) 前記投与は、出血エピソードの80~100%超、80~90%超、85~90%超、90~100%超、90~95%超、または95~100%超を解消する、項目129に記載の方法。 (項目131) 前記投与は、外科手術を必要とする対象の集団において、恒常性(homeostatis)を維持する、項目103~130のいずれかに記載の方法。 (項目132) 前記長時間作用型FVIIIタンパク質は、前記外科手術前、その間、またはその後に投与される、項目103~131のいずれかに記載の方法。 (項目133) 前記外科手術は、小手術、大手術、抜歯、扁桃摘出術、鼠径部ヘルニア切開術、滑膜切除術、人工膝関節全置換術、開頭術、骨接合術、外傷外科手術、頭蓋内手術、腹腔内手術、胸腔内手術、または関節置換手術である、項目132に記載の方法。 (項目134) 前記外科手術は、緊急手術である、項目131~133のいずれかに記載の方法。 (項目135) 前記長期間作用型FVIIIタンパク質は、FVIIIからなるポリペプチドよりも長い半減期を有する、項目103~134のいずれかに記載の方法。 (項目136) 前記長期間作用型FVIIIタンパク質は、ペグ化、HES化、またはポリシアル化されている、項目103~135のいずれかに記載の方法。 (項目137) 前記長期間作用型FVIIIタンパク質は、FVIII部分および第2の部分を含むキメラタンパク質である、項目103~136のいずれかに記載の方法。 (項目138) 前記第2の部分は、Fc領域、アルブミン、PAS配列、トランスフェリンもしくはCTP(4つのO-グリカンを有するhCGの28アミノ酸C末端ペプチド(CTP))、ポリエチレングリコール(PEG)、ヒドロキシエチルデンプン(HES)、アルブミン結合ポリペプチド、アルブミン結合小分子、またはそれらのいずれかの組み合わせである、項目137に記載の方法。 (項目139) 前記第2の部分は、前記FVIII部分のアミノ末端またはカルボキシ末端と融合する、項目137または138に記載の方法。 (項目140) 前記第2の部分は、前記FVIII部分の2つのアミノ酸間に挿入される、項目137または138に記載の方法。 (項目141) 前記キメラタンパク質は、FVIIIFc単量体二量体ハイブリッドである、項目137~140のいずれかに記載の方法。 (項目142) 前記FVIII部分は、一本鎖である、項目141に記載の方法。 (項目143) 前記FVIII部分は、重鎖および軽鎖を含む、項目142に記載の方法。 (項目144) 前記FVIII部分は、完全長第VIII因子、成熟第VIII因子、またはBドメインの完全もしくは部分欠失を有する第VIII因子を含む、項目137~143のいずれかに記載の方法。 (項目145) 前記FVIII部分は、配列番号2のアミノ酸1~1438または配列番号6のアミノ酸1~2332と少なくとも80%、85%、90%、95%、96%、97%、98%、99%、または100%同一であるアミノ酸配列を含む、項目141に記載の方法。 (項目146) 前記FVIII部分は、配列番号2のアミノ酸1~1438または配列番号6のアミノ酸1~2332を含む、項目145に記載の方法。 (項目147) 前記キメラポリペプチドの前記第2の部分は、配列番号2のアミノ酸1439~1665または配列番号6のアミノ酸2333~2559と少なくとも80%、85%、90%、95%、96%、97%、98%、99%、または100%同一であるFc部分を含む、項目137~146のいずれかに記載の方法。 (項目148) 前記第2の部分は、配列番号2のアミノ酸1439~1665または配列番号6のアミノ酸2333~2559を含む、項目147に記載の方法。 (項目149) 前記長期間作用型FVIIIのポリペプチドは、少なくとも1つの賦形剤を含む薬学的組成物の一部として投与される、項目103~148のいずれか1項に記載の方法。 (項目150) 出血性障害であると診断されている対象を治療する方法であって、前記対象におけるFVIII-Fcの半減期を測定し、正常な対象におけるFVIII-Fcの半減期よりも少なくとも約1.2倍長い半減期が、前記対象が長い投与間隔の候補者であることを示すことと、有効量のFVIII-Fcポリペプチドを少なくとも3日間の投与間隔で投与することと、を含む、方法。 (項目151) 出血性障害であると診断されている対象を治療する方法であって、対象に、有効量のFVIII-Fcポリペプチドを少なくとも3日間の投与間隔で投与することであって、前記対象におけるFVIII-Fcの半減期が、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの半減期よりも少なくとも約1.2倍長い、投与することを含む、方法。 (項目152) 前記対象におけるFVIII-Fcの血漿半減期は、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの血漿半減期よりも少なくとも約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍長い、項目150または151に記載の方法。 (項目153) 前記FVIII-Fcの血漿半減期は、20~40時間である、項目150~152のいずれかに記載の方法。 (項目154) 前記長期間作用型FVIIIタンパク質は、約21時間、22時間、23時間、24時間、25時間、26時間、27時間、28時間、29時間、30時間、31時間、31時間、32時間、33時間、34時間、35時間、36時間、37時間、38時間、39時間、または40時間の半減期を有する、項目153に記載の方法。 (項目155) 前記長期間作用型FVIIIタンパク質は、約20~約27時間の半減期を有する、項目154に記載の方法。 (項目156) 出血性障害であると診断されている対象を治療する方法であって、前記対象に投与される短時間作用型FVIIIの半減期を測定し、平均レベルのVWFを有する対象における前記短時間作用型FVIIIの半減期よりも少なくとも約1.2倍長い半減期が、前記対象が長い投与間隔の候補者であることを示すことと、有効量の長期間作用型FVIII-Fcポリペプチドを少なくとも3日間の投与間隔で投与することと、を含む、方法。 (項目157) 前記対象における前記短時間作用型FVIIIの半減期は、平均レベルのVWFを有する対象に投与される場合の短時間作用型FVIIIの半減期よりも少なくとも約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍長い、項目156に記載の方法。 (項目158) 前記対象は、ヒトである、項目150~157のいずれかに記載の方法。 (項目159) 前記対象は、小児対象である、項目150~158のいずれかに記載の方法。 (項目160) 前記対象は、血友病Aを有する、項目150~159のいずれかに記載の方法。 (項目161) 前記対象は、血清型A、B、またはABを有する、項目150~160のいずれかに記載の方法。 (項目162) 前記長期間作用型FVIII-Fcは、少なくとも約20IU/kg、少なくとも約25IU/kg、少なくとも約30IU/kg、少なくとも約35IU/kg、少なくとも約40IU/kg、少なくとも約45IU/kg、少なくとも約50IU/kg、少なくとも約55IU/kg、少なくとも約60IU/kg、少なくとも約65IU/kg、少なくとも約70IU/kg、少なくとも約75IU/kg、少なくとも約80IU/kg、少なくとも約85IU/kg、または少なくとも約90IU/kgの有効量で投与される、項目150~161のいずれかに記載の方法。 (項目163) 前記有効量は、少なくとも約65IU/kg~少なくとも約90IU/kgである、項目150~161のいずれかに記載の方法。 (項目164) 前記有効量の前記FVIII-Fcタンパク質は、1週間ごとに約1回、10日ごとに約1回、2週間ごとに約1回、15日ごとに約1回、20日ごとに約1回、3週間ごとに約1回、25日ごとに約1回、4週間ごとに約1回、または1ヶ月ごとに約1回投与される、項目150~163のいずれかに記載の方法。 (項目165) 前記投与は、出血エピソードの5~20%超、5~15%超、5~10%超、10~20%超、または10~15%超を解消する、項目150~164のいずれかに記載の方法。 (項目166) 前記対象において、血漿第VIII因子:Cのトラフレベルは、1~3または3~5IU/dLを超えて維持される、項目150~165のいずれかに記載の方法。 (項目167) 前記投与は、前記対象における出血エピソードを予防する、項目150~166のいずれかに記載の方法。 (項目168) 前記出血エピソードは、突発的である、項目167に記載の方法。 (項目169) 前記投与は、出血エピソードの80~100%超、80~90%超、85~90%超、90~100%超、90~95%超、または95~100%超を解消する、項目167に記載の方法。 (項目170) 前記投与は、外科手術を必要とする対象の集団において、恒常性を維持する、項目150~169のいずれかに記載の方法。 (項目171) 前記FVIII-Fcタンパク質は、前記外科手術前、その間、またはその後に投与される、求項150~170のいずれかに記載の方法。 (項目172) 前記外科手術は、小手術、大手術、抜歯、扁桃摘出術、鼠径部ヘルニア切開術、滑膜切除術、人工膝関節全置換術、開頭術、骨接合術、外傷外科手術、頭蓋内手術、腹腔内手術、胸腔内手術、または関節置換手術である、項目170または171に記載の方法。 (項目173) 前記外科手術は、緊急手術である、項目170~172のいずれかに記載の方法。 (項目174) 前記FVIII-Fcタンパク質は、FVIIIからなるポリペプチドよりも長い半減期を有する、項目150~173のいずれかに記載の方法。 (項目175) 前記FVIII-Fcタンパク質は、ペグ化、HES化、またはポリシアル化されている、項目150~174のいずれかに記載の方法。 (項目176) 前記FVIII-Fcタンパク質は、FVIIIFc単量体二量体ハイブリッドである、項目174または175に記載の方法。 (項目177) 前記FVIII部分は、一本鎖である、項目172~176のいずれかに記載の方法。(項目178) 前記FVIII部分は、重鎖および軽鎖を含む、項目177に記載の方法。 (項目179) 前記FVIII部分は、完全長第VIII因子、成熟第VIII因子、またはBドメインの完全もしくは部分欠失を有する第VIII因子を含む、項目174~178のいずれかに記載の方法。 (項目180) 前記FVIII部分は、配列番号2のアミノ酸1~1438または配列番号6のアミノ酸1~2332と少なくとも80%、85%、90%、95%、96%、97%、98%、99%、または100%同一であるアミノ酸配列を含む、項目179に記載の方法。 (項目181) 前記FVIII部分は、配列番号2のアミノ酸1~1438または配列番号6のアミノ酸1~2332を含む、項目180に記載の方法。 (項目182) 前記キメラポリペプチドの前記第2の部分は、配列番号2のアミノ酸1439~1665または配列番号6のアミノ酸2333~2559と少なくとも80%、85%、90%、95%、96%、97%、98%、99%、または100%同一であるFc部分を含む、項目174~181のいずれかに記載の方法。 (項目183) 前記第2の部分は、配列番号2のアミノ酸1439~1665または配列番号6のアミノ酸2333~2559を含む、項目182に記載の方法。 (項目184) 前記FVIII-Fcポリペプチドは、少なくとも1つの賦形剤を含む薬学的組成物の一部として投与される、項目150~183のいずれかに記載の方法。 (項目185) 出血性障害であると診断されている対象が、長時間作用型FVIIIポリペプチドを用いた長い投与間隔の候補者であるかどうかを決定するための方法であって、血漿VWFの発現レベルを測定し、少なくとも100IU/dLのVWF発現レベルが、前記対象が長時間作用型FVIIIポリペプチドを用いた長い投与間隔の候補者であることを示すことを含む、方法。 (項目186) 前記VWF発現レベルは、少なくとも約110IU/dL、約120IU/dL、約130IU/dL、約140IU/dL、約150IU/dL、約160IU/dL、約170IU/dL、約180IU/dL、約190IU/dL、または約200IU/dLである、項目185に記載の方法。 (項目187) 出血性障害であると診断されている対象が、長時間作用型FVIIIポリペプチドの長い投与間隔の候補者であるかどうかを決定するための方法であって、前記対象におけるFVIII-Fcの半減期を測定し、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの半減期よりも少なくとも約1.2倍長い半減期が、前記対象が長い投与間隔の候補者であることを示す、方法。 (項目188) FVIII-Fcの半減期は、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの半減期よりも少なくとも約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍長い、項目187に記載の方法。 (項目189) 出血性障害であると診断されている対象が、長時間作用型FVIIIポリペプチドの長い投与間隔の候補者であるかどうかを決定するための方法であって、前記対象における短時間作用型FVIIIの半減期を測定し、平均レベルのVWFを有する対象に投与される場合の短時間作用型FVIIIの半減期よりも少なくとも約1.2倍長い半減期が、前記対象が長い投与間隔の候補者であることを示す、方法。 (項目190) 前記半減期は、平均レベルのVWFを有する対象に投与される場合のFVIII-Fcの半減期よりも少なくとも約1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、または2.5倍長い、項目189に記載の方法。 (項目191) 前記キメラポリペプチドは、Fc部分を含む、項目1~38、73~87、94~100、および102~190のいずれかに記載の方法。 (項目192) 前記投与間隔は、前記第VIII因子部分からなる等価用量のポリペプチドに必要とされる投与間隔よりも少なくとも約1.5~6倍長い、1.5~5倍長い、1.5~4倍長い、1.5~3倍長い、または1.5~2倍長い、項目191に記載の方法。 (項目193) 前記投与間隔は、前記第VIII因子部分からなる等価用量のポリペプチドに必要とされる投与間隔よりも少なくとも約1.5、2、2.5、3、3.5、4、4.5、5、5.5、または6倍長い、項目192に記載の方法。 (項目194) 前記キメラポリペプチドの前記投与間隔は、約3、4、5、6、7、8、9、10、11、12、13、もしくは14日ごと、またはそれ以上である、項目1~38、73~87、94~100、および102~193のいずれかに記載の方法。 (項目195) 前記対象は、予防的処置を必要とする、項目1~38、73~87、94~100、および102~194のいずれかに記載の方法。 (項目196) 前記対象は、オンデマンドを必要とする、項目1~38、73~87、94~100、および102~193のいずれかに記載の方法。 (項目197) 前記対象は、出血エピソードの治療を必要とする、項目196に記載の方法。 (項目198) 前記対象は、関節血症、筋肉出血、口の出血、出血、筋肉への出血、口腔出血、外傷、頭部外傷、胃腸出血、頭蓋内出血、腹腔内出血、胸腔内出血、骨折、中枢神経系の出血、咽頭後隙の出血、後腹膜腔内出血、または腸腰筋鞘内の出血の治療を必要とする、項目197に記載の方法。 (項目199) 前記対象は、外科的予防、手術中管理、または外科手術のための処置を必要とする、項目196に記載の方法。 (項目200) 前記外科手術は、小手術、大手術、抜歯、扁桃摘出術、鼠径部ヘルニア切開術、滑膜切除術、人工膝関節全置換術、開頭術、骨接合術、外傷外科手術、頭蓋内手術、腹腔内手術、胸腔内手術、または関節置換手術である、項目199に記載の方法。 (項目201) 前記キメラポリペプチドの前記投与間隔は、24~36、24~48、24~72、24~96、24~120、24~144、24~168、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、もしくは72時間ごと、またはそれ以上で約1回である、項目196~200のいずれかに記載の方法。 (項目202) 前記治療用量は、10~100IU/kgである、項目1~38、73~87、94~100、および102~201のいずれかに記載の方法。 (項目203) 前記治療用量は、10~20、20~30、30~40、40~50、50~60、60~70、70~80、80~90、または90~100IU/kgである、項目201に記載の方法。 (項目204) 前記治療用量は、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、または100IU/kgである、項目202に記載の方法。 (項目205) 前記第VIII因子は、ヒト第VIII因子である、項目1~38、73~87、94~100、または102~204のいずれかに記載の方法。 (項目206) 前記第VIII因子は、前記Bドメインの完全または部分欠失を有する、項目1~38、73~87、94~100、および102~205のいずれかに記載の方法。 (項目207) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457、配列番号6のアミノ酸20~2351)と少なくとも90%または95%同一である、項目205に記載の方法。 (項目208) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と同一である、項目207に記載の方法。 (項目209) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と少なくとも90%または95%同一である、項目205に記載の方法。 (項目210) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と同一である、項目209に記載の方法。 (項目211) 前記キメラポリペプチドの前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と少なくとも90%または95%同一である、項目205または206に記載の方法。 (項目212) 前記キメラポリペプチドの前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と同一である、項目211に記載の方法。 (項目213) 前記キメラポリペプチドは、前記キメラポリペプチドと結合した第2のポリペプチドを含むハイブリッドの形態であり、前記第2のポリペプチドは、Fcから本質的になる、項目1~38、73~87、94~100、および102~212のいずれかに記載の方法。 (項目214) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目213に記載の方法。 (項目215) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と同一である配列を含む、項目214に記載の方法。 (項目216) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む、表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目213~215のいずれかに記載の方法。 (項目217) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と同一であるか、またはシグナル配列を含む、表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と同一である配列から本質的になる、項目216に記載の方法。 (項目218) 前記キメラポリペプチドは、少なくとも1つの賦形剤を含む薬学的組成物の一部として投与される、項目1~38、73~87、94~100、および102~217のいずれかに記載の方法。 (項目219) 第VIII因子を、それを必要とするヒト対象に投与する方法であって、前記対象に、第VIII因子部分および第2の部分を含む治療用量のキメラポリペプチドを投与して、前記第VIII因子部分からなる等価用量のポリペプチドによって得られる血漿濃度対時間曲線下面積(AUC)よりも少なくとも約1.25倍大きいAUCを得ることを含む、方法。 (項目220) 前記キメラポリペプチドは、約3、4、5、6、7、8、9、10、11、12、13、もしくは14日ごと、またはそれ以上の投与間隔で投与される、項目219に記載の方法。 (項目221) 前記対象は、予防的処置を必要とする、項目219または220に記載の方法。 (項目222) 前記対象は、オンデマンド治療を必要とする、項目219または220に記載の方法。(項目223) 前記対象は、出血エピソードの治療を必要とする、項目219~222のいずれかに記載の方法。 (項目224) 前記対象は、関節血症、筋肉出血、口の出血、出血、筋肉への出血、口腔出血、外傷、頭部外傷、胃腸出血、頭蓋内出血、腹腔内出血、胸腔内出血、骨折、中枢神経系の出血、咽頭後隙の出血、後腹膜腔内出血、または腸腰筋鞘内の出血の治療を必要とする、項目219~222のいずれかに記載の方法。 (項目225) 前記対象は、外科的予防、手術中管理、または外科手術のための処置を必要とする、項目222に記載の方法。 (項目226) 前記外科手術は、小手術、大手術、抜歯、扁桃摘出術、鼠径部ヘルニア切開術、滑膜切除術、人工膝関節全置換術、開頭術、骨接合術、外傷外科手術、頭蓋内手術、腹腔内手術、胸腔内手術、または関節置換手術である、項目225に記載の方法。 (項目227) 前記キメラポリペプチドの前記投与間隔は、24~36、24~48、24~72、24~96、24~120、24~144、24~168、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、もしくは72時間ごと、またはそれ以上で約1回である、項目219~226のいずれかに記載の方法。 (項目228) 前記治療用量は、10~100IU/kgである、項目219~227のいずれかに記載の方法。 (項目229) 前記治療用量は、10~20、20~30、30~40、40~50、50~60、60~70、70~80、80~90、または90~100IU/kgである、項目228に記載の方法。 (項目230) 前記治療用量は、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、または100IU/kgである、項目229に記載の方法。 (項目231) 前記第VIII因子は、ヒト第VIII因子である、項目219~230のいずれかに記載の方法。 (項目232) 前記第VIII因子は、前記Bドメインの完全または部分欠失を有する、項目219~231のいずれかに記載の方法。 (項目233) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457、配列番号6のアミノ酸20~2351)と少なくとも90%または95%同一である、項目231に記載の方法。 (項目234) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と同一である、項目233に記載の方法。 (項目235) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と少なくとも90%または95%同一である、項目231に記載の方法。 (項目236) 前記キメラポリペプチドの前記第VIII因子部分は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と同一である、項目235に記載の方法。 (項目237) 前記キメラポリペプチドの前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と少なくとも90%または95%同一である、項目231または232に記載の方法。 (項目238) 前記キメラポリペプチドの前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と同一である、項目237に記載の方法。 (項目239) 前記キメラポリペプチドは、前記キメラポリペプチドと結合した第2のポリペプチドを含むハイブリッドの形態であり、前記第2のポリペプチドは、Fcから本質的になる、項目219~238のいずれかに記載の方法。 (項目240) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目239に記載の方法。 (項目241) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目240に記載の方法。 (項目242) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目239~241のいずれかに記載の方法。 (項目243) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と同一であるか、またはシグナル配列を含む表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と同一である配列から本質的になる、項目242に記載の方法。 (項目244) 前記キメラポリペプチドは、少なくとも1つの賦形剤を含む薬学的組成物の一部として投与される、項目219~243のいずれかに記載の方法。 (項目245) 第VIII因子を、それを必要とする対象に投与する方法であって、 前記対象に、約3、4、5、6、7、8、9、10、11、12、13、もしくは14日ごと、またはそれ以上の投与間隔で、第VIII因子およびFcを含む治療用量のポリペプチドを投与することを含む、方法。 (項目246) 前記対象は、予防的処置を必要とする、項目245に記載の方法。 (項目247) 前記治療用量は、10~100IU/kgである、項目245または246に記載の方法。 (項目248) 前記治療用量は、10~20、20~30、30~40、40~50、50~60、60~70、70~80、80~90、または90~100IU/kgである、項目247に記載の方法。 (項目249) 前記治療用量は、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、または100IU/kgである、項目248に記載の方法。 (項目250) 前記第VIII因子は、ヒト第VIII因子である、項目245~249のいずれかに記載の方法。 (項目251) 前記第VIII因子は、前記Bドメインの完全または部分欠失を有する、項目245~250のいずれかに記載の方法。 (項目252) 前記第VIII因子は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と少なくとも90%または95%同一である、項目250に記載の方法。 (項目253) 前記第VIII因子は、シグナル配列を含まない表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸20~1457または配列番号6のアミノ酸20~2351)と同一である、項目252に記載の方法。 (項目254) 前記第VIII因子は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と少なくとも90%または95%同一である、項目250に記載の方法。 (項目255) 前記第VIII因子は、シグナル配列を含む表2に示される第VIII因子アミノ酸配列(配列番号2のアミノ酸1~1457または配列番号6のアミノ酸1~2351)と同一である、項目250に記載の方法。 (項目256) 前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と少なくとも90%または95%同一である、項目250または251に記載の方法。 (項目257) 前記第2の部分は、表2に示されるFcアミノ酸配列(配列番号2のアミノ酸1458~1684または配列番号6のアミノ酸2352~2578)と同一である、項目256に記載の方法。 (項目258) 前記キメラポリペプチドは、前記キメラポリペプチドと結合した第2のポリペプチドを含むハイブリッドの形態である第VIII因子-Fcキメラポリペプチドであり、前記第2のポリペプチドは、Fcから本質的になる、項目245~257のいずれかに記載の方法。 (項目259) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目258に記載の方法。 (項目260) 前記キメラポリペプチドは、シグナル配列を含まない表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸20~1684)と同一であるか、またはシグナル配列を含む表2A(i)に示される第VIII因子およびFcアミノ酸配列(配列番号2のアミノ酸1~1684)と少なくとも90%もしくは95%同一である配列を含む、項目259に記載の方法。 (項目261) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と少なくとも90%もしくは95%同一であるか、またはシグナル配列を含む表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目258~260のいずれかに記載の方法。 (項目262) 前記第2のポリペプチドは、シグナル配列を含まない表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸21~247)と同一であるか、またはシグナル配列を含む表2A(ii)に示されるアミノ酸配列(配列番号4のアミノ酸1~247)と少なくとも90%もしくは95%同一である配列から本質的になる、項目261に記載の方法。(項目263) 前記ポリペプチドは、少なくとも1つの賦形剤を含む薬学的組成物の一部として投与される、項目1~38、73~87、94~100、および102~262のいずれかに記載の方法。 (項目264) 前記キメラポリペプチドは、第VIII因子活性を有する、項目1~38、73~87、94~100、または102~263のいずれか1項に記載の方法。
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Abstract
Description
[Technical field]
[0001] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the Sequence Listing submitted electronically in an ASCII text file with this application (Name: sequencelisting_ascii.txt, Size: ____ bytes, and Creation Date: ____) are hereby incorporated by reference in their entirety.
[0002] FIELD OF THEINVENTION The present invention relates generally to the field of drugs for the treatment of hemostatic disorders. [Background technology]
[0003] Hemophilia A is an X-linked bleeding disorder caused by mutations and / or deletions in the factor VIII (FVIII) gene that result in deficient FVIII activity (Peyvandi, F. et al. Haemophilia 12:82-89 (2006)). The disease is characterized by episodic hemorrhages and excessive bleeding after trauma. Eventually, repeated bleeding into muscles and joints, often beginning in early childhood, leads to hemophilic arthropathy and irreversible joint damage. This damage is progressive and can lead to very limited joint mobility, muscle atrophy, and chronic pain (Rodriguez-Merchan, EC, Semin. Thromb. Hemost. 29:87-96 (2003)), which is incorporated herein by reference in its entirety).
[0004] The A2 domain is necessary for the procoagulant activity of the factor VIII molecule. Studies have shown that porcine factor VIII has six times higher procoagulant activity than human factor VIII (Lollar, P., and ET Parker, J. Biol. Chem. 266:12481-12486 (1991)), and the difference in clotting activity between human and porcine factor VIII appears to be due to differences in amino acid sequence between one or more residues in the A2 domain of human and porcine (Lollar, P., et al., J. Biol. Chem. 267:23652-23657 (1992)). These documents are incorporated herein by reference in their entirety.
[0005] Treatment of hemophilia A is by replacement therapy, which aims to restore FVIII activity to 1-5% of normal levels and prevent spontaneous bleeding (Mannucci, PM, et al., N. Engl. J. Med. 344:1773-1779 (2001)), which is incorporated herein by reference in its entirety). There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on demand or to prevent them from occurring by treating prophylactically. However, based on the short half-life of these products, e.g., 8-12 hours, treatment regimens require frequent administration of intravenous injections. Such frequent administration is painful and inconvenient. Reduction in mortality, prevention of joint damage, and improved quality of life are important outcomes of the development of plasma-derived and recombinant factor VIII. Long-term protection from bleeding would represent another important advance in the treatment of patients with hemophilia A. However, to date no products have been developed that allow for long-term hemostatic protection. Thus, there is a need for improved methods of treating hemophilia due to factor VIII deficiency that are more tolerable, longer-lasting, and more effective than current therapies. [Prior art documents] [Non-patent literature]
[0006] [Non-Patent Document 1] Peyvandi, F. et al. Haemophilia 12:82-89 (2006) [Non-Patent Document 2] Rodriguez-Merchan,EC,Semin.Thromb.Hemost.29:87-96(2003) [Non-Patent Document 3] Lollar, P., and ETParker, J. Biol. Chem. 266:12481-12486 (1991) [Non-Patent Document 4] Lollar, P., et al., J. Biol. Chem. 267:23652-23657 (1992) [Non-Patent Document 5] Mannucci, PM, et al., N. Engl. J. Med. 344:1773-1779 (2001) Summary of the Invention [Means for solving the problem]
[0007] The present invention provides methods of administering factor VIII, methods of administering chimeric polypeptides comprising factor VIII and hybrids of such chimeric polypeptides, chimeric polypeptides comprising factor VIII and hybrids of such chimeric polypeptides, polynucleotides encoding such chimeric and hybrid polypeptides, cells comprising such polynucleotides, and methods of producing such chimeric and hybrid polypeptides using such cells.
[0008] The present invention provides a method of administering factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric factor VIII polypeptide, e.g., a chimeric factor VIII-Fc polypeptide, at an administration interval that is at least about 1.5 times longer than the administration interval required for an equivalent dose of the factor VIII (a polypeptide consisting of the factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion.
[0009] The dosing interval can be at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent dose of the factor VIII (polypeptide comprising the factor VIII portion) that does not include a non-factor VIII portion, such as an Fc portion. The dosing interval can be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent dose of the factor VIII (polypeptide comprising the factor VIII portion) that does not include a non-factor VIII portion, such as an Fc portion. The dosing interval can be about every 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or longer.
[0010] The interval between doses can be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days, or more.
[0011] The present invention also provides a method of administering factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric factor VIII polypeptide, e.g., a chimeric factor VIII-Fc polypeptide, to obtain an area under the plasma concentration versus time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained with an equivalent dose of the factor VIII (a polypeptide consisting of the factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion.
[0012] The present invention also provides a method of administering factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a polypeptide comprising factor VIII and Fc about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more.
[0013] The methods of the invention can be practiced on subjects in need of preventative treatment or on-demand treatment.
[0014] On-demand treatments include treatments for bleeding episodes, hemarthrosis, muscle bleeding, mouth bleeding, bleeding, bleeding into muscle, oral cavity bleeding, trauma, head trauma (trauma capitis), gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal bleeding, or bleeding into the illiopsoas sheath. The subject may require treatment for surgical prophylaxis, perioperative management, or surgery. Such surgery includes, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery.
[0015] For on-demand treatment, the chimeric polypeptide is administered about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more.
[0016] Therapeutic doses that may be used in the methods of the present invention are about 10 to about 100 IU / kg, more specifically, about 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg, more specifically, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg.
[0017] Therapeutic doses that may be used in the methods of the present invention are about 10 to about 150 IU / kg, more specifically, about 100-110, 110-120, 120-130, 130-140, 140-150 IU / kg, more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU / kg.
[0018] The subject in the method of the present invention is a human subject. The determination of the dosing interval and AUC can be performed in a single subject or in a population of subjects.
[0019] The factor VIII (or the factor VIII portion of the chimeric polypeptide) is human factor VIII. The factor VIII (or the factor VIII portion of the chimeric polypeptide) can have a complete or partial deletion of the B domain.
[0020] The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 4 to 2351 of SEQ ID NO:6) without the signal sequence. The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be identical to a factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20 to 2351 of SEQ ID NO:6) without the signal sequence.
[0021] The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2, including the signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6). The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be identical to a factor VIII amino acid sequence shown in Table 2, including the signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6).
[0022] The Fc portion (or the Fc portion of a chimeric polypeptide) can be at least 90% or 95% identical to an Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc portion (or the Fc portion of a chimeric polypeptide) can be identical to an Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6).
[0023] The chimeric polypeptide may comprise a sequence that is at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) without the signal sequence (amino acids 20 to 1684 of SEQ ID NO:2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) with the signal sequence (amino acids 1 to 1684 of SEQ ID NO:2). The chimeric polypeptide may comprise a sequence that is identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) without the signal sequence (amino acids 20 to 1684 of SEQ ID NO:2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) with the signal sequence (amino acids 1 to 1684 of SEQ ID NO:2).
[0024] The chimeric polypeptide may be in the form of a hybrid comprising a second polypeptide linked to the chimeric polypeptide, the second polypeptide comprising or consisting essentially of an Fc.
[0025] The second polypeptide may comprise or essentially consist of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21-247 of SEQ ID NO:4), or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids 1-247 of SEQ ID NO:4). The second polypeptide may comprise or essentially consist of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21-247 of SEQ ID NO:4), or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids 1-247 of SEQ ID NO:4).
[0026] The chimeric polypeptide or hybrid can be administered as part of a pharmaceutical composition that includes at least one excipient.
[0027] The present invention also provides the above-described chimeric and hybrid polypeptides themselves, the polynucleotides encoding them, cultured human embryonic cells containing the polynucleotides, methods of producing such chimeric and hybrid polypeptides, and the polypeptides produced by such methods.
[0028] The present invention also provides a chimeric polypeptide having factor VIII activity comprising a factor VIII portion and a second portion, the factor VIII portion being processed factor VIII comprising two chains, a first chain comprising a heavy chain and a second chain comprising a light chain, the first chain and the second chain being associated by metal binding, for example, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the factor VIII portion of the chimeric polypeptide is processed factor VIII.
[0029] In addition, the present invention includes chimeric polypeptides having factor VIII activity, wherein the factor VIII portion is single-chain factor VIII. In one aspect, the single-chain factor VIII can contain an intact intracellular processing site. In one embodiment, at least about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII. In another embodiment, at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII. In another aspect, the single-chain FVIII does not contain an intracellular processing site. For example, the single-chain FVIII includes a substitution or mutation at the amino acid position corresponding to arginine 1645, a substitution or mutation at the amino acid position corresponding to arginine 1648, or a substitution or mutation at the amino acid positions corresponding to arginine 1645 and arginine 1648 in full-length factor VIII. The amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, e.g., alanine.
[0030] In some embodiments, the chimeric polypeptide comprising single-chain factor VIII has a level of factor VIII activity comparable to that of a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties, when factor VIII activity is measured in vitro by a chromogenic assay. In other embodiments, the chimeric polypeptide comprising single-chain factor VIII has a factor VIII activity in vivo comparable to that of a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. In yet other embodiments, the chimeric polypeptide comprising single-chain factor VIII has a factor Xa incidence comparable to that of a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. In certain embodiments, the single-chain factor VIII in the chimeric polypeptide is inactivated by activated protein C at a level comparable to that of the processed factor VIII in the chimeric polypeptide consisting of two Fc portions and the processed factor VIII. In yet other embodiments, the single-chain factor VIII in the chimeric polypeptide has an interaction rate of factor IXa comparable to that of the processed factor VIII in the chimeric polypeptide consisting of two Fc portions and the processed factor VIII. In further embodiments, the single-chain factor VIII in the chimeric polypeptide binds to von Willebrand factor at a level comparable to that of the processed factor VIII in the chimeric polypeptide consisting of two Fc portions and the processed factor VIII.
[0031] The present invention further includes compositions comprising a chimeric polypeptide having factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the polypeptide comprises a factor VIII portion that is a single-chain factor VIII, and a second portion, the single-chain factor VIII being at least 90% or 95% identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20-1457 of SEQ ID NO:2 or amino acids 20-2351 of SEQ ID NO:6) without the signal sequence. In one embodiment, the second portion can be Fc. In another embodiment, the polypeptide is in the form of a hybrid comprising a second polypeptide, the second polypeptide consisting essentially of Fc. In other embodiments, the polypeptide has a half-life that is at least 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than that of the polypeptide consisting of factor VIII.
[0032] Also provided is a method of treating a bleeding condition comprising administering a therapeutically effective amount of the composition. The treatment can be prophylactic or on-demand treatment or during surgery. The bleeding clotting disorder can be hemophilia. In one embodiment, the subject being treated is a pediatric subject.
[0033] The present invention is also directed to a method of preventing, reducing, or treating a bleeding episode in a subject, comprising administering to the subject an effective amount of a long-acting factor VIII (FVIII) protein, the subject expressing a high level of von Willebrand factor (VWF) in plasma. In one embodiment, the subject is identified as expressing a high level of VWF in plasma. The present invention is also directed to a method of preventing, reducing, or treating a bleeding episode in a subject, the method comprising: (a) identifying a subject having a high level of VWF by measuring a VWF level in the subject's plasma, wherein a VWF level of at least about 100 IU / dL identifies the subject as having a high level of VWF; and (b) administering to the subject an effective amount of a long-acting FVIII protein.
[0034] In one embodiment, the subject is a human. In another embodiment, the subject is a pediatric subject. In another embodiment, the subject has hemophilia A.
[0035] In one embodiment, the high level of VWF is at least about 100 IU / dL. In another embodiment, the high level of VWF is about 100 IU / dL to about 200 IU / dL. In another embodiment, the high level of VWF is about 110 IU / dL, about 120 IU / dL, about 130 IU / dL, about 140 IU / dL, about 150 IU / dL, about 160 IU / dL, about 170 IU / dL, about 180 IU / dL, about 190 IU / dL, or about 200 IU / dL.
[0036] In one embodiment, the subject has serotype A, B, or AB.
[0037] In one embodiment, the long-acting FVIII protein has a half-life of about 20 to about 40 hours in the subject. In another embodiment, the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In another embodiment, the long-acting FVIII protein has a half-life of about 20 to about 27 hours. In another embodiment, the long-acting FVIII protein has a half-life at least about 1.2 times longer than the half-life of the long-acting FVIII protein when administered to an individual with an average level of VWF. In another embodiment, the long-acting FVIII protein has a half-life that is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of said long-acting FVIII protein when administered to an individual with an average level of VWF.
[0038] In one embodiment, the effective amount of the long-acting FVIII protein administered is at least about 20 IU / kg, at least about 25 IU / kg, at least about 30 IU / kg, at least about 35 IU / kg, at least about 40 IU / kg, at least about 45 IU / kg, at least about 50 IU / kg, at least about 55 IU / kg, at least about 60 IU / kg, at least about 65 IU / kg, at least about 70 IU / kg, at least about 75 IU / kg, at least about 80 IU / kg, at least about 85 IU / kg, or at least about 90 IU / kg. In another embodiment, the effective amount is at least about 65 IU / kg to at least about 90 IU / kg. In another embodiment, the effective amount is 80 IU / kg.
[0039] In one embodiment, the long-acting FVIII protein is administered every 72 hours or more. In another embodiment, the long-acting FVIII protein is administered about once a week or more. In another embodiment, the long-acting FVIII protein is administered about once every 10 days, about once every 2 weeks, about once every 15 days, about once every 20 days, about once every 3 weeks, about once every 25 days, about once every 4 weeks, or about once a month.
[0040] In one embodiment, the long-acting FVIII is administered at a dose of 80 IU / kg once every 72 hours. In a further embodiment, the long-acting FVIII is administered to a pediatric subject at a dose of 80 IU / kg once every 72 hours.
[0041] In one embodiment, administration of the long-acting FVIII protein eliminates more than 5-20%, more than 5-15%, more than 5-10%, more than 10-20%, or more than 10-15% of bleeding episodes. In one embodiment, in the subject, plasma Factor VIII:C trough levels are maintained above 1-3 or 3-5 IU / dL. In one embodiment, administration prevents a bleeding episode in the subject. In another embodiment, the bleeding episode is episodic. In another embodiment, administration eliminates more than 80-100%, more than 80-90%, more than 85-90%, more than 90-100%, more than 90-95%, or more than 95-100% of bleeding episodes.
[0042] In one embodiment, administration maintains homeostasis in a population of subjects who require surgery.In another embodiment, the long-acting FVIII protein is administered before, during, or after surgery.In another embodiment, the surgery is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery.In another embodiment, the surgery is emergency surgery.
[0043] In one embodiment, the long-acting FVIII protein has a longer half-life than the polypeptide consisting of FVIII. In another embodiment, the long-acting FVIII protein is pegylated, HESylated or polysialylated.
[0044] In one embodiment, the long-acting FVIII protein is a chimeric protein comprising a FVIII portion and a second portion. In another embodiment, the second portion is an Fc region, albumin, a PAS sequence, transferrin, CTP (28 amino acid C-terminal peptide (CTP) of hCG with four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin-binding polypeptide, an albumin-binding small molecule, or a combination of two or more thereof. In another embodiment, the second portion is fused to the amino or carboxy terminus of the FVIII portion. In another embodiment, the second portion is inserted between two amino acids of the FVIII portion. In another embodiment, the chimeric protein is a FVIIIFc monomer-dimer hybrid. In another embodiment, the FVIII portion is a single chain. In another embodiment, the FVIII portion comprises a heavy chain and a light chain. In another embodiment, the FVIII portion comprises full-length factor VIII, mature factor VIII, or factor VIII with a complete or partial deletion of the B domain. In another embodiment, the FVIII portion comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In another embodiment, the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In another embodiment, the chimeric polypeptide comprises an Fc portion that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In another embodiment, the second portion comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In another embodiment, the long-acting FVIII polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient.
[0045] The present invention also provides a method of treating a subject diagnosed with a bleeding disorder, comprising measuring the half-life of FVIII-Fc in the subject, where a half-life that is at least about 1.2-fold longer than the half-life of FVIII-Fc in a normal subject indicates that the subject is a candidate for a longer dosing interval, and administering an effective amount of a FVIII-Fc polypeptide with a dosing interval of at least 3 days.
[0046] The present invention also provides a method of treating a subject diagnosed with a bleeding disorder, comprising administering to the subject an effective amount of a FVIII-Fc polypeptide with an administration interval of at least 3 days, wherein the half-life of FVIII-Fc in the subject is at least about 1.2 times longer than the half-life of FVIII-Fc when administered to a subject with an average level of VWF.
[0047] In one embodiment, the plasma half-life of FVIII-Fc in the subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the plasma half-life of FVIII-Fc when administered to a subject with an average level of VWF. In another embodiment, the plasma half-life of FVIII-Fc is 20 to 40 hours. In another embodiment, the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In another embodiment, the long-acting FVIII protein has a half-life of about 20 to about 27 hours.
[0048] The invention also provides a method of treating a subject diagnosed with a bleeding disorder, comprising measuring the half-life of a short-acting FVIII administered to the subject, where a half-life at least about 1.2 times longer than the half-life of the short-acting FVIII in subjects with average levels of VWF indicates the subject is a candidate for a long dosing interval, and administering an effective amount of a long-acting FVIII-Fc polypeptide with a dosing interval of at least 3 days. In one embodiment, the half-life of the short-acting FVIII in the subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of the short-acting FVIII when administered to subjects with average levels of VWF.
[0049] In one embodiment, the subject is a human. In another embodiment, the subject is a pediatric subject. In another embodiment, the subject has hemophilia A. In another embodiment, the subject has serotype A, B, or AB.
[0050] In one embodiment, the long-acting FVIII-Fc is administered in an effective amount of at least about 20 IU / kg, at least about 25 IU / kg, at least about 30 IU / kg, at least about 35 IU / kg, at least about 40 IU / kg, at least about 45 IU / kg, at least about 50 IU / kg, at least about 55 IU / kg, at least about 60 IU / kg, at least about 65 IU / kg, at least about 70 IU / kg, at least about 75 IU / kg, at least about 80 IU / kg, at least about 85 IU / kg, or at least about 90 IU / kg. In another embodiment, the effective amount is at least about 65 IU / kg to at least about 90 IU / kg.
[0051] In one embodiment, an effective amount of FVIII-Fc protein is administered about once per week, about once every 10 days, about once every 2 weeks, about once every 15 days, about once every 20 days, about once every 3 weeks, about once every 25 days, about once every 4 weeks, or about once every month.
[0052] In one embodiment, administration eliminates more than 5-20%, more than 5-15%, more than 5-10%, more than 10-20%, or more than 10-15% of bleeding episodes. In one embodiment, plasma Factor VIII:C trough levels are maintained at greater than 1-3 or 3-5 IU / dL in the subject.
[0053] In one embodiment, the administration prevents a bleeding episode in the subject. In one embodiment, the bleeding episode is episodic. In one embodiment, the administration eliminates more than 80-100%, more than 80-90%, more than 85-90%, more than 90-100%, more than 90-95%, or more than 95-100% of the bleeding episodes. In one embodiment, the administration maintains homeostasis in a population of subjects requiring surgery. In one embodiment, the FVIII-Fc protein is administered before, during, or after a surgical procedure. In one embodiment, the surgical procedure is a minor surgery, a major surgery, a tooth extraction, a tonsillectomy, an inguinal herniotomy, a synovectomy, a total knee replacement, a craniotomy, an osteosynthesis, a trauma surgery, an intracranial surgery, an intraperitoneal surgery, an intrathoracic surgery, or a joint replacement surgery. In one embodiment, the surgical procedure is an emergency surgery.
[0054] In one embodiment, the FVIII-Fc protein has a longer half-life than a polypeptide consisting of FVIII. In one embodiment, the FVIII-Fc protein is pegylated, HESylated, or polysialylated. In one embodiment, the FVIII-Fc protein is a FVIIIFc monomer-dimer hybrid. In one embodiment, the FVIII portion is a single chain. In one embodiment, the FVIII portion comprises a heavy chain and a light chain. In one embodiment, the FVIII portion comprises full-length factor VIII, mature factor VIII, or factor VIII having a full or partial deletion of the B domain. In one embodiment, the FVIII portion comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1-1438 of SEQ ID NO:2 or amino acids 1-2332 of SEQ ID NO:6. In one embodiment, the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. In one embodiment, the second portion of the chimeric polypeptide comprises an Fc portion that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. In one embodiment, the second portion comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6.
[0055] In one embodiment, the FVIII-Fc polypeptide is administered as part of a pharmaceutical composition that includes at least one excipient.
[0056] The present invention also provides a method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long dosing interval with a long-acting FVIII polypeptide, comprising measuring the expression level of plasma VWF, and a VWF expression level of at least 100 IU / dL indicates that the subject is a candidate for a long dosing interval with a long-acting FVIII polypeptide. In one embodiment, the VWF expression level is at least about 110 IU / dL, about 120 IU / dL, about 130 IU / dL, about 140 IU / dL, about 150 IU / dL, about 160 IU / dL, about 170 IU / dL, about 180 IU / dL, about 190 IU / dL, or about 200 IU / dL.
[0057] The invention also provides a method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long dosing interval of a long-acting FVIII polypeptide, comprising measuring the half-life of FVIII-Fc in the subject, wherein a half-life that is at least about 1.2 times longer than the half-life of FVIII-Fc when administered to a subject with an average level of VWF indicates that the subject is a candidate for a long dosing interval. In one embodiment, the half-life of FVIII-Fc is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of FVIII-Fc when administered to a subject with an average level of VWF.
[0058] The present invention also provides a method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long dosing interval of a long-acting FVIII polypeptide, comprising measuring the half-life of the short-acting FVIII in the subject, wherein a half-life that is at least about 1.2 times longer than the half-life of the short-acting FVIII when administered to a subject with an average level of VWF indicates that the subject is a candidate for a long dosing interval. In one embodiment, the half-life is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of FVIII-Fc when administered to a subject with an average level of VWF. In certain embodiments, for example, the following are provided: (Item 1) 1. A method of administering factor VIII to a human subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric polypeptide comprising a factor VIII portion and a second portion at an administration interval at least about 1.5 times longer than the administration interval required for an equivalent dose of a polypeptide consisting of said factor VIII portion, wherein said factor VIII portion comprises processed factor VIII having two chains, a first chain comprising a heavy chain and a second chain comprising a light chain, and wherein said first chain and said second chain are associated by metal binding. (Item 2) 2. The method of claim 1, wherein at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% of the Factor VIII portion of the chimeric polypeptide is processed Factor VIII. (Item 3) 3. The method of claim 2, wherein about 100% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 4) 3. The method of any one of items 1 or 2, wherein the factor VIII portion is single chain and comprises single-chain factor VIII. (Item 5) 5. The method of claim 4, wherein the single-chain factor VIII contains an intact intracellular processing site. (Item 6) 6. The method of claim 5, wherein the intracellular processing site corresponds to arginine at residue 1648 of full-length factor VIII (arginine residue 1667 at amino acids 20 to 2351 of SEQ ID NO: 6). (Item 7) 7. The method of any one of items 3 to 6, wherein at least about 1%, about 5%, about 10%, about 15%, about 20%, or about 25% of the Factor VIII portion of the chimeric polypeptide is single-chain Factor VIII. (Item 8) 8. The method of claim 7, wherein about 25% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 75% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 9) 8. The method of claim 7, wherein about 20% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 80% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 10) 8. The method of claim 7, wherein about 15% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 85% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 11) 8. The method of claim 7, wherein about 10% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 90% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 12) 8. The method of claim 7, wherein about 5% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 95% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 13) 8. The method of claim 7, wherein about 1% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 99% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 14) 7. The method of any one of items 3 to 6, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% of the Factor VIII portion of the chimeric polypeptide is single-chain Factor VIII. (Item 15) 15. The method of claim 14, wherein about 30% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 70% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 16) 15. The method of claim 14, wherein about 40% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 60% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 17) 15. The method of claim 14, wherein about 50% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 50% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 18) 15. The method of claim 14, wherein about 60% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 40% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 19) 15. The method of claim 14, wherein about 70% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 30% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 20) 15. The method of claim 14, wherein about 80% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 20% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 21) 15. The method of claim 14, wherein about 85% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 15% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 22) 15. The method of claim 14, wherein about 90% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 10% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 23) 15. The method of claim 14, wherein about 95% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 5% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 24) 15. The method of claim 14, wherein about 99% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 1% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 25) 15. The method of claim 14, wherein about 100% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII. (Item 26) 26. The method according to any one of items 4 to 25, wherein the chimeric polypeptide comprising single-chain factor VIII has factor VIII activity at a level comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties, when factor VIII activity is measured in vitro by a chromogenic assay. (Item 27) 27. The method according to any one of items 4 to 26, wherein the chimeric polypeptide comprising single-chain factor VIII has in vivo factor VIII activity comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. (Item 28) 28. The method according to any one of items 4 to 27, wherein the chimeric polypeptide comprising single-chain factor VIII has a frequency of factor Xa comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. (Item 29) 29. The method according to any one of items 4 to 28, wherein the single-chain factor VIII in the chimeric polypeptide is inactivated by activated protein C at a level comparable to that of processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. (Item 30) 30. The method according to any one of items 4 to 29, wherein the single-chain factor VIII in the chimeric polypeptide has an interaction rate of factor IXa comparable to that of processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. (Item 31) 31. The method according to any one of items 4 to 30, wherein the single-chain factor VIII in the chimeric polypeptide binds to von Willebrand factor at a level comparable to processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. (Item 32) The chimeric polypeptide comprises: an ability to interact with phospholipid vesicles comparable to the ability of a polypeptide consisting of said Factor VIII portion; an ability to form an Xase complex that activates factor X that is comparable to the ability of a polypeptide consisting of said factor VIII portion; the ability to be activated by alpha-thrombin within 5 minutes comparable to that of a polypeptide comprising the factor VIII portion; and an ability to interact with factor VIIIa that is comparable to the ability of the polypeptide consisting of the factor VIII portion. (Item 33) The chimeric polypeptide comprises: forming an Xase complex that activates factor X with a Km that is within about 1, about 1.5, or about 2 standard deviations of the Km of a polypeptide consisting of said factor VIII portion, the Km being measured as a function of factor X concentration; forming an Xase complex that activates factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of a polypeptide consisting of said factor VIII portion, where Vmax is measured as a function of factor X concentration; forming an Xase complex that activates factor X with a Kd that is within about 1, about 1.5, or about 2 standard deviations of the Kd of a polypeptide consisting of said factor VIII portion, where the Kd is measured as a function of the concentration of factor IXa; and forming an Xase complex that activates factor X with a Vmax within about 1, about 1.5, or about 2 standard deviations of the Vmax of the polypeptide consisting of the factor VIII portion, where Vmax is measured as a function of the concentration of factor IXa. (Item 34) 34. The method according to any of items 1 to 33, wherein the dose has a mean incremental recovery (K value) of greater than 1.38 IU / dL per IU / kg (activity; observed value). (Item 35) 35. The method of claim 34, wherein the dose has a mean incremental recovery (K value) of at least about 1.5, at least about 1.85, or at least about 2.46 IU / dL per IU / kg (activity; observed value). (Item 36) 36. The method according to any of items 1 to 35, wherein the chimeric polypeptide exhibits, in the patient population or in the subject, one or more pharmacokinetic parameters selected from the group consisting of: C in the subject administered 25 IU / kg of the chimeric polypeptide max In subjects administered 25 IU / kg of the polypeptide consisting of full-length mature factor VIII, the C-OBS is measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay. max _Comparable to OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 60.5 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 53.1 to 69 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 119 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 103 to 136 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 76.5 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 64.9 to 90.1 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 182 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 146 to 227 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, A mean clearance (CL) (activity) in said patient population of less than or equal to about 2.33±1.08 mL / hr / kg; A mean clearance (CL) (activity) in said patient population of about 1.8 to 2.69 mL / hr / kg; a mean clearance (CL) (activity) in said patient population that is about 65% of the clearance of a polypeptide consisting of said factor VIII portion; A mean clearance (CL) in said subject of about 1.22 to 5.19 mL / hour / kg (activity); the clearance (CL) (activity) in the subject is 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or 0.70 fold lower than the clearance in a subject administered the same amount of a polypeptide consisting of the full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay; a clearance (CL) (activity) in the subject of about 1.68 mL / hr / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.31 to 2.15 mL / hr / kg, as measured by a one-stage (aPTT) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 2.32 mL / hr / kg, as measured by a one-stage (aPTT) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.64 to 3.29 mL / hr / kg, as measured by a one-stage (aPTT) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.49 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.16 to 1.92 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.52 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.05 to 2.20 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; A mean mean residence time (MRT) (activity) in said patient population of at least about 26.3±8.33 hours; A mean MRT (activity) in said patient population of about 25.9 to 26.5 hours; a mean MRT (activity) in said patient population that is about 1.5 times longer than the mean MRT of a polypeptide consisting of said factor VIII portion; A mean residence time (MRT) (activity) in the subject of about 14 to 41.3 hours; the MRT in said subject, when measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay, is at least 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, or more preferably ... 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, or 1.90 times higher, an MRT (activity) in said subject of about 27 hours as measured by a one-stage (aPTT) assay; an MRT (activity) in said subject of about 20.6 to 35.3 hours as measured by a one-stage (aPTT) assay; MRT (activity) in said subject between about 23.9 and 28.5 hours as measured by a two-stage (chromogenic) assay; Two-stage (chromogenic) assay: MRT (activity) in said subject of about 19.8 to 28.9 hours, as measured by a two-stage (chromogenic) assay; Two-stage (chromogenic) assay: MRT (activity) in said subject from about 20.5 to 39.6 hours, as measured by a two-stage (chromogenic) assay; A mean t1 / 2 beta(activity) in said patient population of about 18.3±5.79 hours; a mean t1 / 2 beta (activity) in said patient population of about 18 to 18.4 hours; an average t1 / 2 beta (activity) in said patient population that is about 1.5 times longer than the average t1 / 2 beta of a polypeptide consisting of said factor VIII portion; A mean t1 / 2 beta (activity) in said subject of about 11 to 26.4 hours; t1 / 2 beta (activity) of at least 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 1, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, or 1.87 fold higher t1 / 2 beta (activity) in said subject; a t1 / 2 beta (activity) in said subject of about 18.8 hours as measured by a one-stage (aPTT) assay; a t1 / 2 beta (activity) in said subject of about 14.3 to 24.5 hours as measured by a one-stage (aPTT) assay; a t1 / 2 beta (activity) in said subject of about 16.7 hours as measured by a two-stage (chromogenic) assay; a t1 / 2 beta (activity) in said subject of about 13.8 to 20.1 hours as measured by a two-stage (chromogenic) assay; a t1 / 2 beta (activity) in said subject of about 19.8 hours as measured by a two-stage (chromogenic) assay; a t1 / 2 beta (activity) in said subject of about 14.3 to 27.5 hours as measured by a two-stage (chromogenic) assay; A mean incremental recovery (K value) in said patient population of about 2.01±0.44 IU / dL per IU / kg (activity; observed value); A mean incremental recovery rate (K value) in said patient population of about 1.85 to 2.46 IU / dL per IU / kg (activity; observed value); a mean incremental recovery (K value) in said patient population that is about 90% of the mean incremental recovery of a polypeptide comprising said factor VIII portion (activity; observed value); Incremental recovery in said subject of about 1.38 to 2.88 IU / dL per IU / kg (K value) (activity; observed value); an incremental recovery in said subject that is comparable to the incremental recovery in said subject administered a polypeptide consisting of said full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay; an incremental recovery in the subject of about 2.44 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 2.12 to 2.81 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 1.83 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 65 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 1.59 to 2.10 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 65 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 3.09 IU / dL per IU / kg as measured by a two-stage (chromogenic) assay when about 25 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 2.80 IU / dL per IU / kg as measured by a two-stage (chromogenic) assay when about 65 IU / kg of the chimeric polypeptide is administered; an incremental recovery in the subject of about 2.61 to 3.66 IU / dL per IU / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; and an incremental recovery in the subject of about 2.24 to 3.50 IU / dL per IU / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; A mean Vss(activity) in said patient population of about 55.1±12.3 mL / kg; A mean Vss (activity) in said patient population of about 45.3 to 56.1 mL / kg; A mean Vss (activity) in the subject of about 37.7 to 79.4 mL / kg; a Vss(activity) in said subject that is comparable to the Vss(activity) in said subject administered the same amount of a polypeptide consisting of said full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay; a Vss (activity) in the subject of about 45.5 mL / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 39.3 to 52.5 mL / kg, as measured by a one-stage (aPTT) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 62.8 mL / kg, as measured by a one-stage (aPTT) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 55.2 to 71.5 mL / kg, as measured by a one-stage (aPTT) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 35.9 mL / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 30.4 to 42.3 mL / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 43.4 mL / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 38.2 to 49.2 mL / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; Approximately 49.9±18.2 IU per kg * Mean AUC / dose (activity) in said patient population in h / dL; Approximately 44.8 to 57.6 IU per kg * Mean AUC / dose (activity) in said patient population in h / dL, and Approximately 19.2 to 81.7 IU per kg * AUC / dose in said subject of h / dL; AUC in said subjects administered the same amount of polypeptide consisting of said full-length mature factor VIII as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay INFAUC in the subject that is at least 1.45 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than INF , When about 25 IU / kg of the chimeric polypeptide is administered, there is a median time to pulmonary function of about 1440±316 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 1160 to 1880 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, there is a median time to about 1480 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, there is a median time to immunization of about 2910±1320 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 1980 to 3970 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, there is a median time of about 2800 hours per IU / kg as measured by a one-stage (aPTT) assay. *AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, there is about 1660 hours of activity per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, the patient has a response rate of about 1300 to 2120 hours per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, there is about 4280 hours of cytotoxicity per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, the patient has a response rate of about 2960 to 6190 hours per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF . (Item 37) 37. The method according to any of items 1 to 36, wherein the therapeutic dose is about 10 to about 150, 100 to 110, 110 to 120, 120 to 130, 130 to 140, 140 to 150, 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU / kg. (Item 38) 38. The method according to any of items 1 to 37, wherein the administration interval is 1.5 to 5, 1.5, 2, 3, 4, or 5 days or more. (Item 39) 1. A composition comprising a chimeric polypeptide having Factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said polypeptide comprises a Factor VIII portion that is a single-chain Factor VIII, and a second portion, wherein said single-chain Factor VIII is at least 90% or 95% identical to a Factor VIII amino acid sequence set forth in Table 2 (amino acids 20 to 1457 of SEQ ID NO:2 or amino acids 20 to 2351 of SEQ ID NO:6) without a signal sequence. (Item 40) 40. The composition according to item 39, wherein the single-chain factor VIII is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 41) 1. A composition comprising a chimeric polypeptide having Factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of said polypeptide comprises a Factor VIII portion which is a single-chain Factor VIII, and a second portion, said single-chain Factor VIII being at least 90% or 95% identical to a Factor VIII amino acid sequence set forth in Table 2, including a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6). (Item 42) 42. The composition according to item 41, wherein the single-chain factor VIII is identical to the factor VIII amino acid sequence shown in Table 2 including the signal sequence (amino acids 1 to 1457 of SEQ ID NO: 2 or amino acids 1 to 2351 of SEQ ID NO: 6). (Item 43) 43. The composition according to any of items 39 to 42, wherein the second portion is an Fc region, albumin, a PAS sequence, transferrin or CTP (the 28 amino acid C-terminal peptide of hCG (CTP) having four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding polypeptide, an albumin binding small molecule, or any combination thereof. (Item 44) 44. The composition of claim 43, wherein the second portion is an Fc region. (Item 45) 45. The composition according to item 44, wherein the Fc is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 46) 46. The composition according to item 45, wherein the Fc is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 47) 40. The composition of item 39, wherein the polypeptide comprises a sequence that is at least 90% or 95% identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 48) 40. The composition of item 39, wherein the polypeptide comprises a sequence that is identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 49) The composition according to any one of Items 39 to 48, wherein the polypeptide is in the form of a hybrid containing a second polypeptide, and the second polypeptide essentially consists of Fc. 50. The composition of item 49, wherein the second polypeptide consists essentially of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence set forth in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence set forth in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 51) 51. The composition of item 50, wherein the second polypeptide consists essentially of a sequence that is identical to the amino acid sequence shown in Table 2A(ii) without a signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) including a signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 52) 52. The composition according to any of items 39 to 51, wherein the polypeptide has a half-life that is at least 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than that of the polypeptide consisting of factor VIII. (Item 53) an ability to interact with phospholipid vesicles comparable to the ability of a polypeptide consisting of said Factor VIII portion; an ability to form an Xase complex that activates factor X that is comparable to the ability of a polypeptide consisting of said factor VIII portion; the ability to be activated by alpha-thrombin within 5 minutes comparable to that of a polypeptide comprising the factor VIII portion; and an ability to interact with factor VIIIa comparable to the ability of a polypeptide consisting of said factor VIII portion. (Item 54) 54. The composition according to any of items 39 to 53, wherein about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the factor VIII portion of the polypeptide is processed factor VIII. (Item 55) 55. The composition according to any one of items 39 to 54, wherein the chimeric polypeptide comprising single-chain factor VIII has a factor VIII activity comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII, the processed factor VIII being fused to one Fc of the two Fc moieties, when the factor VIII is measured in vitro by a chromogenic assay. (Item 56) 56. The composition according to any one of items 39 to 55, wherein the chimeric polypeptide comprising single-chain factor VIII has an incidence of factor Xa comparable to processed factor VIII. (Item 57) 57. The composition of any one of items 39 to 56, wherein the chimeric polypeptide comprising single-chain factor VIII has a frequency of factor Xa comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. (Item 58) 58. Any one of items 39 to 57, wherein the single-chain factor VIII in the chimeric polypeptide has an interaction rate with factor Xa comparable to that of processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. The composition described in (Item 59) 59. The composition according to any one of items 39 to 58, wherein the single-chain factor VIII in the chimeric polypeptide is inactivated by activated protein C at a level comparable to the processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. (Item 60) 60. The composition according to any one of items 39 to 59, wherein the single-chain factor VIII in the chimeric polypeptide binds to von Willebrand factor at a level comparable to processed factor VIII in a chimeric polypeptide consisting of two Fc portions and processed factor VIII. (Item 61) 61. The composition according to any one of items 39 to 60, wherein the chimeric polypeptide comprising single-chain factor VIII has in vivo factor VIII activity comparable to a chimeric polypeptide consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties. (Item 62) 62. The composition of any one of items 39-61, wherein about 30% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 70% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 63) 62. The composition of any one of items 39-61, wherein about 40% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 60% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 64) 62. The composition of any one of items 39-61, wherein about 50% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 50% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 65) 62. The composition of any one of items 39-61, wherein about 60% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 40% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 66) 62. The composition of any one of items 39-61, wherein about 70% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 30% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 67) 62. The composition of any one of items 39-61, wherein about 80% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 20% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 68) 62. The composition of any one of items 39-61, wherein about 90% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 10% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 69) 62. The composition of any one of items 39-61, wherein about 95% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 5% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 70) 62. The composition of any one of items 39-61, wherein about 99% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 5% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (Item 71) 62. The composition of any one of items 39-61, wherein about 100% of the Factor VIII portion of the chimeric polypeptide is single-chain Factor VIII. (Item 72) 72. The composition according to any one of items 39 to 71, which is a pharmaceutical composition further comprising an excipient. (Item 73) 73. A method for treating a bleeding condition comprising administering a therapeutically effective amount of the composition according to any one of items 39 to 72. (Item 74) 74. The method of claim 73, wherein the treatment is a prophylactic treatment. (Item 75) 74. The method of claim 73, wherein the treatment is an on-demand treatment. (Item 76) 76. The method according to any one of items 73 to 75, wherein the bleeding condition is selected from the group consisting of bleeding coagulation disorders, hemarthrosis, muscle hemorrhage, bleeding from the mouth, hemorrhage, bleeding into muscle, oral bleeding, trauma, trauma capitis, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, and bleeding into the illiopsoas sheath. (Item 77) 77. The method of claim 76, wherein the bleeding clotting disorder is hemophilia. (Item 78) 78. The method of claim 77, wherein the bleeding clotting disorder is hemophilia A. (Item 79) 80. The method of any one of items 1-38 and 73-78, wherein the administration maintains factor VIII trough levels in at least 90% of the patient population at a level greater than 1%. (Item 80) 80. The method of any one of items 1-38 and 73-78, wherein the administration maintains factor VIII trough levels in at least 60% of the population at a level greater than 3%. (Item 81) A method for maintaining factor VIII in a patient population at a level greater than 1% for at least 1, 2, or 3 days, comprising administering to the population a therapeutically effective amount of rFVIIIFc, wherein the therapeutically effective amount is at least 25 IU / kg of rFVIIIFc, and wherein at least 90% of the population maintains a trough level of factor VIII of at least 1% or greater. (Item 82) A method for maintaining factor VIII in a patient population at a level greater than 3% for at least 1, 2, or 3 days, comprising administering to the population a therapeutically effective amount of rFVIIIFc, wherein the therapeutically effective amount is at least 65 IU / kg of rFVIIIFc, and wherein at least 90% of the population maintains a trough level of factor VIII of at least 3% or greater. (Item 83) 83. The method according to any one of items 4 to 38 and 73 to 82, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1645. (Item 84) 84. The method according to any one of items 4 to 38 and 73 to 83, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1648. (Item 85) 85. The method of any one of items 4 to 38 and 73 to 84, wherein the single-chain factor VIII comprises substitutions or mutations at amino acid positions corresponding to arginine 1645 and arginine 1648 in full-length factor VIII. (Item 86) 86. The method of claim 85, wherein the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. (Item 87) 87. The method according to any one of items 83 to 86, wherein the substitution or mutation is an amino acid other than arginine. (Item 88) 73. The composition of any one of items 39 to 72, wherein the single-chain FVIII comprises an intact intracellular processing site. (Item 89) 73. The composition of any one of items 39 to 72, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1645 in full-length mature factor VIII. (Item 90) 73. The composition of any one of items 39 to 72, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1648 in full-length mature factor VIII. (Item 91) 73. The composition of any one of items 39 to 72, wherein the single-chain factor VIII comprises substitutions or mutations at amino acid positions corresponding to arginine 1645 and arginine 1648 in full-length mature factor VIII. (Item 92) 92. The composition of claim 91, wherein the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. (Item 93) 93. The composition according to any one of items 89 to 92, wherein the substitution or mutation is an amino acid other than arginine. (Item 94) 83. The method of any one of items 73 to 82, wherein the single-chain factor VIII contains an intact intracellular processing site. (Item 95) 83. The method of any one of items 73 to 82, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1645 in full-length mature factor VIII. (Item 96) 83. The method of any one of items 73 to 82, wherein the single-chain factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1648 in full-length mature factor VIII. (Item 97) 83. The method of any one of items 73 to 82, wherein the single-chain factor VIII comprises substitutions or mutations at amino acid positions corresponding to arginine 1645 and arginine 1648 in full-length mature factor VIII. (Item 98) 98. The method of claim 97, wherein the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648 in full-length factor VIII. (Item 99) 99. The method according to any one of items 95 to 98, wherein the substitution or mutation is an amino acid other than arginine. (Item 100) 99. The method according to item 87 or 99, wherein the amino acid residue other than arginine is selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine. (Item 101) 94. The composition of claim 93, wherein the amino acid residue other than arginine is selected from the group consisting of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine. (Item 102) 102. The method of any one of items 1-38, 73-87, or 94-100, wherein the subject is a pediatric subject. (Item 103) 104. A method for preventing, reducing, or treating a bleeding episode in a subject, comprising administering to the subject an effective amount of a long-acting factor VIII (FVIII) protein, the subject expressing high levels of von Willebrand factor (VWF) in plasma. 104. The method of claim 103, wherein the subject is identified as expressing high levels of VWF in plasma. (Item 105) 1. A method of preventing, reducing, or treating a bleeding episode in a subject, comprising: (a) identifying a subject having a high level of VWF by measuring the level of VWF in the subject's plasma, wherein a VWF level of at least about 100 IU / dL identifies the subject as having a high level of VWF; (b) administering to the subject an effective amount of a long-acting FVIII protein. (Item 106) 106. The method according to any one of items 103 to 105, wherein the subject is a human. (Item 107) 107. The method according to any of items 103 to 106, wherein the subject is a pediatric subject. (Item 108) 108. The method according to any of items 103 to 107, wherein the subject has hemophilia A. (Item 109) 109. The method according to any of items 103 to 108, wherein the high level of VWF is at least about 100 IU / dL. (Item 110) 109. The method according to any of items 103 to 109, wherein the high level of VWF is from about 100 IU / dL to about 200 IU / dL. (Item 111) 111. The method of claim 110, wherein the high level of VWF is about 110 IU / dL, about 120 IU / dL, about 130 IU / dL, about 140 IU / dL, about 150 IU / dL, about 160 IU / dL, about 170 IU / dL, about 180 IU / dL, about 190 IU / dL, or about 200 IU / dL. (Item 112) 112. The method according to any one of items 103 to 111, wherein the subject has serotype A, B, or AB. (Item 113) 113. The method according to any of items 103 to 112, wherein the long-acting FVIII protein has a half-life in the subject of from 20 hours to about 40 hours. (Item 114) 14. The method of claim 113, wherein the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. (Item 115) 15. The method according to item 114, wherein the long-acting FVIII protein has a half-life of about 20 to about 27 hours. (Item 116) 116. The method according to any of items 103 to 115, wherein the long-acting FVIII protein has a half-life that is at least about 1.2 times higher than the half-life of the long-acting FVIII protein when administered to an individual with an average level of VWF. (Item 117) 17. The method of claim 116, wherein the long-acting FVIII protein has a half-life that is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times higher than the half-life of the long-acting FVIII protein when administered to an individual with an average level of VWF. (Item 118) The method of any one of items 103-117, wherein the effective amount is at least about 20 IU / kg, at least about 25 IU / kg, at least about 30 IU / kg, at least about 35 IU / kg, at least about 40 IU / kg, at least about 45 IU / kg, at least about 50 IU / kg, at least about 55 IU / kg, at least about 60 IU / kg, at least about 65 IU / kg, at least about 70 IU / kg, at least about 75 IU / kg, at least about 80 IU / kg, at least about 85 IU / kg, or at least about 90 IU / kg. (Item 119) 19. The method according to any of items 103 to 118, wherein the effective amount is at least about 65 IU / kg to at least about 90 IU / kg. (Item 120) Item 120. The method of item 119, wherein the effective amount is 80 IU / kg. (Item 121) 121. The method according to any of items 103 to 120, wherein the long-acting FVIII protein is administered every 72 hours or more. (Item 122) 122. The method of claim 121, wherein the long-acting FVIII protein is administered about once a week or more. (Item 123) 122. The method of item 121, wherein the effective amount of the long-acting FVIII protein is administered about once every 10 days, about once every 2 weeks, about once every 15 days, about once every 20 days, about once every 3 weeks, about once every 25 days, about once every 4 weeks, or about once every month. (Item 124) 122. The method according to item 121, wherein said long-acting FVIII is administered at a dosage of 80 IU / kg once every 72 hours. (Item 125) 125. The method of claim 124, wherein the subject is a pediatric subject. (Item 126) 126. The method of any of items 103-125, wherein said administering eliminates more than 5-20%, more than 5-15%, more than 5-10%, more than 10-20%, or more than 10-15% of bleeding episodes. (Item 127) 127. The method according to any of items 103 to 126, wherein in the subject, the plasma factor VIII:C trough level is maintained above 1 to 3 or 3 to 5 IU / dL. (Item 128) 128. The method of any of items 103 to 127, wherein said administering prevents a bleeding episode in said subject. (Item 129) 129. The method of claim 128, wherein the bleeding episode is spontaneous. (Item 130) 130. The method of claim 129, wherein the administration eliminates more than 80-100%, more than 80-90%, more than 85-90%, more than 90-100%, more than 90-95%, or more than 95-100% of bleeding episodes. (Item 131) 131. The method according to any of items 103 to 130, wherein the administration maintains homeostasis in a population of subjects requiring surgery. (Item 132) 132. The method according to any of items 103 to 131, wherein the long-acting FVIII protein is administered before, during or after the surgery. (Item 133) 133. The method of claim 132, wherein the surgical procedure is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee arthroplasty, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery. (Item 134) 134. The method according to any one of items 131 to 133, wherein the surgical procedure is an emergency procedure. (Item 135) 15. The method according to any of items 103 to 134, wherein the long-acting FVIII protein has a longer half-life than a polypeptide consisting of FVIII. (Item 136) 136. The method according to any of items 103 to 135, wherein the long-acting FVIII protein is pegylated, HESylated, or polysialylated. (Item 137) 137. The method according to any of items 103 to 136, wherein the long-acting FVIII protein is a chimeric protein comprising a FVIII part and a second part. (Item 138) 138. The method of claim 137, wherein the second moiety is an Fc region, albumin, a PAS sequence, transferrin or CTP (the 28 amino acid C-terminal peptide of hCG (CTP) having four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding polypeptide, an albumin binding small molecule, or any combination thereof. (Item 139) 139. The method of claim 137 or 138, wherein the second portion is fused to the amino or carboxy terminus of the FVIII portion. (Item 140) 139. The method of claim 137 or 138, wherein the second portion is inserted between two amino acids of the FVIII portion. (Item 141) 141. The method according to any of items 137 to 140, wherein the chimeric protein is a FVIIIFc monomer-dimer hybrid. (Item 142) 142. The method of claim 141, wherein the FVIII portion is a single chain. (Item 143) 143. The method of claim 142, wherein the FVIII portion comprises a heavy chain and a light chain. (Item 144) 144. The method according to any of items 137 to 143, wherein the FVIII portion comprises full-length factor VIII, mature factor VIII, or factor VIII having a complete or partial deletion of the B domain. (Item 145) 142. The method of claim 141, wherein the FVIII portion comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. (Item 146) 146. The method of item 145, wherein the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. (Item 147) 147. The method according to any of items 137 to 146, wherein the second portion of the chimeric polypeptide comprises an Fc portion that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. (Item 148) 148. The method according to item 147, wherein the second portion comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. (Item 149) 149. The method according to any one of items 103 to 148, wherein the long-acting FVIII polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 150) A method of treating a subject diagnosed with a bleeding disorder, comprising: measuring the half-life of FVIII-Fc in the subject, wherein a half-life that is at least about 1.2 times longer than the half-life of FVIII-Fc in a normal subject indicates that the subject is a candidate for a longer dosing interval; and administering an effective amount of a FVIII-Fc polypeptide at a dosing interval of at least 3 days. (Item 151) A method for treating a subject diagnosed with a bleeding disorder, comprising administering to the subject an effective amount of a FVIII-Fc polypeptide with an administration interval of at least 3 days, wherein the half-life of FVIII-Fc in the subject is at least about 1.2 times longer than the half-life of FVIII-Fc when administered to a subject having an average level of VWF. (Item 152) 152. The method of claim 150 or 151, wherein the plasma half-life of FVIII-Fc in the subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the plasma half-life of FVIII-Fc when administered to a subject having an average level of VWF. (Item 153) 153. The method according to any of items 150 to 152, wherein the plasma half-life of the FVIII-Fc is 20 to 40 hours. (Item 154) 154. The method of claim 153, wherein the long-acting FVIII protein has a half-life of about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. (Item 155) 155. The method according to item 154, wherein the long-acting FVIII protein has a half-life of about 20 to about 27 hours. (Item 156) A method of treating a subject diagnosed with a bleeding disorder, comprising: measuring the half-life of a short-acting FVIII administered to the subject; and a half-life that is at least about 1.2 times longer than the half-life of the short-acting FVIII in subjects with an average level of VWF indicating that the subject is a candidate for a longer dosing interval; and administering an effective amount of a long-acting FVIII-Fc polypeptide at a dosing interval of at least 3 days. (Item 157) 157. The method of claim 156, wherein the half-life of the short-acting FVIII in the subject is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of the short-acting FVIII when administered to a subject having an average level of VWF. (Item 158) 158. The method according to any one of items 150 to 157, wherein the subject is a human. (Item 159) 159. The method according to any of items 150 to 158, wherein the subject is a pediatric subject. (Item 160) 160. The method according to any of items 150 to 159, wherein the subject has hemophilia A. (Item 161) 161. The method according to any of items 150 to 160, wherein the subject has serotype A, B, or AB. (Item 162) The method according to any of items 150 to 161, wherein the long-acting FVIII-Fc is administered in an effective amount of at least about 20 IU / kg, at least about 25 IU / kg, at least about 30 IU / kg, at least about 35 IU / kg, at least about 40 IU / kg, at least about 45 IU / kg, at least about 50 IU / kg, at least about 55 IU / kg, at least about 60 IU / kg, at least about 65 IU / kg, at least about 70 IU / kg, at least about 75 IU / kg, at least about 80 IU / kg, at least about 85 IU / kg, or at least about 90 IU / kg. (Item 163) 162. The method according to any of items 150 to 161, wherein the effective amount is at least about 65 IU / kg to at least about 90 IU / kg. (Item 164) The method according to any of items 150 to 163, wherein the effective amount of the FVIII-Fc protein is administered about once per week, about once per 10 days, about once per 2 weeks, about once per 15 days, about once per 20 days, about once per 3 weeks, about once per 25 days, about once per 4 weeks, or about once per month. (Item 165) 165. The method of any of items 150-164, wherein said administering eliminates more than 5-20%, more than 5-15%, more than 5-10%, more than 10-20%, or more than 10-15% of bleeding episodes. (Item 166) 166. The method according to any of items 150 to 165, wherein in the subject, plasma factor VIII:C trough levels are maintained above 1 to 3 or 3 to 5 IU / dL. (Item 167) 167. The method of any of items 150 to 166, wherein said administering prevents a bleeding episode in said subject. (Item 168) Item 168. The method of item 167, wherein the bleeding episode is spontaneous. (Item 169) 168. The method of claim 167, wherein the administration eliminates more than 80-100%, more than 80-90%, more than 85-90%, more than 90-100%, more than 90-95%, or more than 95-100% of bleeding episodes. (Item 170) 169. The method according to any of items 150 to 169, wherein the administration maintains homeostasis in a population of subjects requiring surgery. (Item 171) 171. The method according to any one of claims 150 to 170, wherein the FVIII-Fc protein is administered before, during, or after the surgery. (Item 172) 172. The method according to item 170 or 171, wherein said surgical procedure is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee arthroplasty, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery. (Item 173) 173. The method according to any of items 170 to 172, wherein the surgical procedure is an emergency surgery. (Item 174) 174. The method according to any of items 150 to 173, wherein the FVIII-Fc protein has a longer half-life than a polypeptide consisting of FVIII. (Item 175) 175. The method according to any one of items 150 to 174, wherein the FVIII-Fc protein is pegylated, HESylated, or polysialylated. (Item 176) 176. The method of claim 174 or 175, wherein the FVIII-Fc protein is a FVIIIFc monomer-dimer hybrid. (Item 177) The method according to any one of items 172 to 176, wherein the FVIII portion is a single chain. 178. The method of claim 177, wherein the FVIII portion comprises a heavy chain and a light chain. (Item 179) 179. The method according to any of items 174 to 178, wherein the FVIII portion comprises full-length factor VIII, mature factor VIII, or factor VIII having a complete or partial deletion of the B domain. (Item 180) 180. The method of claim 179, wherein the FVIII portion comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. (Item 181) 181. The method of item 180, wherein the FVIII portion comprises amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6. (Item 182) 182. The method of any of items 174 to 181, wherein the second portion of the chimeric polypeptide comprises an Fc portion that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. (Item 183) 183. The method according to item 182, wherein the second portion comprises amino acids 1439 to 1665 of SEQ ID NO: 2 or amino acids 2333 to 2559 of SEQ ID NO: 6. (Item 184) 184. The method according to any of items 150 to 183, wherein the FVIII-Fc polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 185) A method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long dosing interval using a long-acting FVIII polypeptide, comprising measuring plasma VWF expression levels and finding that a VWF expression level of at least 100 IU / dL indicates that the subject is a candidate for a long dosing interval using a long-acting FVIII polypeptide. (Item 186) 186. The method of claim 185, wherein the VWF expression level is at least about 110 IU / dL, about 120 IU / dL, about 130 IU / dL, about 140 IU / dL, about 150 IU / dL, about 160 IU / dL, about 170 IU / dL, about 180 IU / dL, about 190 IU / dL, or about 200 IU / dL. (Item 187) A method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long-acting FVIII polypeptide dosing interval, comprising measuring the half-life of FVIII-Fc in the subject, and finding that a half-life that is at least about 1.2 times longer than the half-life of FVIII-Fc when administered to a subject with an average level of VWF indicates that the subject is a candidate for a long dosing interval. (Item 188) 188. The method of claim 187, wherein the half-life of FVIII-Fc is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of FVIII-Fc when administered to a subject having an average level of VWF. (Item 189) A method for determining whether a subject diagnosed with a bleeding disorder is a candidate for a long dosing interval of a long-acting FVIII polypeptide, comprising measuring the half-life of a short-acting FVIII polypeptide in the subject, and a half-life that is at least about 1.2 times longer than the half-life of a short-acting FVIII polypeptide when administered to a subject with an average level of VWF indicates that the subject is a candidate for a long dosing interval. (Item 190) 190. The method of claim 189, wherein the half-life is at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 times longer than the half-life of FVIII-Fc when administered to a subject having an average level of VWF. (Item 191) 191. The method according to any one of items 1 to 38, 73 to 87, 94 to 100, and 102 to 190, wherein the chimeric polypeptide comprises an Fc portion. (Item 192) 192. The method of claim 191, wherein the administration interval is at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the administration interval required for an equivalent dose of the polypeptide consisting of the factor VIII portion. (Item 193) 193. The method of claim 192, wherein the administration interval is at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the administration interval required for an equivalent dose of the polypeptide consisting of the factor VIII portion. (Item 194) 194. The method of any of items 1-38, 73-87, 94-100, and 102-193, wherein the administration interval of the chimeric polypeptide is about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. (Item 195) The method according to any one of items 1 to 38, 73 to 87, 94 to 100, and 102 to 194, wherein the subject is in need of a prophylactic treatment. (Item 196) The method of any one of items 1 to 38, 73 to 87, 94 to 100, and 102 to 193, wherein the subject requires on-demand. (Item 197) 197. The method of claim 196, wherein the subject is in need of treatment for a bleeding episode. (Item 198) 198. The method of claim 197, wherein the subject is in need of treatment for hemarthrosis, muscle bleed, mouth bleed, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding into the iliopsoas sheath. (Item 199) 20. The method of claim 196, wherein the subject is in need of surgical prophylaxis, perioperative management, or treatment for surgery. (Item 200) 200. The method of claim 199, wherein the surgical procedure is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee arthroplasty, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery. (Item 201) 201. The method of any of items 196-200, wherein the administration interval of the chimeric polypeptide is about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more. (Item 202) The method according to any one of items 1 to 38, 73 to 87, 94 to 100, and 102 to 201, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 203) 202. The method according to claim 201, wherein the therapeutic dose is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg. (Item 204) 203. The method of claim 202, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg. (Item 205) 5. The method according to any one of items 1 to 38, 73 to 87, 94 to 100, or 102 to 204, wherein the factor VIII is human factor VIII. (Item 206) 6. The method according to any of items 1 to 38, 73 to 87, 94 to 100, and 102 to 205, wherein the factor VIII has a complete or partial deletion of the B domain. (Item 207) 206. The method of claim 205, wherein the Factor VIII portion of the chimeric polypeptide is at least 90% or 95% identical to a Factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 and amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 208) 208. The method of item 207, wherein the factor VIII portion of the chimeric polypeptide is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 209) 206. The method of claim 205, wherein the Factor VIII portion of the chimeric polypeptide is at least 90% or 95% identical to a Factor VIII amino acid sequence set forth in Table 2 (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6) including a signal sequence. (Item 210) 209. The method of claim 209, wherein the Factor VIII portion of the chimeric polypeptide is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1457 of SEQ ID NO: 2 or amino acids 1 to 2351 of SEQ ID NO: 6) including a signal sequence. (Item 211) 27. The method according to claim 205 or 206, wherein the second portion of the chimeric polypeptide is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 212) 212. The method of claim 211, wherein the second portion of the chimeric polypeptide is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 213) 213. The method according to any of items 1 to 38, 73 to 87, 94 to 100, and 102 to 212, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide linked to the chimeric polypeptide, and the second polypeptide consists essentially of Fc. (Item 214) 214. The method of claim 213, wherein the chimeric polypeptide comprises a sequence that is at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 215) 215. The method of claim 214, wherein the chimeric polypeptide comprises a sequence identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or a sequence identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) with a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 216) 216. The method according to any of items 213 to 215, wherein the second polypeptide essentially consists of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 217) 27. The method of claim 216, wherein the second polypeptide consists essentially of an amino acid sequence identical to the amino acid sequence set forth in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO:4) or identical to the amino acid sequence set forth in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO:4). (Item 218) The method of any of items 1-38, 73-87, 94-100, and 102-217, wherein the chimeric polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 219) A method for administering factor VIII to a human subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric polypeptide comprising a factor VIII portion and a second portion to obtain an area under the plasma concentration versus time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained by an equivalent dose of a polypeptide consisting of the factor VIII portion. (Item 220) 220. The method of claim 219, wherein the chimeric polypeptide is administered at an administration interval of about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more. (Item 221) 221. The method of claim 219 or 220, wherein the subject is in need of a prophylactic treatment. (Item 222) The method of claim 219 or 220, wherein the subject is in need of on-demand treatment. 223. The method according to any of items 219 to 222, wherein the subject is in need of treatment for a bleeding episode. (Item 224) The method according to any of items 219 to 222, wherein the subject is in need of treatment for hemarthrosis, muscle bleed, mouth bleed, bleeding, bleeding into muscle, oral cavity bleed, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal space bleeding, retroperitoneal bleeding, or bleeding into the iliopsoas sheath. (Item 225) 23. The method of claim 222, wherein the subject is in need of surgical prophylaxis, perioperative management, or treatment for surgery. (Item 226) 226. The method of claim 225, wherein the surgical procedure is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee arthroplasty, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery. (Item 227) 227. The method of any of items 219-226, wherein the administration interval of the chimeric polypeptide is about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more. (Item 228) 228. The method according to any of items 219 to 227, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 229) 29. The method of claim 228, wherein the therapeutic dose is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg. (Item 230) 230. The method of claim 229, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg. (Item 231) 231. The method according to any of items 219 to 230, wherein the factor VIII is human factor VIII. (Item 232) 232. The method according to any of items 219 to 231, wherein the factor VIII has a complete or partial deletion of the B domain. (Item 233) 232. The method of claim 231, wherein the factor VIII portion of the chimeric polypeptide is at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 and amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 234) 234. The method of claim 233, wherein the factor VIII portion of the chimeric polypeptide is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 235) 232. The method of claim 231, wherein the factor VIII portion of the chimeric polypeptide is at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1457 of SEQ ID NO:2 or amino acids 1 to 2351 of SEQ ID NO:6), including a signal sequence. (Item 236) 26. The method of claim 235, wherein the Factor VIII portion of the chimeric polypeptide is identical to the Factor VIII amino acid sequence shown in Table 2 (amino acids 1 to 1457 of SEQ ID NO: 2 or amino acids 1 to 2351 of SEQ ID NO: 6) including a signal sequence. (Item 237) 233. The method according to claim 231 or 232, wherein the second portion of the chimeric polypeptide is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 238) 238. The method of item 237, wherein the second portion of the chimeric polypeptide is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 6 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 239) 29. The method according to any of items 219 to 238, wherein the chimeric polypeptide is in the form of a hybrid comprising a second polypeptide linked to the chimeric polypeptide, the second polypeptide consisting essentially of Fc. (Item 240) 239. The method of claim 239, wherein the chimeric polypeptide comprises a sequence that is at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 241) 241. The method of claim 240, wherein the chimeric polypeptide comprises a sequence that is identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 242) 242. The method according to any of items 239 to 241, wherein the second polypeptide essentially consists of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence set out in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence set out in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 243) 243. The method according to item 242, wherein the second polypeptide consists essentially of an amino acid sequence identical to the amino acid sequence set forth in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or identical to the amino acid sequence set forth in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 244) 244. The method of any of items 219 to 243, wherein the chimeric polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 245) 1. A method of administering factor VIII to a subject in need thereof, comprising: A method comprising administering to the subject a therapeutic dose of a polypeptide comprising Factor VIII and Fc about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more between doses. (Item 246) 246. The method of claim 245, wherein the subject is in need of a prophylactic treatment. (Item 247) 27. The method according to item 245 or 246, wherein the therapeutic dose is 10 to 100 IU / kg. (Item 248) 28. The method of claim 247, wherein the therapeutic dose is 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg. (Item 249) 29. The method of claim 248, wherein the therapeutic dose is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg. (Item 250) 249. The method according to any of items 245 to 249, wherein the factor VIII is human factor VIII. (Item 251) 251. The method according to any of items 245 to 250, wherein the factor VIII has a complete or partial deletion of the B domain. (Item 252) 251. The method of item 250, wherein the factor VIII is at least 90% or 95% identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) without the signal sequence. (Item 253) 253. The method according to item 252, wherein the factor VIII is identical to the factor VIII amino acid sequence shown in Table 2 (amino acids 20 to 1457 of SEQ ID NO: 2 or amino acids 20 to 2351 of SEQ ID NO: 6) without a signal sequence. (Item 254) 251. The method of item 250, wherein the factor VIII is at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2 including a signal sequence (amino acids 1 to 1457 of SEQ ID NO: 2 or amino acids 1 to 2351 of SEQ ID NO: 6). (Item 255) 251. The method according to item 250, wherein the factor VIII is identical to the factor VIII amino acid sequence shown in Table 2 including a signal sequence (amino acids 1 to 1457 of SEQ ID NO: 2 or amino acids 1 to 2351 of SEQ ID NO: 6). (Item 256) 252. The method according to item 250 or 251, wherein the second part is at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 257) 257. The method according to item 256, wherein the second portion is identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO: 2 or amino acids 2352 to 2578 of SEQ ID NO: 6). (Item 258) 258. The method according to any of items 245 to 257, wherein the chimeric polypeptide is a Factor VIII-Fc chimeric polypeptide in the form of a hybrid comprising a second polypeptide linked to the chimeric polypeptide, the second polypeptide consisting essentially of Fc. (Item 259) 259. The method of item 258, wherein the chimeric polypeptide comprises a sequence that is at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to a factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 260) 259. The method of claim 259, wherein the chimeric polypeptide comprises a sequence that is identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) without a signal sequence (amino acids 20 to 1684 of SEQ ID NO: 2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence set forth in Table 2A(i) including a signal sequence (amino acids 1 to 1684 of SEQ ID NO: 2). (Item 261) 261. The method according to any of items 258 to 260, wherein the second polypeptide consists essentially of a sequence that is at least 90% or 95% identical to the amino acid sequence set out in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence set out in Table 2A(ii) including the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). (Item 262) The method according to Item 261, wherein the second polypeptide essentially consists of an amino acid sequence identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21 to 247 of SEQ ID NO: 4) or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids 1 to 247 of SEQ ID NO: 4). The method of any of items 1-38, 73-87, 94-100, and 102-262, wherein the polypeptide is administered as part of a pharmaceutical composition comprising at least one excipient. (Item 264) 4. The method according to any one of items 1 to 38, 73 to 87, 94 to 100, or 102 to 263, wherein the chimeric polypeptide has factor VIII activity. [Brief description of the drawings]
[0059] [Figure 1] Schematic diagram showing the rFVIIIFc monomer. [Diagram 2]A-B. Non-reducing and reducing SDS-PAGE analysis of rFVIIIFc (processed or single chain). C. Structure of rFVIIIFc analyzed by LC / UV and LC / MS. D. Total ion current (TIC) chromatogram (LC / MS map) of rFVIIIFc after thrombin cleavage. Major digestion products are indicated. E. Deconvoluted mass spectra of the A2 domain of rFVIIIFc and rBDDFVIII. Major products and their cognate masses are indicated, corresponding to the thrombin-cleaved A2 domain (S373-R740) and two cleavage products, S373-Y729 and S373-E720. [Diagram 3] Biochemical properties of rFVIII-Fc: A. Factor X activation as a function of phospholipid vesicle concentration. B. Factor X activation as a function of factor FX concentration. C. Factor X activation as a function of factor IXa concentration. [Figure 4] Activation of factor X after cleavage by activated protein C. [Diagram 5] Observed group mean FVIII activity (± standard error) profiles versus time, categorized by dose level and grouped by compound (one-stage assay, 25 IU / kg (A) or 65 IU / kg (B); and chromogenic assay, 25 IU / kg (C) or 65 IU / kg (D)). [Figure 6] Observed group mean FVIII activity (± standard error) profiles versus time, grouped by dose level and compound (one-stage assay (A) or chromogenic assay (B)). [Figure 7]In vivo efficacy of single-chain FVIII:Fc in the hemophilia A mouse tail vein transection model. (A) Doses of single-chain rFVIII:Fc are indicated by squares and doses of processed rFVIII:Fc are indicated by circles. (B) Percent survival following tail vein transection for 4.6 μg / kg, 1.38 μg / kg, and 0.46 μg / kg rFVIIIFc or SC rFVIIIFc. (C) Percentage of bleeding free following tail vein transection for 4.6 μg / kg (black circles or inverted triangles), 1.38 μg / kg (triangles or diamonds), and 0.46 μg / kg (squares and grey circles) rFVIIIFc or SC rFVIIIFc, respectively. [Figure 8] Study Design. Figure 8 shows the study design of a Phase 1 / 2a trial, which was a dose-escalation sequential design to evaluate the safety and PK of rFVIIIFc compared to ADVATE® following a single intravenous dose of either 25 IU / kg (low-dose cohort A) or 65 IU / kg (high-dose cohort B). [Figure 9] Correlation of rFVIII activity by one-stage (aPTT) and chromogenic assays. Correlation between the results of one-stage clotting (aPTT) and chromogenic assays measuring FVIII activity (IU / mL) after injection of ADVATE® (◆) and rFVIIIFc (□). [Figure 10] Pharmacokinetic profiles of group mean plasma FVIII activity for low and high dose cohorts. Plasma FVIII activity (one-stage aPTT assay) versus time curves after a single intravenous injection of rFVIIIFc or ADVATE® are shown for (A) 25 IU / kg (low dose cohort, n=6) and (B) 65 IU / kg (high dose cohort, n=10 [ADVATE®], n=9 [rFVIIIFc]). Results presented are group mean ± standard error of the mean (SEM). [Figure 11]Effect of VWF antigen levels on Cl and t1 / 2 of FVIII activity after injection of ADVATE® or rFVIIIFc. Correlation between VWF antigen levels and (A) weight-adjusted Cl for ADVATE® (R2=0.5415 and p=0.0012) and rFVIIIFc (R2=0.5492 and p=0.0016) and (B) t1 / 2 for ADVATE® (R2=0.7923 and p<0.0001) and rFVIIIFc (R2=0.6403 and p=0.0003). Each point represents an individual subject. [Figure 12] Ex vivo whole blood ROTEM results for individual subjects following injection of ADVATE® or rFVIIIFc. Blood was sampled from subjects pre- and post-treatment at the indicated time points with (A) 25 IU / kg ADVATE® and rFVIIIFc, and (B) 65 IU / kg ADVATE® and rFVIIIFc doses. Clotting times were determined by NATEM initiated with Ca++ in a ROTEM® instrument. Results shown are the mean ± standard error (SEM) from triplicate channel readings for each individual sample. [Figure 13] Comparison of activity in thrombin generation assay (TGA). (A) SC rFVIIIFc showed reduced endogenous thrombin potential (ETP) and (B) reduced peak thrombin compared to rFVIIIFc. [Figure 14] In vitro ROTEM data. ROTEM (NATEM) results (mean ± standard deviation) for different concentrations of XYNTHA, ADVATE, and rFVIIIFc spiked into pooled whole blood obtained from naive hemophilia A mice. (A) Mean clotting time (CT) (FIG. 14A), (B) clot formation time (CFT), and (C) alpha angle. [Figure 15]Ex vivo ROTEM data. ROTEM (NATEM) results (mean ± SD) from hemophilia A mice 5 min, 24, 48, 72, and 96 h after a single intravenous dose of 50 IU / kg XYNTHA, ADVATE, or rFVIIIFc. (A) Mean clotting time (CT), (B) clot formation time (CFT), and (C) alpha angle. [Figure 16A] Real-time assessment of the interaction of rFVIIIFc and single-chain (SC) rFVIIIFc with vWF and the thrombin-mediated release of SC rFVIIIFc from rFVIIIFc and vWF. (A) Surface plasmon resonance (SPR) analysis of rFVIIIFc and SC rFVIIIFc affinity for vWF. Binding curves and a 1:1 fitted interaction model are shown. The x-axis shows time in seconds and the y-axis shows response in response units (RU). [Figure 16B] Real-time assessment of the interaction of rFVIIIFc and single-chain (SC) rFVIIIFc with vWF and thrombin-mediated release of SC rFVIIIFc from rFVIIIFc and vWF. (B) Reference subtracted sensograms of thrombin-mediated release of activated rFVIIIFc, SC rFVIIIFc, and B-domain deleted, Fc-moiety-depleted rFVIII (rBDD FVIII) at 25° C. (top) and 37° C. (bottom). The x-axis shows time in seconds and the y-axis shows response in response units (RU). Individual lines show response at different α-thrombin concentrations. The top line is the reaction at 0 U / mL α-thrombin and each subsequent line is displayed for α-thrombin concentration in the order 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5, 10, and 20 U / mL. [Figure 16C]Real-time assessment of the interaction of rFVIIIFc and single-chain (SC) rFVIIIFc with vWF and thrombin-mediated release of SC rFVIIIFc from rFVIIIFc and vWF. (C) Double-baseline division sensorgrams of the thrombin-mediated release phase for rFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C. (top) and 37° C. (bottom). The x-axis shows time in seconds and the y-axis shows response in response units (RU). Individual lines show response at different α-thrombin concentrations. The top line is the reaction at 0 U / mL α-thrombin and each subsequent line is displayed for α-thrombin concentration in the order 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.3, 2.5, 5.0, 10, and 20 U / mL. [Figure 16D] Real-time assessment of the interaction of rFVIIIFc and single-chain (SC) rFVIIIFc with vWF and thrombin-mediated release of SC rFVIIIFc from rFVIIIFc and vWF. (D) Thrombin-mediated release rate as a function of time for rFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C. (top) and 37° C. (bottom). The x-axis shows time in seconds and the y-axis shows response in response units (RU). Individual lines show response at different α-thrombin concentrations. The top line is the reaction at 20 U / mL of α-thrombin and each subsequent line is displayed for α-thrombin concentration in the order of 10, 5, 2.5, 1.3, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, 0.01, and 0.005 U / mL. [Figure 16E]Real-time assessment of the interaction of rFVIIIFc and single-chain (SC) rFVIIIFc with vWF and thrombin-mediated release of SC rFVIIIFc from rFVIIIFc and vWF. (E) Peak thrombin-mediated release rate as a function of thrombin concentration for rFVIIIFc, SC rFVIIIFc, and rBDD FVIII at 25° C. (top) and 37° C. (bottom). EC50 is the half-maximal effective concentration. The x-axis is α-thrombin concentration in U / mL and the y-axis is maximum release rate in RU / sec. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0060] The present invention provides methods of treating hemophilia A with factor VIII (processed, single chain, or combinations thereof) with longer dosing intervals and / or greater AUC than is possible with currently known factor VIII products. The present invention also provides improved chimeric factor VIII polypeptides and methods of production.
[0061] Treatment of hemophilia A is with replacement therapy aimed at restoring FVIII activity to 1-5% of normal levels and preventing spontaneous bleeding episodes (Mannucci, PM et al., N. Engl. J. Med. 344:1773-9 (2001), which is incorporated herein by reference in its entirety). There are plasma-derived and recombinant FVIII products available to treat bleeding episodes on demand or to prevent bleeding episodes by treating prophylactically. Based on the short half-life (8-12 hours) of these products (White GC, et al., Thromb. Haemost. 77:660-7 (1997); Morfini, M., Haemophilia 9(suppl 1):94-99; Discussion 100 (2003)), treatment regimens generally require frequent intravenous administration, 2-3 times per week for prophylaxis and 1-3 times per day for on-demand treatment (Manco-Johnson, MJ, et al., N. Engl. J. Med. 357:535-544 (2007)), each of which is incorporated herein by reference in its entirety. Such frequent administration is painful and inconvenient.
[0062] The present invention provides a method of administering factor VIII to a human subject (e.g., a human patient) in need thereof, comprising administering to the subject a therapeutic dose of a chimeric factor VIII polypeptide, e.g., a chimeric factor VIII-Fc polypeptide, or a hybrid of such polypeptides, at an administration interval at least about 1.5 times longer than that required for an equivalent dose of said factor VIII (a polypeptide consisting of said factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. The present invention is also directed to a method of increasing the administration interval for factor VIII to a human subject in need thereof, comprising administering a chimeric factor VIII polypeptide.
[0063] The dosing interval can be at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent dose of the factor VIII (polypeptide comprising the factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. The dosing interval can be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, or 6 times longer than the dosing interval required for an equivalent dose of the factor VIII (polypeptide comprising the factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. The dosing interval can be about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more.
[0064] The interval between doses can be at least about 1.5 to 5, 1.5, 2, 3, 4, or 5 days, or more.
[0065] The present invention also provides a method of administering factor VIII to a human subject in need thereof, comprising administering to the subject a therapeutic dose of a chimeric factor VIII polypeptide, e.g., a chimeric factor VIII-Fc polypeptide, or a hybrid of such polypeptides, to obtain an area under the plasma concentration versus time curve (AUC) that is at least about 1.25-fold greater than the AUC obtained by an equivalent dose of the factor VIII (a polypeptide consisting of the factor VIII portion) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. Thus, the present invention includes a method of increasing or broadening the AUC of factor VIII activity in a human patient in need thereof, comprising administering a chimeric factor VIII polypeptide.
[0066] The present invention also provides a method of administering factor VIII to a subject in need thereof, comprising administering to the subject a therapeutic dose of a polypeptide comprising factor VIII and Fc at a dosing interval of about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or more.
[0067] The methods of the invention can be practiced on subjects in need of preventative treatment or on-demand treatment.
[0068] "Administering" as used herein means giving a pharma- ceutically acceptable Factor IX polypeptide of the present invention to a subject by a pharma-ceutically acceptable route.The route of administration can be intravenous, for example, intravenous injection and intravenous infusion.Additional routes of administration include, for example, subcutaneous, intramuscular, oral, nasal, and pulmonary administration.The chimeric polypeptide and hybrid protein may be administered as part of a pharmaceutical composition that includes at least one excipient.
[0069] "Area under the plasma concentration versus time curve" used herein is the same as the terminology in pharmacology, and is based on the rate and extent of absorption of factor VIII after administration.AUC is determined over a specific time period, for example, 12, 18, 24, 36, 48, or 72 hours, or at infinity using extrapolation based on the slope of the curve.Unless otherwise stated herein, AUC is determined at infinity.Determination of AUC can be performed in a single subject, or in a population of subjects for which the average is calculated.
[0070] The "B domain" of factor VIII as used herein is the same as the B domain known in the art, which is defined by internal amino acid sequence identity and proteolytic cleavage site by thrombin, e.g., residues Ser741-Arg1648 of full-length human factor VIII. Other human factor VIII domains are defined by amino acid residues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the factor VIII light chain activation peptide. The location of the boundaries for all of the domains, including the B domain, for porcine, mouse, and canine factor VIII are also known in the art. In one embodiment, the B domain of factor VIII is deleted ("B domain deleted factor VIII" or "BDD FVIII"). One example of a BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the factor VIII portion of the sequence in Table 2A(i) (amino acids 1-1457 or 20-1457 of SEQ ID NO:2). In another embodiment, the B domain deleted factor VIII contains an intact intracellular processing site corresponding to the arginine at residue 754 of B domain deleted factor VIII, which corresponds to arginine residue 773 of SEQ ID NO:2, or at residue 1648 of full length factor VIII, which corresponds to arginine residue 1667 of SEQ ID NO:6. Unless otherwise indicated, sequence residue numbers used herein that are not referred to in any SEQ ID NO correspond to the factor VIII sequence without its signal peptide sequence (19 amino acids). For example, S743 / Q1638 of full-length factor VIII corresponds to S762 / Q1657 of SEQ ID NO:6 due to the 19 amino acid signal peptide sequence.In other embodiments, the B-domain deleted factor VIII comprises a substitution or mutation at the amino acid position corresponding to arginine 1645, a substitution or mutation at the amino acid position corresponding to arginine 1648, or a substitution or mutation at the amino acid positions corresponding to arginine 1645 and arginine 1648 in full-length factor VIII. In some embodiments, the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, e.g., alanine.
[0071] A "B domain deleted factor VIII" may have complete or partial deletions as disclosed in U.S. Patent Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, 6,458,563 (each of which is incorporated herein by reference in its entirety). In some embodiments, the B-domain deleted factor VIII sequences of the invention comprise any one of the deletions disclosed in U.S. Patent No. 6,316,226, column 4, line 4 to column 5, line 28 and Examples 1-5 (or also in U.S. Patent No. 6,346,513). In some embodiments, the B-domain deleted factor VIII sequences of the invention have deletions disclosed in U.S. Patent No. 5,789,203, column 2, lines 26-51 and Examples 5-8 (or also in U.S. Patent Nos. 6,060,447, 5,595,886, and 6,228,620). In some embodiments, the B-domain deleted factor VIII can be prepared as described in U.S. Pat. No. 5,972,885, column 1, line 25 to column 2, line 40; U.S. Pat. No. 6,048,720, column 6, lines 1-22 and Example 1; U.S. Pat. No. 5,543,502, column 2, lines 17-46; U.S. Pat. No. 5,171,844, column 4, line 22 to column 5, line 36; U.S. Pat. No. 5,112,950, column 2, lines 17-46; No. 4,868,112; column 19, line 21 and Table 2 of U.S. Pat. No. 4,041,635; column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39 of U.S. Pat. No. 7,041,635; or column 4, lines 25 to 53 of U.S. Pat. No. 6,458,563.In some embodiments, the B domain deleted factor VIII has most of the B domain deleted, but still contains the amino terminal sequence of the B domain that is essential for in vivo proteolytic processing of the primary translation product into two polypeptide chains (i.e., intracellular processing sites) as disclosed in International Publication WO 91 / 09122, which is incorporated by reference in its entirety. In some embodiments, the B domain deleted factor VIII comprises a deletion of amino acids 747-1638, i.e., a substantially complete deletion of the B domain. Hoeben RC, et. al. J. Biol. Chem. 265(13):7318-7323 (1990), which is incorporated herein by reference in its entirety. B-domain deleted factor VIII may also include deletion of amino acids 771-1666 or amino acids 868-1562 of factor VIII. Meulien P., et al. Protein Eng. 2(4):301-6 (1988), which is incorporated herein by reference in its entirety. Additional B domain deletions that are part of the present invention include, for example, a deletion of amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), a deletion of 797-1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)), a deletion of 741-1646 (Kaufman (PCT Published Application WO 87 / 04187)), a deletion of 747-1560 (Sarver et al., DNA 6:553-564 (1987)), the 741 to 1648 deletion (Pasek (PCT Application No. 88 / 00831)), the 816 to 1598 or 741 to 1689 deletion (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, European Patent No. EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions can be made in any factor VIII sequence.
[0072] In one embodiment, the B domain deleted factor VIII portion in the chimeric polypeptide is processed into two chains connected (or linked) by metal binding, the first chain comprising a heavy chain (A1-A2-partially B) and the second chain comprising a light chain (A3-C1-C2). In another embodiment, the B domain deleted factor VIII portion is single chain factor VIII. Single chain factor VIII can comprise an intracellular processing site corresponding to the arginine at residue 754 (residue 773 of SEQ ID NO:2) of B domain deleted factor VIII or at residue 1648 (residue 1657 of SEQ ID NO:6) of full length factor VIII.
[0073] The metal bond between the heavy and light chains can be any metal known in the art. For example, metals useful in the present invention can be divalent metal ions. Metals that can be used to bond the heavy and light chains include Ca. 2+ , Mn 2+ , or Cu 2+ These include, but are not limited to, Fatouros et al., Intern. J. Pharm. 155(1):121-131(1997), Wakabayashi et al., JBC. 279(13):12677-12684(2004).
[0074] As used herein, "chimeric polypeptide" refers to a polypeptide that contains at least two polypeptides (or subsequences or peptides) therein that are derived from different sources. A chimeric polypeptide may contain, for example, two, three, four, five, six, seven, or more polypeptides derived from different sources, such as different genes, different cDNAs, or different animals or other species. A chimeric polypeptide may contain, for example, one or more linkers linking different subsequences. Thus, the subsequences may be directly linked within a single chimeric polypeptide, or may be indirectly linked through a linker, or both. A chimeric polypeptide may contain, for example, additional peptides, such as signal sequences, and sequences such as 6His and FLAG that aid in the purification or detection of the protein. In addition, a chimeric polypeptide may have amino acid or peptide additions to the N- and / or C-terminus.
[0075] In some embodiments, the chimeric polypeptide comprises a factor VIII portion and a non-factor VIII portion. Exemplary non-factor VIII portions include, for example, Fc, XTEN, albumin, PAS sequence, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin binding polypeptides, and albumin binding small molecules. Exemplary chimeric polypeptides of the present invention include, for example, chimeric Factor VIII-Fc polypeptides, chimeric Factor VIII-XTEN polypeptides, chimeric Factor VIII-albumin polypeptides, chimeric Factor VIII-PAS polypeptides, chimeric Factor VIII-transferrin polypeptides, chimeric Factor VIII-CTP polypeptides, chimeric Factor VIII-PEG polypeptides, chimeric Factor VIII-HES polypeptides, chimeric Factor VIII-albumin binding polypeptides, and chimeric Factor VIII-albumin binding small molecule polypeptides.
[0076] Exemplary chimeric Factor VIII-Fc polypeptides include, for example, the chimeric Fc polypeptides of SEQ ID NO: 2 or 6 (Table 2), and SEQ ID NO: 4 (Table 2), with or without their signal sequences.
[0077] The chimeric polypeptide may comprise a sequence that is at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) without the signal sequence (amino acids 20-1684 of SEQ ID NO:2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) with the signal sequence (amino acids 1-1684 of SEQ ID NO:2), which has factor VIII activity. Factor VIII activity can be measured by activated partial thromboplastin time (aPPT) assay, chromogenic assay, or other known methods. The chimeric polypeptide may comprise a sequence that is at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) without the signal sequence (amino acids 20-1684 of SEQ ID NO:2) or at least 90% or 95% identical to the factor VIII and Fc amino acid sequence shown in Table 2A(i) with the signal sequence (amino acids 1-1684 of SEQ ID NO:2).
[0078] As mentioned above, exemplary chimeric polypeptides include factor VIII fused to one or more XTEN polypeptides. Schellenburger et al., Nat. Biotech. 27:1186-90 (2009), which is incorporated herein by reference in its entirety. XTEN polypeptides can be fused to either the N-terminus of FVIII or the C-terminus of FVIII. A protease site can be included between the XTEN portion and the factor VIII portion to allow such processing. XTEN polypeptides include, for example, those disclosed in WO 2009 / 023270, WO 2010 / 091122, WO 2007 / 103515, US 2010 / 0189682, and US 2009 / 0092582, each of which is incorporated herein by reference in its entirety.
[0079] As mentioned above, exemplary chimeric polypeptides also include factor VIII fused with one or more albumin polypeptides, albumin-binding polypeptides, or albumin-binding small molecules.In one embodiment, albumin is human albumin.Albumin or albumin-binding protein can be fused with either the N-terminus of FVIII or the C-terminus of FVIII, or can be inserted between two amino acids in FVIII.Examples of albumin, such as fragments thereof, that can be used in the present invention are known, for example, in U.S. Patent No. 7,592,010, U.S. Patent No. 6,686,179, and Schulte, Thrombosis Res.124 Suppl.2:S6-S8(2009), each of which is incorporated herein by reference in its entirety.
[0080] Albumin-binding polypeptides can include, but are not limited to, bacterial albumin-binding domains, albumin-binding peptides, or albumin-binding antibody fragments that can bind to albumin. Domain 3 from streptococcal protein G, disclosed by Kraulis et al., FEBS Lett. 378: 190-194 (1996) and Linhult et al., Protein Sci. 11: 206-213 (2002), is an example of a bacterial albumin-binding domain. Examples of albumin-binding peptides include a series of peptides with the core sequence DICLPRWGCLW (SEQ ID NO: 7). See, for example, Dennis et al., J. Biol. Chem. 2002, 277: 35035-35043 (2002). Examples of albumin-binding antibody fragments are disclosed in Muller and Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007), Rooverset et al., Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot. Eng. Design Sci., 21:283-288 (2008), which are incorporated by reference in their entireties.
[0081] In certain embodiments, the recombinant FVIII polypeptide of the present invention comprises at least one attachment site for a non-polypeptide small molecule, variant, or derivative thereof that can bind to albumin. An example of such an albumin binding moiety is 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate ("Albu" tag), disclosed by Trusselet et al., Bioconjugate Chem. 20:2286-2292 (2009).
[0082] As mentioned above, exemplary chimeric polypeptides also include factor VIII fused to at least one β subunit of the C-terminal peptide (CTP) of human chorionic gonadotropin, or a fragment, variant, or derivative thereof. The CTP can be fused to factor VIII at either the N-terminus of FVIII or the C-terminus of FVIII, or can be inserted between two amino acids in FVIII. It is known that one or more CTP peptides fused to or inserted into a recombinant protein increase the in vivo half-life of the protein. See, for example, U.S. Patent No. 5,712,122, which is incorporated herein by reference in its entirety. Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ ID NO: 8) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ (SEQ ID NO: 9). See, for example, U.S. Patent Application Publication No. US 2009 / 0087411 A1, which is incorporated by reference.
[0083] As mentioned above, the exemplary chimeric polypeptide also includes factor VIII fused with at least one PAS sequence, or a fragment, variant, or derivative thereof. The PAS sequence can be fused to either the N-terminus of FVIII or the C-terminus of FVIII, or can be inserted between two amino acids in FVIII. As used herein, PAS peptide or PAS sequence refers to an amino acid sequence that forms a random coil structure under physiological conditions, mainly comprising alanine and serine residues, or mainly comprising alanine, serine, and proline residues. Thus, the PAS sequence is a building block, amino acid polymer, or sequence cassette that comprises, consists essentially of, or consists of alanine, serine, and proline, which can be used as part of the heterologous moiety in the chimeric protein. The amino acid polymer can also form a random coil structure when residues other than alanine, serine, and proline are added as minor components in the PAS sequence. By "minor components" is meant that amino acids other than alanine, serine, and proline can be added to the PAS sequence to a certain extent, for example, up to about 12%, i.e., about 12, up to about 10%, up to about 9%, up to about 8%, about 6%, about 5%, about 4%, about 3%, i.e., about 2%, or about 1% of the 100 amino acids of the PAS sequence. The amino acids different from alanine, serine, and proline can be selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. Under physiological conditions, the PAS peptide forms a random coil structure, thereby mediating increased in vivo and / or in vitro stability to the recombinant protein of the invention, which has procoagulant activity.
[0084] Non-limiting examples of PAS peptides include ASPAAPAPASPAAPAPSAPA (SEQ ID NO: 10), AAPASPAPAAPSAPAAPS (SEQ ID NO: 11), APSSPSPSAPSSPSPASPSS (SEQ ID NO: 12), APSSPSPSAPSSPSPSPASPS (SEQ ID NO: 13), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 14), AASPAAPSAPPAAASPAAPSAPPA (SEQ ID NO: 15), ASAAAAPAAASAAASAPSAAA (SEQ ID NO: 16), or any variant, derivative, fragment, or combination thereof. Further examples of PAS sequences are known from U.S. Patent Publication No. 2010 / 0292130 A1 and PCT Publication No. WO 2008 / 155134 A1, European Patent Application No. 2173890.
[0085] As mentioned above, the exemplary chimeric polypeptide also includes factor VIII fused with at least one transferrin peptide, or its fragment, variant, or derivative. At least one transferrin peptide can be fused to either the N-terminus of FVIII or the C-terminus of FVIII, or can be inserted between two amino acids in FVIII. Any transferrin can be fused to or inserted into the recombinant FVIII protein of the present invention. As an example, wild-type human Tf (Tf) is a 679 amino acid protein of about 75 KDa (not composed of glycosylation) with two major domains, N (about 330 amino acids) and C (about 340 amino acids), which appear to be derived from gene duplication. See GenBank Accession Nos. NM001063, XM002793, M12530, XM039845, XM039847, and S95936 (www.ncbi.nlm.nih.gov), all of which are incorporated by reference in their entireties.
[0086] Transferrin transports iron through transferrin receptor (TfR)-mediated endocytosis. After iron is released into endosomal compartments and the Tf-TfR complex is recycled to the cell surface, Tf is released back into the extracellular space for the next cycle of iron transport. Tf has a long half-life of more than 14-17 days (Li et al., Trends Pharmacol. Sci. 23:206-209 (2002)). Transferrin fusion proteins have been investigated for extended half-life, targeted delivery for cancer therapy, oral delivery and sustained activation of proinsulin (Brandsma et al., Biotechnol. Adv., 29:230-238 (2011); Bai et al., Proc. Natl. Acad. Sci. USA 102:7292-7296 (2005); Kim et al., J. Pharmacol. Exp. Ther., 334:682-692 (2010); Wang et al., J. Controlled Release 155:386-392 (2011)).
[0087] As noted above, exemplary chimeric polypeptides also include Factor VIII fused to at least one polyethylene glycol (PEG) moiety.
[0088] PEGylated FVIII may refer to a conjugate formed between FVIII and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a wide variety of molecular weights and average molecular weight ranges. Typical examples of average molecular weight ranges of PEG include, but are not limited to, about 200, about 300, about 400, about 600, about 1000, about 1300-1600, about 1450, about 2000, about 3000, about 3000-3750, about 3350, about 3000-7000, about 3500-4500, about 5000-7000, about 7000-9000, about 8000, about 10000, about 8500-11500, about 16000-24000, about 35000, about 40000, about 60000, and about 80000 Daltons. These average molecular weights are provided merely as examples and are not meant to be limiting in any way.
[0089] The recombinant FVIII protein of the present invention can be PEGylated to include mono- or poly (e.g., 2-4) PEG moieties. PEGylation can be carried out by any PEGylation reaction known in the art. Methods for preparing PEGylated protein products generally include (i) reacting a polypeptide with polyethylene glycol (a reactive ester or aldehyde derivative of PEG) under conditions in which the peptide of the present invention is conjugated with one or more PEG groups, and (ii) obtaining the reaction product(s). In general, optimal reaction conditions for the reaction are determined individually based on known parameters and desired results.
[0090] For example, Malik F et al., Exp. Hematol. 20:1028-35 (1992), Francis, Focus on Growth Factors 3(2):4-10(1992), European Patent Publication Nos. 0401384, 0154316, and 0401384; and International Patent Application Publication Nos. WO 92 / 16221 and WO 95 / 34326. As a non-limiting example, FVIII variants can contain cysteine substitutions at one or more insertion positions in FVIII, and the cysteine can be further conjugated to a PEG polymer. See Mei et al., Blood 116:270-279(2010) and U.S. Patent No. 7,632,921, which are incorporated herein by reference in their entirety.
[0091] As mentioned above, exemplary chimeric polypeptides also include factor VIII fused to at least one hydroxyethyl starch (HES) polymer. HES is a derivative of natural amylopectin and is degraded by alpha-amylase in the body. HES exhibits advantageous biological properties and is used in hemodilution therapy in the clinic as a blood volume replacement agent. See, e.g., Sommermeyer et al., Krankenhauspharmazie 8:271-278 (1987), and Weidler et al., Arzneim.-Forschung / Drug Res. 41:494-498 (1991).
[0092] HES is primarily characterized by its molecular weight distribution and degree of substitution. HES has an average molecular weight (weight average) of 1-300 kD, 2-200 kD, 3-100 kD, or 4-70 kD. Hydroxyethyl starch can further exhibit a molar degree of substitution of 0.1-3, 0.1-2, 0.1-0.9, or 0.1-0.8, and a C2:C6 substitution ratio in the range of 2-20 with respect to the hydroxylethyl groups. HES with an average molecular weight of about 130 kD is Voluven® from Fresenius. Voluven® is an artificial colloid used as a replacement fluid for therapeutic indications, for example, the treatment and prevention of hypovolemia. There are many methods of HES attachment available to the skilled artisan, for example the same PEG attachment method described above.
[0093] In some embodiments, the chimeric polypeptide comprising a factor VIII portion has an increased half-life (t1 / 2) over a polypeptide consisting of the same factor VIII portion without a non-factor VIII portion. The chimeric factor VIII polypeptide having an increased t1 / 2 may be referred to herein as a long-acting factor VIII. Long-acting chimeric factor VIII polypeptides include, for example, factor VIII fused to Fc (including chimeric factor VIII polypeptides in the form of hybrids such as FVIIIFc monomer-dimer hybrids; see Example 1, FIG. 1, and Table 2A; and U.S. Patent Nos. 7,404,956 and 7,348,004), factor VIII fused to XTEN, and factor VIII fused to albumin.
[0094] As used herein, "Culture," "to culture," and "culturing" refer to incubating cells or maintaining cells in a viable state under in vitro conditions that permit cell growth or cell division. As used herein, "cultured cells" refers to cells that are grown in vitro.
[0095] "Factor VIII" as used herein means a functional factor VIII polypeptide in its normal role in blood clotting, unless otherwise specified. Thus, the term factor VIII includes functional variant polypeptides. Factor VIII proteins can be human, porcine, canine, and murine factor VIII proteins. As described in the Background section, full-length polypeptide and polynucleotide sequences are known, as well as many functional fragments, mutants, and modified versions. Examples of human factor VIII sequences are shown as subsequences in SEQ ID NO: 2 or 6 (Table 2). Factor VIII polypeptides include, for example, full-length factor VIII, full-length factor VIII without Met at the N-terminus, mature factor VIII (without signal sequence), mature factor VIII with an additional Met at the N-terminus, and / or factor VIII with a complete or partial deletion of the B domain. Factor VIII variants include B domain deletions, whether partial or complete deletions.
[0096] As mentioned above and below, a large number of functional factor VIII variants are known.In addition, hundreds of non-functional mutations in factor VIII have been identified in hemophilia patients, and it has been found that the effect of these mutations on factor VIII function is due to where they are located in the three-dimensional structure of factor VIII, rather than the nature of the substitution (Cutler et al., Hum.Mutat.19:274-8(2002)), which is incorporated herein by reference in its entirety.In addition, comparison of factor VIII from humans and other species has identified conserved residues that may be required for function (Cameron et al., Thromb.Haemost.79:317-22(1998), US Patent US6,251,632), which is incorporated herein by reference in its entirety.
[0097] The human factor VIII gene has been isolated and expressed in mammalian cells (Toole, JJ, et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, WI, et al., Nature 312:330-337 (1984); Vehar, GA, et al., Nature 312:337-342 (1984); International Publication Nos. WO 87 / 04187, WO 88 / 08035, WO 88 / 03558; U.S. Pat. No. 4,757,006) (each of which is incorporated herein by reference in its entirety), and the amino acid sequence has been deduced from the cDNA. Capon et al., U.S. Patent No. 4,965,199 (incorporated herein in its entirety by reference) discloses recombinant DNA methods for the production of factor VIII in mammalian host cells and the purification of human factor VIII. Human factor VIII expression in CHO (Chinese Hamster Ovary) cells and BHKC (Baby Hamster Kidney cells) has been reported. Human factor VIII has been modified to delete part or all of the B domain (U.S. Patent Nos. 4,994,371 and 4,868,112, each of which is incorporated herein in its entirety by reference), and a replacement of human factor VIII B domain with human factor V B domain has been performed (U.S. Patent No. 5,004,803, incorporated herein in its entirety by reference). The cDNA sequence encoding human factor VIII and the predicted amino acid sequence are shown in SEQ ID NOs: 1 and 2, respectively, in U.S. Patent Application Publication No. 2005 / 0100990 (incorporated herein in its entirety by reference).
[0098] U.S. Patent No. 5,859,204, Lollar, JS (incorporated herein in its entirety by reference) reports functional mutations of factor VIII with reduced antigenicity and reduced immunoreactivity. U.S. Patent No. 6,376,463, Lollar, JS (incorporated herein in its entirety by reference) also reports mutations of factor VIII with reduced immunoreactivity. U.S. Patent Application Publication No. 2005 / 0100990, Saenko et al. (incorporated herein in its entirety by reference) reports functional mutations in the A2 domain of factor VIII.
[0099] A number of functional Factor VIII molecules containing B domain deletions have been disclosed in the following patents: U.S. Patent Nos. 6,316,226 and 6,346,513, both assigned to Baxter; U.S. Patent No. 7,041,635, assigned to In2Gen; U.S. Patent No. 5,789,203, U.S. Patent No. 6,060,447, U.S. Patent No. 5,595,886, and U.S. Patent No. 6,228,620, assigned to Chiron; U.S. Patent No. 5,972,885 and U.S. Patent No. 6,048,720, assigned to Biovitrum; U.S. Patent No. 5,543,502 and U.S. Patent No. 5,610,278, assigned to Novo Nordisk; U.S. Patent No. 5,171,844, assigned to Immuno Ag; U.S. Patent No. 5,112,950, assigned to Transgene SA; and U.S. Patent No. 4,868,112, assigned to Genetics. Institute, each of which is incorporated herein by reference in its entirety.
[0100] The porcine factor VIII sequence has been published (Toole, JJ, et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), which is incorporated herein by reference in its entirety, and the complete porcine cDNA sequence obtained by PCR amplification of the factor VIII sequence from a porcine pancreatic cDNA library has been reported (Healey, JF et al., Blood 88:4209-4214 (1996), which is incorporated herein by reference in its entirety). Hybrid human / porcine factor VIII with all domains, all subunits, and specific amino acid sequence substitutions was disclosed in U.S. Patent No. 5,364,771 by Lollar and Runge, and in WO 93 / 20093, which is incorporated herein by reference in its entirety. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine factor VIII and chimeric factor VIII in which the porcine A1 and / or A2 domains are replaced with the corresponding human domains were reported in WO 94 / 11503 (incorporated herein by reference in its entirety). U.S. Patent No. 5,859,204, Lollar, JS also discloses porcine cDNA and deduced amino acid sequences. U.S. Patent No. 6,458,563, assigned to Emory (incorporated herein by reference in its entirety), discloses a B-domain deleted porcine factor VIII.
[0101] The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2 without the signal sequence (amino acids 20 to 1457 of SEQ ID NO:2 and amino acids 20 to 2351 of SEQ ID NO:6), and the factor VIII portion has factor VIII activity. The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be identical to a factor VIII amino acid sequence shown in Table 2 without the signal sequence (amino acids 20 to 1457 of SEQ ID NO:2 and amino acids 20 to 2351 of SEQ ID NO:6).
[0102] The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be at least 90% or 95% identical to a factor VIII amino acid sequence shown in Table 2, including a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351 of SEQ ID NO:6), and the factor VIII portion has factor VIII activity. The factor VIII (or the factor VIII portion of the chimeric polypeptide) can be identical to a factor VIII amino acid sequence shown in Table 2, including a signal sequence (amino acids 1 to 1457 of SEQ ID NO:2 and amino acids 1 to 2351 of SEQ ID NO:6).
[0103] As used herein, "equivalent dose" refers to the same dose of factor VIII activity, expressed in international units, regardless of the molecular weight of the polypeptide in question. One international unit (IU) of factor VIII activity corresponds approximately to the amount of factor VIII in one milliliter of normal human plasma. Several assays are available for measuring factor VIII activity, including the European Pharmacopoeia chromogenic substrate assay and the one-stage clotting assay.
[0104] As used herein, "Fc" refers to a functional neonatal Fc receptor (FcRn) binding partner unless otherwise specified. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor, resulting in active transport of the FcRn binding partner by the FcRn receptor. Thus, the term Fc includes any variant of IgG Fc that is functional. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al., Nature 372:379 (1994), incorporated herein by reference in its entirety). The main contact region of FcRn and Fc is near the junction of the CH2 and CH3 domains. All Fc-FcRn contacts are within a single Ig heavy chain. FcRn binding partners include, for example, whole IgG, Fc fragments of IgG, and other fragments of IgG that contain the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 in the CH2 domain, and amino acid residues 385-387, 428, and 433-436 in the CH3 domain. All references made to amino acid numbering of immunoglobulins or immunoglobulin fragments or regions are based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, USDepartment of Public Health, Bethesda; MD, which is incorporated herein by reference in its entirety. (FcRn receptors have been isolated from several mammalian species, including humans. The sequences of human FcRn, rat FcRn, and mouse FcRn are known (Story et al., J. Exp. Med. 180:2377 (1994), incorporated herein by reference in its entirety). Fc may comprise immunoglobulin CH2 and CH3 domains with or without the immunoglobulin hinge region. Exemplary Fc variants are provided in WO 2004 / 101740 and WO 2006 / 074199, incorporated herein by reference in their entireties.
[0105] The Fc (or the Fc portion of a chimeric polypeptide) may contain one or more mutations, and combinations of mutations.
[0106] The Fc (or the Fc portion of the chimeric polypeptide) may be an increased half-life-conferring mutant such as M252Y, S254T, T256E, and combinations thereof, disclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is incorporated by reference in its entirety, and H433K, N434F, and combinations thereof, disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporated by reference in its entirety, the mutants disclosed in paragraphs
[0012] on pages 1-2, and in Examples 9 and 10 of U.S. Patent No. 2009 / 0264627 A1, which is incorporated by reference in its entirety, and the mutants disclosed in U.S. Patent No. 20090163699, which is incorporated by reference in its entirety. A1, page 2, paragraphs
[0014] to
[0021] (hereby incorporated by reference in its entirety).
[0107] The Fc (or the Fc portion of the chimeric polypeptide) may also include, for example, the following mutations: The Fc region of IgG can also be modified according to well-recognized techniques, such as site-directed mutagenesis, to obtain modified IgG or Fc fragments or portions thereof that will be bound by FcRn. Such modifications include modifications distant from the FcRn contact site, as well as modifications within the contact site that retain or even enhance binding to FcRn. For example, human IgG1 One of the following amino acid residues in Fc (Fcy1) can be substituted without significant loss of Fc binding affinity to FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S 298A, Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K3 17A, E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, A330S, P33 1A, P331S, E333A, K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345 A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A D376A, A378Q, E380A, E382A, S383A, N384A, Q386A, E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where, for example, P238A indicates that the wild-type proline at position 238 is replaced by alanine. In addition to alanine, other amino acids can also be substituted for the wild-type amino acids at the positions specified above.Mutations can be introduced into Fc alone to generate over 100 FcRn binding partners that are different from the native Fc. Furthermore, combinations of two, three or more of these individual mutations can be introduced together to generate hundreds more FcRn binding partners. Some of these mutations can also confer new functionality to FcRn binding partners. For example, one embodiment incorporates N297A, removing a highly conserved N-glycosylation site. The effect of this mutation is to reduce immunogenicity, thereby enhancing the circulating half-life of the FcRn binding partner, as well as to render it unable to bind FcyRI, FcyRIIA, FcyRIIB, and FcyRIIIA without compromising affinity for FcRn (Routledge et al. 1995, Transplantation 60:847, incorporated herein by reference in its entirety; Friend et al. 1999, Transplantation 68:1632, incorporated herein by reference in its entirety; Shields et al. 1995, J. Biol. Chem. 276:6591, incorporated herein by reference in its entirety). Furthermore, at least three human Fc gamma receptors appear to recognize a binding site on IgG within the lower hinge region, generally amino acids 234-237. Thus, another example of new functions and potentially reduced immunogenicity can result from mutations in this region, for example by replacing amino acids 233-236 of human IgG1 "ELLG" with the corresponding sequence from IgG2 "PVA" (with one amino acid deletion). FcyRI, FcyRII, and FcyRIII, which mediate various effector functions, have been shown not to bind IgG1 when such mutations are introduced (Ward and Ghetie, Therapeutic Immunology 2:77 (1995), incorporated herein by reference in their entireties; and Armour et al., Eur. J. Immunol. 29:2613 (1999), incorporated herein by reference in their entireties).As another example of the new functionality resulting from the above mutations, in some cases, the affinity for FcRn can be increased from that of wild type. This increased affinity can reflect an increase in "on" rate, a decrease in "off" rate, or both an increase in "on" rate and a decrease in "off" rate. Mutations that are thought to confer increased affinity for FcRn include, for example, T256A, T307A, E380A, and N434A (Shields et al., J.Biol.Chem.276:6591 (2001), which is incorporated herein by reference in its entirety).
[0108] The Fc (or the Fc portion of the chimeric polypeptide) can be at least 90% or 95% identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO:2 or amino acids 2352 to 2578 of SEQ ID NO:6). The Fc (or the Fc portion of the chimeric polypeptide) can be identical to the Fc amino acid sequence shown in Table 2 (amino acids 1458 to 1684 of SEQ ID NO:2 and amino acids 2352 to 2578 of SEQ ID NO:6).
[0109] "Hybrid" polypeptides and proteins as used herein refer to a combination of a chimeric polypeptide with a second polypeptide. The chimeric polypeptide and the second polypeptide in the hybrid may be bound to each other by protein-protein interactions, such as charge-charge interactions or hydrophobic interactions. The chimeric polypeptide and the second polypeptide in the hybrid may be bound to each other by disulfide bonds or other covalent bond(s). Hybrids are described in WO 2004 / 101740 and WO 2006 / 074199, each of which is incorporated herein by reference in its entirety. See also U.S. Patent Nos. 7,404,956 and 7,348,004, each of which is incorporated herein by reference in its entirety. The second polypeptide may be a second copy of the same chimeric polypeptide or a non-identical chimeric polypeptide. See, for example, FIG. 1, Example 1, and Table 2. In one embodiment, the second polypeptide is a polypeptide comprising an Fc. In another embodiment, the chimeric polypeptide is a chimeric Factor VIII-Fc polypeptide and the second polypeptide is a hybrid polypeptide of Example 1, which is a rFVIIIFc recombinant fusion protein consisting essentially of Fc, e.g., one molecule of recombinant B-domain deleted human FVIII (BDD-rFVIII) fused to a dimeric Fc domain of human IgG1, without an intervening linker sequence. This hybrid polypeptide is referred to herein as FVIIIFc monomer Fc fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIIFc hybrid, and FVIIIFc monomer-dimer. See Example 1, Figure 1, and Table 2A. The Examples provide preclinical and clinical data for this hybrid polypeptide.
[0110] The second polypeptide in the hybrid may comprise or essentially consist of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21-247 of SEQ ID NO:4), or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids 1-247 of SEQ ID NO:4). The second polypeptide may comprise or essentially consist of an amino acid sequence that is at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) without the signal sequence (amino acids 21-247 of SEQ ID NO:4), or at least 90% or 95% identical to the amino acid sequence shown in Table 2A(ii) with the signal sequence (amino acids 1-247 of SEQ ID NO:4).
[0111] FIG. 1 is a schematic diagram showing the structure of the B-domain deleted factor VIII-Fc chimeric polypeptide and its association with a second polypeptide, which is an Fc polypeptide. To obtain this hybrid, the coding sequence of human recombinant B-domain deleted FVIII was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver polyA RNA (Clontech) using FVIII specific polymers. The FVIII sequence contains the native signal sequence of FVIII. The B-domain deletion was a total of 2682 bp deletion from serine 743 (S743; 2287 bp) to glutamine 1638 (Q1638; 4969 bp). The coding sequence of human recombinant Fc was then obtained by RT-PCR from a human leukocyte cDNA library (Clontech) using Fc specific polymers. Primers were designed such that the B-domain deleted FVIII sequence was fused directly to the N-terminus of the Fc sequence without an intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under the control of the CMV promoter. A second identical Fc sequence containing the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of a second promoter, EF1α, in the expression vector pBUDCE4.1.
[0112] The rFVIIIFc expression vector was transfected into human embryonic kidney 293 cells (HEK293H; Invitrogen) using Lipofectamine2000 transfection reagent (Invitrogen). Stable clonal cell lines were generated by selection with Zeocin (Invitrogen). One clonal cell line, 3C4-22, was used to generate FVIIIFc for in vivo characterization. Recombinant FVIIIFc was produced and purified at Biogen Idec (Cambridge, MA) (McCue et al. 2009). The transfection strategy described above was expected to yield three products, namely, monomeric rFVIIIFc hybrid, dimeric rFVIIIFc hybrid, and dimeric Fc. However, essentially no dimeric rFVIIIFc was detected in the conditioned medium from these cells. Rather, the conditioned medium contained Fc and monomeric rFVIIIFc. The size of the dimeric rFVIIIFc may have been too large and prevented adequate secretion from cells. This result was informative because it made purification of the monomer less complicated than if all three proteins were present. The material used in these studies had a specific activity of approximately 9000 IU / mg.
[0113] In one embodiment, the polypeptide of the present invention is administered to a patient expressing high levels of von Willebrand factor (VWF). As used herein, "subject" or "patient" refers to a human individual. The subject may be a patient currently suffering from a bleeding disorder or expected to require such treatment. "Subject" may include an adult or pediatric subject. A pediatric subject may be a pediatric patient under the age of 12. As used herein, the term "pediatrics" is a department that deals with the care of infants and children and the treatment of their diseases. In one embodiment, the subject is a pediatric patient who has been diagnosed with severe hemophilia A. In certain embodiments, a pediatric subject is treated with a long-acting factor VIII polypeptide of the present invention.
[0114] VWF is a plasma protein with a multimeric structure whose molecular weight in its various forms varies from about 230 kDa for each monomeric subunit to more than 20 million Da in the larger molecular weight multimeric forms, thus forming the largest soluble protein known. Its plasma concentration is approximately around 5-10 μg / mL (Siedlecki et al., Blood, vol 88:2939-2950 (1996)), with the smaller size plasma form corresponding to a dimer with a size of about 500 kDa.
[0115] VWF plays an important role in primary hemostasis, involved in the adhesion of platelets to damaged vascular surfaces and thus the formation of platelet plugs on which the mechanisms of formation of fibrin aggregates unfold. It has been suggested that higher molecular weight multimers more efficiently support platelet adhesion mechanisms to the subendothelium, and the clinical efficacy of VWF concentrates has been related to the concentration of these higher molecular weight multimers (Metzner et al., Haemophilia 4:25-32 (1998)).
[0116] Thus, subjects expressing high levels of VWF will require less frequent administration of FVIII compared to subjects expressing lower or normal levels of VWF. The average range of VWF in plasma is about 50 IU / dL to about 200 IU / dL. In one embodiment, the average level of VWF in plasma is about 50 IU / dL. In another embodiment, a VWF level in plasma of at least about 100 IU / dL is considered to be a high VWF level. In another embodiment, a high level of VWF in plasma is about 100 IU / dL to about 200 IU / dL. In another embodiment, the high level of VWF in plasma is at least about 110 IU / dL, about 120 IU / dL, about 130 IU / dL, about 140 IU / dL, about 150 IU / dL, about 160 IU / dL, about 170 IU / dL, about 180 IU / dL, about 190 IU / dL, or about 200 IU / dL.
[0117] Thus, in one embodiment, the long-acting FVIII polypeptide of the present invention is administered in a long interval dosing regimen to a subject expressing at least about 100 IU / dL of plasma VWF.In one embodiment, the long-acting FVIII polypeptide is administered at a dosing interval of at least about 3 days.In another embodiment, the long-acting FVIII polypeptide is administered at a dosing interval of at least about once per week, about once per 2 weeks, about once per 15 days, about once per 20 days, about once per 3 weeks, about once per 25 days, about once per 4 weeks, or about once per month.
[0118] In one embodiment, the subject has previously been identified as having high levels of VWF. In certain embodiments, subjects with serotypes other than O (i.e., A, B, or AB) require less frequent administration of long-acting FVIII, since long-acting FVIII has a longer half-life in these subjects. In these subjects, the increased half-life is due to their elevated VWF levels.
[0119] Furthermore, pharmacokinetic data, defined as a study of the time course of drug absorption, distribution, metabolism, and excretion, can be used as a discriminator of subjects who are suitable for longer or shorter dosing intervals using the long-acting FVIII polypeptides of the present invention. Clinical pharmacokinetics is the application of pharmacokinetic principles to the therapeutic management of drug safety and efficacy in individual patients. The primary objectives of clinical pharmacokinetics include enhancing the efficacy of patients' drug therapies and reducing their toxicity. The occurrence of strong correlations between drug concentrations and their pharmacological responses allows clinicians to apply pharmacokinetic principles to real patient situations.
[0120] Thus, in one embodiment, the half-life of the FVIII-Fc polypeptide of the present invention is used to identify patients expressing high levels of VWF. The half-life of FVIII-Fc ranges from about 10 to about 40 hours, differing at least in part in the level of VWF present. However, on average, the half-life of FVIII-Fc is about 18 hours. In general, FVIII-Fc exhibits at least about 1.2-fold increased half-life in patients with high levels of VWF compared to the half-life of FVIII-Fc when administered to individuals with average levels of VWF. In one embodiment, FVIII-Fc exhibits at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold increased half-life compared to the half-life of FVIII-Fc when administered to individuals with average levels of VWF. In one embodiment, the half-life of FVIII-Fc in a subject expressing high levels of VWF is at least about 20 hours to about 40 hours. In another embodiment, the half-life of FVIII-Fc is at least about 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, or 40 hours. In one embodiment, the half-life of FVIII-Fc in a subject expressing high levels of VWF is about 20 to about 27 hours. Thus, in one embodiment, an increased half-life of FVIII-Fc compared to the mean value indicates a subject suitable for longer administration intervals using a long-acting FVIII polypeptide of the present invention.
[0121] In another embodiment, the half-life of the short-acting FVIII polypeptide is used to identify patients who express high levels of VWF. As used herein, the term "short-acting FVIII" refers to a FVIII polypeptide without the addition of a half-life extension element. In one embodiment, the short-acting FVIII polypeptide consists of full-length or B-domain deleted FVIII. Examples of short-acting FVIII polypeptides are Advate® and ReFacto®.
[0122] Since the half-life of short-acting FVIII also depends at least in part on the VWF level, the short-acting FVIII polypeptide can also be used to identify patients who are suitable for longer administration intervals of the long-acting FVIII polypeptide of the present invention.In one embodiment, the short-acting FVIII shows at least about 1.2-fold increased half-life in individuals expressing high levels of VWF compared to the half-life of the short-acting FVIII when administered to individuals with average levels of VWF.In another embodiment, the short-acting FVIII shows at least about 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5-fold increased half-life in individuals expressing high levels of VWF compared to the half-life of the short-acting FVIII when administered to individuals with average levels of VWF. Thus, when an individual is administered a short-acting FVIII, an individual that exhibits at least about a 1.2-fold half-life is eligible for longer dosing intervals with a long-acting FVIII polypeptide of the present invention.
[0123] As used herein, "dosing interval" refers to the time that elapses between multiple doses administered to a subject. Comparison of dosing intervals can be performed in a single subject or in a population of subjects, and the average obtained in the population can then be calculated.
[0124] The dosing interval when administering a chimeric factor VIII polypeptide, e.g., a chimeric factor VIII-Fc polypeptide of the invention (a polypeptide comprising factor VIII or a hybrid) can be at least about 1.5 times longer than the dosing interval required for an equivalent dose of the factor VIII (a polypeptide comprising the factor VIII) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. The dosing interval can be at least about 1.5 to 6 times longer, 1.5 to 5 times longer, 1.5 to 4 times longer, 1.5 to 3 times longer, or 1.5 to 2 times longer than the dosing interval required for an equivalent dose of the factor VIII (a polypeptide comprising the factor VIII) that does not include a non-factor VIII portion, e.g., does not include an Fc portion. The dosing interval can be at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5.5.5, or 6 times longer than the dosing interval required for an equivalent dose of the factor VIII (polypeptide consisting of the factor VIII) that does not include the non-factor VIII portion, e.g., does not include the Fc portion. The dosing interval can be about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or more. The dosing interval can be at least about 1.5-5, 1.5, 2, 3, 4, or 5 days, or more. For on-demand treatment, the chimeric polypeptide or hybrid is administered about once every 24-36, 24-48, 24-72, 24-96, 24-120, 24-144, 24-168, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours or more.
[0125] In one embodiment, the effective amount is 25-80 IU / kg (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 IU / kg) and the dosing interval is once every 3-5, 3-6, 3-7, 3, 4, 5, 6, 7, or 8 days or more, or 3 times per week, or no more than 3 times per week. In one embodiment, the effective amount is 80 IU / kg and the dosing interval is once every 3 days. In a further embodiment, an effective amount of 80 IU / kg given at a dosing interval of every 3 days is administered to a pediatric subject. In another embodiment, the effective amount is 65 IU / kg and the dosing interval is once a week or once every 6-7 days. This dose can be repeated as long as necessary (e.g., for at least 10, 20, 28, 30, 40, 50, 52, or 57 weeks, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years).
[0126] In certain embodiments, the effective amount for on-demand treatment is 20-50 IU / Kg (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 IU / kg). On-demand treatment can be a one-time administration or repeated administration. For repeated administration, the administration interval can be every 12-24 hours, every 24-36 hours, every 24-48 hours, every 36-48 hours, or every 48-72 hours.
[0127] A "long-acting factor VIII" is a factor VIII having an increased half-life (referred to herein as t1 / 2, t1 / 2 beta, elimination half-life, and HL) over a reference factor VIII. The increased half-life of a long-acting factor VIII can be due to fusion to one or more non-factor VIII polypeptides, such as Fc, XTEN, albumin, PAS sequence, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of hCG with four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, etc., or a combination of two or more thereof. The increased half-life can be due to one or more modifications, such as, for example, pegylation. Exemplary long-acting factor VIII polypeptides include, for example, chimeric factor VIII polypeptides comprising Fc, chimeric factor VIII polypeptides comprising XTEN, and chimeric factor VIII polypeptides comprising albumin. Further exemplary long-acting factor VIII polypeptides include, for example, pegylated factor VIII.
[0128] In the case of a long-acting chimeric Factor VIII polypeptide, the "reference" polypeptide is a polypeptide that consists essentially of the Factor VIII portion of the chimeric polypeptide, e.g., the same Factor VIII portion without the Fc portion, the XTEN portion, or the albumin portion. Similarly, in the case of a modified Factor VIII, the reference polypeptide is the same Factor VIII without modification, e.g., non-pegylated Factor VIII.
[0129] In some embodiments, the long-acting factor VIII has one or more of the following properties when administered to a subject: Mean residence time (MRT) (activity) in the subject of approximately 14 to 41.3 hours; Clearance (CL) in the subject of about 1.22 to 5.19 mL / hr / kg (activity); t1 / 2 beta (activity) in the subject of approximately 11 to 26.4 hours; Incremental recovery rate (K value) in the subject of approximately 1.38 to 2.88 IU / dL per IU / kg (activity; observed value); A Vss (activity) in the subject of about 37.7 to 79.4 mL / kg; and Approximately 19.2 to 81.7 IU per kg * AUC / dose in the subject in h / dL.
[0130] In some embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: Mean incremental recovery (K value) of more than 1.38 IU / dL per IU / kg (activity; observed value); a mean incremental recovery (K value) of at least about 1.5, at least about 1.85, or at least about 2.46 IU / dL per IU / kg (activity; observed value); A mean clearance (CL) in this patient population of about 2.33±1.08 mL / hr / kg or less (activity); Mean clearance (CL) in this patient population of approximately 1.8 to 2.69 mL / hr / kg (activity); a mean clearance (CL) (activity) in the patient population that is about 65% of the clearance of the unmodified factor VIII-containing polypeptide; A mean mean residence time (MRT) (activity) in the patient population of at least about 26.3±8.33 hours; an average MRT (activity) in the patient population of about 25.9 to 26.5 hours, which is about 1.5 times longer than the average MRT of the polypeptide containing factor VIII without modification; Mean t1 / 2 beta (activity) in this patient population of approximately 18.3 ± 5.79 hours; The mean t1 / 2 beta (activity) in this patient population is approximately 18 to 18.4 hours; a mean t1 / 2 beta (activity) in said patient population that is about 1.5 times longer than the mean t1 / 2 beta of said polypeptide containing factor VIII without modification; A mean incremental recovery (K value) in this patient population of approximately 2.01 ± 0.44 IU / dL per IU / kg (activity; observed value); A mean incremental recovery rate (K value) in this patient population of approximately 1.85 to 2.46 IU / dL per IU / kg (activity; observed value); a mean incremental recovery (K value) in the patient population that is about 90% of the mean incremental recovery of the polypeptide containing the factor VIII without modification (activity; observed value); Mean Vss(activity) in this patient population of approximately 55.1±12.3 mL / kg; Mean Vss (activity) in this patient population of approximately 45.3 to 56.1 mL / kg; Approximately 49.9±18.2 IU per kg * Mean AUC / dose (activity) in the patient population in h / dL; Approximately 44.8 to 57.6 IU per kg * Mean AUC / dose (activity) in the patient population in h / dL.
[0131] In other embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: C in the subject to which the chimeric polypeptide was administered max When OBS is measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay, the C max _Comparable to OBS, When about 25 IU / kg of the chimeric polypeptide is administered, a C in the subject of about 60.5 IU / dL, about 60.5±1 IU / dL, about 60.5±2 IU / dL, about 60.5±3 IU / dL, about 60.5±4 IU / dL, about 60.5±5 IU / dL, about 60.5±6 IU / dL, about 60.5±7 IU / dL, about 60.5±8 IU / dL, about 60.5±9 IU / dL, or about 60.5±10 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 53.1 to 69 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the following concentrations are measured by a one-stage (aPTT) assay: about 119 IU / dL, about 119±1 IU / dL, about 119±2 IU / dL, about 119±3 IU / dL, about 119±4 IU / dL, about 119±5 IU / dL, about 119±6 IU / dL, about 119±7 IU / dL, about 119± C in the subject of about 8 IU / dL, about 119±9 IU / dL, about 119±10 IU / dL, about 119±11 IU / dL, about 119±12 IU / dL, about 119±13 IU / dL, about 119±14 IU / dL, about 119±15 IU / dL, about 119±16 IU / dL, about 119±17 IU / dL, or about 119±18 IU / dL max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 103 to 136 IU / dL as measured by a one-stage (aPTT) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, a C in the subject of about 76.5 IU / dL, about 76.5±1 IU / dL, about 76.5±2 IU / dL, about 76.5±3 IU / dL, about 76.5±4 IU / dL, about 76.5±5 IU / dL, about 76.5±6 IU / dL, about 76.5±7 IU / dL, about 76.5±8 IU / dL, about 76.5±9 IU / dL, about 76.5±10 IU / dL, about 76.5±11 IU / dL, about 76.5±12 IU / dL, about 76.5±13 IU / dL, about 76.5±14 IU / dL, or about 76.5±15 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, When about 25 IU / kg of the chimeric polypeptide is administered, the subject has a C of about 64.9 to 90.1 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, When about 65 IU / kg of the chimeric polypeptide is administered, a C in the subject of about 182 IU / dL, about 182±2 IU / dL, about 182±4 IU / dL, about 182±6 IU / dL, about 182±8 IU / dL, about 182±10 IU / dL, about 182±12 IU / dL, about 182±14 IU / dL, about 182±16 IU / dL, about 182±18 IU / dL, or about 182±20 IU / dL as measured by a two-stage (chromogenic) assay. max _OBS, or When about 65 IU / kg of the chimeric polypeptide is administered, the C in the subject is about 146-227 IU / dL, about 146±5 IU / dL, about 146±10 IU / dL, about 227±5 IU / dL, or about 146±10 IU / dL, as measured by a two-stage (chromogenic) assay. max _OBS.
[0132] In certain embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: When measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay, it is at least 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2. 62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, or 1.90 times higher t1 / 2 beta (activity) in said subject; a t1 / 2 beta (activity) in the subject of about 18.8 hours, 18.8±1 hour, 18.8±1 hour, 18.8±2 hours, 18.8±3 hours, 18.8±4 hours, 18.8±5 hours, 18.8±6 hours, 18.8±7 hours, 18.8±8 hours, 18.8±9 hours, 18.8±10 hours, or 18.8±11 hours as measured by a one-stage (aPTT) assay; a t1 / 2 beta (activity) in the subject of about 14.3 to 24.5 hours as measured by a one-stage (aPTT) assay; a t1 / 2 beta (activity) in the subject of about 16.7 hours, 16.7±1 hour, 16.7±2 hours, 16.7±3 hours, 16.7±4 hours, 16.7±5 hours, 16.7±6 hours, 16.7±7 hours, 16.7±8 hours, 16.7±9 hours, 16.7±10 hours, or 16.7±11 hours, as measured by a two-stage (chromogenic) assay; a t1 / 2 beta (activity) in the subject of about 13.8 to 20.1 hours, as measured by a two-stage (chromogenic) assay; a t1 / 2 beta (activity) in the subject of about 19.8 hours, 19.8±1 hour, 19.8±2 hours, 19.8±3 hours, 19.8±4 hours, 19.8±5 hours, 19.8±6 hours, 19.8±7 hours, 19.8±8 hours, 19.8±9 hours, 19.8±10 hours, or 19.8±11 hours, as measured by a two-stage (chromogenic) assay; or t1 / 2 beta (activity) in the subject of approximately 14.3 to 27.5 hours as measured by a two-stage (chromogenic) assay.
[0133] In certain embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: the clearance (CL) (activity) in the subject, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay, is 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, or 0.70-fold lower than the clearance in a subject administered the same amount of a polypeptide consisting of full-length mature factor VIII; a clearance (CL) (activity) in the subject of about 1.68 mL / hr / kg, 1.68±0.1 mL / hr / kg, 1.68±0.2 mL / hr / kg, 1.68±0.3 mL / hr / kg, 1.68±0.4 mL / hr / kg, 1.68±0.5 mL / hr / kg, 1.68±0.6 mL / hr / kg, or 1.68±0.7 mL / hr / kg, as measured by a one-stage (aPTT) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.31 to 2.15 mL / hr / kg, as measured by a one-stage (aPTT) assay, when about 25 IU / kg of the chimeric polypeptide is administered; When about 65 IU / kg of the chimeric polypeptide is administered, the clearance (CL) (activity) in the subject is about 2.32 mL / hr / kg, 2.32 ± 0.1 mL / hr / kg, 2.32 ± 0.2 mL / hr / kg, 2.32 ± 0.3 mL / hr / kg, 2.32 ± 0.4 mL / hr / kg, 2.32 ± 0.5 mL / hr / kg, 2.32 ± 0.6 mL / hr / kg, or 2.32 ± 0.7 mL / hr / kg as measured by a one-stage (aPTT) assay; when about 65 IU / kg of the chimeric polypeptide is administered, the clearance (CL) (activity) in the subject is about 1.64 to 3.29 mL / hr / kg as measured by a one-stage (aPTT) assay; a clearance (CL) (activity) in the subject of about 1.49 mL / hr / kg, 1.49±0.1 mL / hr / kg, 1.49±0.2 mL / hr / kg, 1.49±0.3 mL / hr / kg, 1.49±0.4 mL / hr / kg, 1.49±0.5 mL / hr / kg, 1.49±0.6 mL / hr / kg, or 1.49±0.7 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.16 to 1.92 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; a clearance (CL) (activity) in the subject of about 1.52 mL / hr / kg, 1.52±0.1 mL / hr / kg, 1.52±0.2 mL / hr / kg, 1.52±0.3 mL / hr / kg, 1.52±0.4 mL / hr / kg, 1.52±0.5 mL / hr / kg, 1.52±0.6 mL / hr / kg, or 1.52±0.7 mL / hr / kg, as measured by a two-stage (chromogenic) assay, when about 65 IU / kg of the chimeric polypeptide is administered; or When about 65 IU / kg of the chimeric polypeptide is administered, the clearance (CL) (activity) in the subject is about 1.05-2.20 mL / hr / kg, as measured by a two-stage (chromogenic) assay.
[0134] In some embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: The MRT in the subject, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay, is at least 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2. 60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, or 1.93 times higher, MRT (activity) in the subject at about 27 hours, 27±1 hours, 27±2 hours, 27±3 hours, 27±4 hours, 27±5 hours, 27±6 hours, 27±7 hours, 27±8 hours, 27±9 hours, or 27±10 hours, as measured by a one-stage (aPTT) assay; MRT (activity) in the subject of about 20.6 to 35.3 hours, as measured by a one-stage (aPTT) assay; MRT (activity) in the subject of about 23.9 to 28.5 hours, as measured by a two-stage (chromogenic) assay; an MRT (activity) in the subject of about 19.8 to 28.9 hours, as measured by a two-stage (chromogenic) assay; or MRT (activity) in the subject of approximately 20.5-39.6 hours, as measured by a two-stage (chromogenic) assay.
[0135] In other embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: an incremental recovery in a subject that is comparable to the incremental recovery in a subject administered the same amount of a polypeptide consisting of full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay; When about 25 IU / kg of the chimeric polypeptide is administered, the IL-10 levels are approximately 2.44 IU / dL per IU / kg, 2.44±0.1 IU / dL per IU / kg, 2.44±0.2 IU / dL per IU / kg, 2.44±0.3 IU / dL per IU / kg, 2.44±0.4 IU / dL per IU / kg, 2.44±0.5 IU / dL per IU / kg, and 2.44±0.6 IU / dL per IU / kg, as measured by a one-stage (aPTT) assay. dL, 2.44±0.6 IU / dL per IU / kg, 2.44±0.7 IU / dL per IU / kg, 2.44±0.8 IU / dL per IU / kg, 2.44±0.9 IU / dL per IU / kg, 2.44±1.0 IU / dL per IU / kg, 2.44±1.1 IU / dL per IU / kg, or 2.44±1.2 IU / dL per IU / kg; an incremental recovery in the subject of about 2.12 to 2.81 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; When about 65 IU / kg of the chimeric polypeptide is administered, there are about 1.83 IU / dL per IU / kg, 1.83±0.1 IU / dL per IU / kg, 1.83±0.2 IU / dL per IU / kg, 1.83±0.3 IU / dL per IU / kg, 1.83±0.4 IU / dL per IU / kg, and 1.83±0.5 IU / dL per IU / kg, as measured by a one-stage (aPTT) assay. an incremental recovery in the subject of 1.83 ± 0.5 IU / dL per IU / kg, 1.83 ± 0.6 IU / dL per IU / kg, 1.83 ± 0.7 IU / dL per IU / kg, 1.83 ± 0.8 IU / dL per IU / kg, 1.83 ± 0.9 IU / dL per IU / kg, 1.83 ± 1.0 IU / dL per IU / kg, or 1.83 ± 1.1 IU / dL per IU / kg; an incremental recovery in the subject of about 1.59 to 2.10 IU / dL per IU / kg as measured by a one-stage (aPTT) assay when about 65 IU / kg of the chimeric polypeptide is administered; When about 25 IU / kg of the chimeric polypeptide is administered, the IL-10 levels are approximately 3.09 IU / dL per IU / kg, 3.09±0.1 IU / dL per IU / kg, 3.09±0.2 IU / dL per IU / kg, 3.09±0.3 IU / dL per IU / kg, 3.09±0.4 IU / dL per IU / kg, 3.09±0.5 IU / dL per IU / kg, and 3.09±0.6 IU / dL per IU / kg, as measured by the two-stage (chromogenic) assay. an incremental recovery in the subject of 3.09±0.6 IU / dL, 3.09±0.7 IU / dL per IU / kg, 3.09±0.8 IU / dL per IU / kg, 3.09±0.9 IU / dL per IU / kg, 3.09±1.0 IU / dL per IU / kg, 3.09±1.1 IU / dL per IU / kg, 3.09±1.2 IU / dL per IU / kg, or 3.09±1.3 IU / dL per IU / kg; When about 65 IU / kg of the chimeric polypeptide is administered, the IL-10 levels are approximately 2.80 IU / dL per IU / kg, 2.80±0.1 IU / dL per IU / kg, 2.80±0.2 IU / dL per IU / kg, 2.80±0.3 IU / dL per IU / kg, 2.80±0.4 IU / dL per IU / kg, 2.80±0.5 IU / dL per IU / kg, and 2.80±0.6 IU / dL per IU / kg, as measured by the two-stage (chromogenic) assay. L, 2.80±0.6 IU / dL per IU / kg, 2.80±0.7 IU / dL per IU / kg, 2.80±0.8 IU / dL per IU / kg, 2.80±0.9 IU / dL per IU / kg, 2.80±1.0 IU / dL per IU / kg, 2.80±1.1 IU / dL per IU / kg, or 2.80±1.2 IU / dL per IU / kg; an incremental recovery in the subject of about 2.61 to 3.66 IU / dL per IU / kg, as measured by a two-stage (chromogenic) assay, when about 25 IU / kg of the chimeric polypeptide is administered; and When about 65 IU / kg of the chimeric polypeptide is administered, the incremental recovery in the subject is about 2.24-3.50 IU / dL per IU / kg as measured by a two-stage (chromogenic) assay.
[0136] In yet other embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: a Vss(activity) in the subject that is comparable to the Vss(activity) in a subject administered the same amount of a polypeptide consisting of full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay; a Vss (activity) in the subject of about 45.5 mL / kg, 45.5±1 mL / kg, 45.5±2 mL / kg, 45.5±3 mL / kg, 45.5±4 mL / kg, 45.5±5 mL / kg, 45.5±6 mL / kg, 45.5±7 mL / kg, 45.5±8 mL / kg, 45.5±9 mL / kg, 45.5±10 mL / kg, or 45.5±11 mL / kg as measured by a one-stage (aPTT) assay when about 25 IU / kg of the chimeric polypeptide is administered; a Vss (activity) in the subject of about 39.3 to 52.5 mL / kg, as measured by a one-stage (aPTT) assay, when about 25 IU / kg of the chimeric polypeptide is administered; When about 65 IU / kg of the chimeric polypeptide is administered, a Vss (activity) in the subject of about 62.8 mL / kg, 62.8±1 mL / kg, 62.8±2 mL / kg, 62.8±3 mL / kg, 62.8±4 mL / kg, 62.8±5 mL / kg, 62.8±6 mL / kg, 62.8±7 mL / kg, 62.8±8 mL / kg, 62.8±9 mL / kg, 62.8±10 mL / kg, 62.8±11 mL / kg, 62.8±12 mL / kg, 62.8±13 mL / kg, 62.8±14 mL / kg, 62.8±15 mL / kg, or 62.8±16 mL / kg as measured by a one-stage (aPTT) assay; When about 65 IU / kg of the chimeric polypeptide is administered, a Vss (activity) in the subject of about 55.2 to 71.5 mL / kg as measured by a one-stage (aPTT) assay; when about 25 IU / kg of the chimeric polypeptide is administered, a Vss (activity) in the subject of about 35.9 mL / kg, 35.9±1 mL / kg, 35.9±2 mL / kg, 35.9±3 mL / kg, 35.9±4 mL / kg, 35.9±5 mL / kg, 35.9±6 mL / kg, 35.9±7 mL / kg, 35.9±8 mL / kg, 35.9±9 mL / kg, 35.9±10 mL / kg, 35.9±11 mL / kg, 35.9±12 mL / kg, or 35.9±13 mL / kg, as measured by a two-stage (chromogenic) assay; When about 25 IU / kg of the chimeric polypeptide is administered, a Vss (activity) in the subject of about 30.4 to 42.3 mL / kg, as measured by a two-stage (chromogenic) assay; or a Vss (activity) in the subject of about 43.4 mL / kg, 43.4±1 mL / kg, 43.4±2 mL / kg, 43.4±3 mL / kg, 43.4±4 mL / kg, 43.4±5 mL / kg, 43.4±6 mL / kg, 43.4±7 mL / kg, 43.4±8 mL / kg, 43.4±9 mL / kg, 43.4±10 mL / kg, 43.4±11 mL / kg, 43.4±12 mL / kg, 43.4±13 mL / kg, 43.4±14 mL / kg, 43.4±15 mL / kg, or 43.4±16 mL / kg as measured by a two-stage (chromogenic) assay when about 65 IU / kg of the chimeric polypeptide is administered; or When about 65 IU / kg of the chimeric polypeptide is administered, the Vss (activity) in the subject is about 38.2-49.2 mL / kg as measured by the two-stage (chromogenic) assay.
[0137] In yet other embodiments, the long-acting factor VIII has one or more of the following properties when administered to a patient population: AUC in subjects administered the same amount of polypeptide consisting of full-length mature factor VIII, as measured by a one-stage (aPTT) assay or a two-stage (chromogenic) assay INFAUC in the subject that is at least 1.45 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90 times higher than INF , When about 25 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 1440±316 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 1160-1880 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, there is a median response rate of about 1480 hours per IU / kg as measured by a one-stage (aPTT) assay. * IU / dL, 1480±100 hours per IU / kg * IU / dL, 1480±200 hours per IU / kg * IU / dL, 1480±300 hours per IU / kg * IU / dL, 1480±400 hours per IU / kg * IU / dL, 1480±500 hours per IU / kg * IU / dL, 1480±600 hours per IU / kg * IU / dL, 1480±700 hours per IU / kg * IU / dL, 1480±800 hours per IU / kg * IU / dL, 1480±900 hours per IU / kg * 1480±1000 hours per IU / dL or 1 IU / kg *AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 2910±1320 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, the patient has a prognosis of about 1980-3970 hours per IU / kg as measured by a one-stage (aPTT) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, there is a median time of about 2800 hours per IU / kg as measured by a one-stage (aPTT) assay. * IU / dL, 2800±100 hours per IU / kg * IU / dL, 2800±200 hours per IU / kg * IU / dL, 2800±300 hours per IU / kg * IU / dL, 2800±400 hours per IU / kg * IU / dL, 2800±500 hours per IU / kg * IU / dL, 2800±600 hours per IU / kg * IU / dL, 2800±700 hours per IU / kg * IU / dL, 2800±800 hours per IU / kg * IU / dL, 2800±900 hours per IU / kg * 2800±1000 hours per IU / dL or 1 IU / kg * AUC in the subject in IU / dL INF , When about 25 IU / kg of the chimeric polypeptide is administered, there is about 1660 hours of activity per IU / kg as measured by a two-stage (chromogenic) assay. * IU / dL, 1660±100 hours per IU / kg * IU / dL, 1660±200 hours per IU / kg * IU / dL, 1660±300 hours per IU / kg *IU / dL, 1660±400 hours per IU / kg * IU / dL, 1660±500 hours per IU / kg * IU / dL, 1660±600 hours per IU / kg * IU / dL, 1660±700 hours per IU / kg * IU / dL, 1660±800 hours per IU / kg * IU / dL, 1660±900 hours per IU / kg * 1660±1000 hours per IU / dL or 1 IU / kg * AUC in the subject in IU / dL INF , When about 25 IU / kg of chimeric polypeptide is administered, the patient has approximately 1300-2120 hours of activity per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF , When about 65 IU / kg of the chimeric polypeptide is administered, there is about 4280 hours of activity per IU / kg as measured by a two-stage (chromogenic) assay. * IU / dL, 4280±100 hours per IU / kg * IU / dL, 4280±200 hours per IU / kg * IU / dL, 4280±300 hours per IU / kg * IU / dL, 4280±400 hours per IU / kg * IU / dL, 4280±500 hours per IU / kg * IU / dL, 4280±600 hours per IU / kg * IU / dL, 4280±700 hours per IU / kg * IU / dL, 4280±800 hours per IU / kg * IU / dL, 4280±900 hours per IU / kg * IU / dL, 4280±1000 hours per IU / kg * IU / dL, 4280±1100 hours per IU / kg * IU / dL, 4280±1200 hours per IU / kg *IU / dL, 4280±1300 hours per IU / kg * IU / dL, 4280±1400 hours per IU / kg * IU / dL, 4280±1500 hours per IU / kg * 4280±1600 hours per IU / dL or 1 IU / kg * AUC in the subject in IU / dL INF ,or When about 65 IU / kg of chimeric polypeptide is administered, the patient has approximately 2960-6190 hours of activity per IU / kg as measured by a two-stage (chromogenic) assay. * AUC in the subject in IU / dL INF .
[0138] As used herein, "on-demand treatment" refers to treatment intended to be administered for a short period of time, in response to a current situation, such as a bleeding episode, or to a recognized need, such as a planned surgery. Conditions that may require on-demand treatment include, for example, bleeding episodes, hemarthrosis, muscle bleeding, bleeding from the mouth, bleeding into muscles, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding into the iliopsoas sheath. The subject may require treatment for surgical prophylaxis, perioperative management, or surgery. Such surgeries include, for example, minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee replacement, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery.
[0139] In one embodiment, the on-demand treatment resolves greater than 80% (greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% of the bleeding (e.g., spontaneous bleeding) in a single dose. In another embodiment, greater than 80% (greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or 100%) or 80-100%, 80-90%, 85-90%, 90-100%, 90-95%, or 95-100% of the bleeding episodes are rated as excellent or good by a physician following on-demand treatment. In other embodiments, more than 5% of bleeding episodes (more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%) or 5-20%, 5-15%, 5-10%, 10-20%, or 10-15% are rated as acceptable by a physician following on-demand treatment.
[0140] "Polypeptide," "peptide," and "protein" are used interchangeably and refer to polymeric compounds made up of covalently linked amino acid residues.
[0141] "Polynucleotide" and "nucleic acid" are used interchangeably and refer to a polymeric compound composed of covalently linked nucleotide residues. A polynucleotide can be DNA, cDNA, RNA, single-stranded or double-stranded, a vector, a plasmid, a phage, or a virus. Polynucleotides include those in Table 1 that encode a polypeptide of Table 2 (see Table 1). Polynucleotides also include, for example, fragments of the polynucleotides of Table 1, such as those that encode fragments of the polypeptides of Table 2, such as Factor VIII, Fc, signal sequence, 6His, and other fragments of the polypeptides of Table 2.
[0142] As used herein, "prophylactic treatment" refers to administering a factor VIII polypeptide to a subject in multiple doses over a long period of time to increase the level of factor VIII activity in the subject's plasma. The increased level may be sufficient to reduce the incidence of spontaneous bleeding or to prevent bleeding in the event of, for example, unexpected injury. During prophylactic treatment, the plasma protein level in the subject is not lowered below the subject's baseline level or is not lowered below the factor VIII level (<1 IU / dL [1%]) that characterizes severe hemophilia.
[0143] In one embodiment, the prophylaxis regimen is "tailored" to the individual patient, for example, by determining PK data for each patient and administering the factor VIII of the present invention at a dosing interval that maintains a trough level of FVIII activity of 1-3%. Adjustments can be made if the subject experiences unacceptable bleeding episodes, defined as 2 or more spontaneous bleeding episodes over a 2-month period. In this case, adjustments are made to target a trough level of 3-5%. In another embodiment, prophylactic treatment results in prevention and control of bleeding, sustained control of bleeding, sustained protection from bleeding, and / or sustained benefit. Prevention, e.g., sustained protection, can be indicated by increased AUC to last measured time point (AUC-LAST) and decreased clearance, resulting in increased terminal t1 / 2 compared to short-acting FVIII. Prevention can be indicated by better Cmax, better Tmax, and / or better mean residence time for short-acting FVIII. In some embodiments, prevention is achieved within about 24, 36, 48, 72, or 96 hours (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours) without a spontaneous bleeding episode.In certain embodiments, the prophylaxis results in an average reduction in annual bleeding episodes of more than 30% (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 96, 87, 88, 89, or 90%, e.g., more than 50%) with once weekly dosing (e.g., at 65 IU / kg). "Therapeutic dose" as used herein means a dose that achieves the therapeutic goal described herein. The calculation of the required dose of factor VIII is based on the empirical finding that, on average, 1 IU of factor VIII per kg of body weight increases plasma factor VIII activity by about 2 IU / dL. The required dose is determined by the following formula: Amount of units needed = body weight (kg) x desired factor VIII increase (IU / dL or % of normal) x 0.5 (IU / kg per IU / dL)
[0144] Therapeutic doses that may be used in the methods of the present invention are from about 10 to about 100 IU / kg, more specifically, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 IU / kg, more specifically, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 IU / kg.
[0145] Further therapeutic doses that may be used in the methods of the present invention are about 10 to about 150 IU / kg, more specifically, about 100-110, 110-120, 120-130, 130-140, 140-150 IU / kg, more specifically, about 110, 115, 120, 125, 130, 135, 140, 145, or 150 IU / kg.
[0146] As used herein, "variant" refers to a polynucleotide or polypeptide that differs from the original polynucleotide or polypeptide but retains its basic properties, such as factor VIII clotting activity or Fc (FcRn binding) activity. Generally, variants are overall very similar and in many regions identical to the original polynucleotide or polypeptide. Variants include, for example, polypeptide and polynucleotide fragments, deletions, insertions, and modified versions of the original polypeptide.
[0147] A variant polynucleotide may comprise or consist of a nucleotide sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleotide coding sequence (Factor VIII portion, Fc portion, individually or together) in SEQ ID NO: 1, 3, or 5, or the complementary strand thereto, the nucleotide coding sequence or the complementary strand thereto of known mutant and recombinant Factor VIII or Fc, such as those disclosed in the publications and patents referenced herein, the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2, 4, or 6 (Factor VIII portion, Fc portion, individually or together), and / or a polynucleotide fragment of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides that hybridize to these nucleic acid molecules under stringent or low stringency hybridization conditions are also included as variants, as are the polypeptides encoded by these polynucleotides, so long as they are functional.
[0148] A variant polypeptide may comprise or consist of, for example, an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence set forth in SEQ ID NO: 2, 4, or 6 (Factor VIII portion, Fc portion, individually or together), and / or a polypeptide fragment of any of these polypeptides (e.g., the fragments described herein).
[0149] A nucleic acid having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the nucleic acid is identical to the reference sequence, except that the nucleotide sequence may contain up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a nucleic acid having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or multiple nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be, for example, the entire sequence shown in SEQ ID NO: 1 or 3, the ORF (open reading frame), or any fragment specified as described herein.
[0150] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence or polypeptide of the present invention can be determined by using known computer programs.The method for determining the best overall match between a query sequence (reference or original sequence) and a subject sequence, which is called global sequence alignment, can be determined by using the FASTDB computer program based on the algorithm of Brutlag et al., Comp.App Biosci.6:237-245 (1990), which is incorporated herein by reference in its entirety.In sequence alignment, both the query and subject sequences are DNA sequences.By converting U to T, RNA sequences can be compared.The result of the global sequence alignment is expressed as percent identity. In another embodiment, the parameters used for FASTDB alignment of DNA sequences to calculate percent identity are: matrix=single, k-tuple=4, mismatch penalty=1, joining penalty=30, randomization group length=0, cutoff score=1, gap penalty=5, gap size penalty 0.05, window size=500 or the length of the subject nucleotide sequence, whichever is shorter.
[0151] If the subject sequence is shorter than the query sequence due to 5' or 3' deletions, but not due to internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not take into account the 5' and 3' truncation of the subject sequence when calculating the percent identity. For subject sequences that are truncated at the 5' or 3' end, the percent identity is corrected by calculating the number of bases of the query sequence that are not matched / aligned with the query sequence and are 5' and 3' of the subject sequence as a percentage of the total bases of the query sequence. Whether a nucleotide is matched / aligned is determined by the result of FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the above FASTDB program with the specified parameters to produce a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence shown by the FASTDB alignment that are not matched / aligned with the query sequence are calculated for the purpose of manual adjustment of the percent identity score.
[0152] For example, a 90-base subject sequence is aligned with a 100-base query sequence to determine percent identity. The deletion occurs at the 5' end of the subject sequence, so the FASTDB alignment does not show a match / alignment of the first 10 bases at the 5' end. Since the 10 unpaired bases are 10% of the sequence (number of bases at the 5' and 3' ends that do not match / total number of bases in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched, the final percent identity would be 90%. In another example, a 90-base subject sequence is compared with a 100-base query sequence. This time, since the deletion is an internal deletion, there are no bases at the 5' or 3' end of the subject sequence that do not match / align with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only bases 5' and 3' to those in the subject sequence that are not matched / aligned with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.
[0153] A polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to a query amino acid sequence of the present invention is intended to mean that the amino acid sequence of the subject polypeptide is identical to the query sequence, except that the subject polypeptide sequence may contain up to 5 amino acid modifications per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (lost), or replaced with another amino acid. These modifications of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, either individually between residues in the reference sequence, or interspersed in one or more consecutive groups within the reference sequence.
[0154] As a practical matter, whether any particular polypeptide is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the amino acid sequence of SEQ ID NO: 2 (factor VIII portion, Fc portion, individually or together) or 4, or a known factor VIII or Fc polypeptide sequence, can be conventionally determined using known computer programs. In one embodiment, the method for determining the best overall match between a query sequence (reference or original sequence) and a subject sequence, referred to as global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., Comp. App. Biosci. 6:237-245 (1990), which is incorporated herein by reference in its entirety. In sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is expressed as percent identity. In another embodiment, the parameters used for FASTDB amino acid alignment are: matrix=PAM 0, k-tuple=2, mismatch penalty=1, joining penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject amino acid sequence, whichever is shorter.
[0155] If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, but not due to internal deletions, manual corrections must be made to the results. This is because the FASTDB program does not consider the N- and C-terminal truncations of the subject sequence when calculating the global identity percentage. For N- and C-terminally truncated subject sequences, the identity percentage is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence that are not matched / aligned with the corresponding subject residues compared to the query sequence as a percentage of the total bases of the query sequence. Whether a residue is matched / aligned is determined by the result of FASTDB sequence alignment. This percentage is then subtracted from the identity percentage calculated by the above FASTDB program with specific parameters to produce a final identity percentage score. This final identity percentage score is the one used for the purposes of the present invention. Only the residues at the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the identity percentage score. That is, only query residues are aligned outside the farthest N- and C-terminal residues of the subject sequence.
[0156] For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, so the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since the 10 unpaired residues are 10% of the sequence (number of residues at the N-terminus and C-terminus that do not match / total number of residues in the query sequence), 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time, since the deletion is an internal deletion, there are no residues at the N-terminus and C-terminus of the subject sequence that are not matched / aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only residue positions outside the N- and C-termini of the subject sequence shown in the FASTDB alignment that are not matched / aligned with the query sequence are manually corrected. No other manual corrections are made for the purposes of the present invention.
[0157] Polynucleotide variants may contain modifications in coding regions, non-coding regions, or both. In one embodiment, polynucleotide variants contain modifications that result in silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In another embodiment, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. In other embodiments, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination. Polynucleotide variants can be produced for a variety of reasons, such as, for example, optimizing codon expression for a particular host (e.g., changing the codons in human mRNA to another, such as a bacterial host, such as E. coli).
[0158] Naturally occurring variants are called "allelic variants" and refer to one of several alternative forms of a gene occupying a given locus on the chromosome of an organism (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants may differ at either the polynucleotide and / or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
[0159] Using known methods of protein engineering and recombinant DNA technology, mutants can be made to improve or modify the characteristics of a polypeptide. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of a secreted protein without substantial loss of biological function. Ron et al., J. Biol. Chem. 268:2984-2988 (1993), incorporated herein by reference in its entirety, reported mutant KGF proteins that retained heparin-binding activity even after deletion of 3, 8, or 27 amino acid-terminal amino acid residues. Similarly, interferon gamma showed up to 10-fold increased activity after deletion of 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. Biotechnology 7:199-216 (1988), incorporated herein by reference in its entirety).
[0160] Moreover, ample evidence indicates that mutants often retain biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J.Biol.Chem 268:22105-22111 (1993), incorporated herein by reference in its entirety) performed a large-scale mutational analysis of the human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants with an average of 2.5 amino acid changes per mutant across the entire length of the molecule. Multiple mutations were explored at every possible amino acid position. The researchers found that "large" portions of the molecule could be altered with little effect on either "binding or biological activity." (See Abstract). In fact, of the over 3,500 nucleotide sequences explored, only 23 unique amino acid sequences produced proteins with activity significantly different from the wild type.
[0161] As discussed above, polypeptide variants include, for example, modified polypeptides. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, oxidation, pegylation (Mei et al., Blood 116:270-79 (2010), incorporated herein by reference in its entirety), proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins (e.g., arginylation), and ubiquitination. In some embodiments, factor VIII is modified, e.g., pegylated, at any convenient position. In some embodiments, factor VIII is pegylated at a surface-exposed amino acid of factor VIII, e.g., at a surface-exposed cysteine, which may be an engineered cysteine. See above. In some embodiments, the modified factor VIII, e.g., pegylated factor VIII, is a long-acting factor VIII.
[0162] As used herein, "volume of distribution at steady state (Vss)" has the same meaning as the term is used in pharmacology: the apparent space (volume) into which a drug is distributed. Vss = amount of drug in the body divided by the steady state plasma concentration.
[0163] When used herein in relation to ranges, "about" modifies both ends of the range, so "about 10 to 20" means "about 10 to about 20."
[0164] The chimeric polypeptide as used herein may comprise processed factor VIII or single chain factor VIII or a combination thereof. As used herein, "processed factor VIII" refers to factor VIII cleaved at arginine 1648 (for full-length factor VIII) or arginine 754 (for B-domain deleted factor VIII), i.e., at an intracellular processing site. Upon cleavage at an intracellular processing site, processed factor VIII comprises two polypeptide chains, the first chain being a heavy chain and the second chain being a light chain. For example, processed factor VIII-Fc fusion protein (i.e., heavy and light chains fused to Fc) migrates at about 90 kDa and 130 kDa, respectively, on non-reducing SDS-PAGE, and 90 kDa and 105 kDa, respectively, on reducing SDS-PAGE. Thus, in one embodiment, at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII moieties in the chimeric polypeptide are processed Factor VIII. In another embodiment, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII moieties in the chimeric polypeptide are processed Factor VIII. In certain embodiments, the chimeric polypeptide comprising processed factor VIII is purified (or isolated) from a chimeric polypeptide comprising single-chain factor VIII, and at least about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the factor VIII moieties in the chimeric polypeptide are processed factor VIII.
[0165] As used herein, "single-chain factor VIII," "SC factor VIII," or "SCFVIII" refers to factor VIII that is not truncated at the arginine site (residue 1648 (i.e., residue 1667 of SEQ ID NO:6) for full-length factor VIII, or residue 754 (i.e., residue 773 of SEQ ID NO:2) for B-domain deleted factor VIII. Thus, as used herein, single-chain factor VIII in a chimeric polypeptide comprises a single chain. In one embodiment, single-chain factor VIII contains an intact intracellular processing site. In another embodiment, the single-chain factor VIII of the invention comprises a substitution or mutation at the amino acid position corresponding to arginine 1645, a substitution or mutation at the amino acid position corresponding to arginine 1648, or a substitution or mutation at arginine 1645 and an amino acid position corresponding to arginine 1648 in full-length factor VIII. The single-chain Factor VIII-Fc fusion protein comprises a substitution or mutation at the amino acid position corresponding to arginine 1648. In other embodiments, the amino acid substituted at the amino acid position corresponding to arginine 1645 is a different amino acid than the amino acid substituted at the amino acid position corresponding to arginine 1648. In certain embodiments, the substitution or mutation is an amino acid other than arginine, such as isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, selenocysteine, serine, tyrosine, histidine, ornithine, pyrrolysine, or taurine. The single-chain Factor VIII-Fc fusion protein migrates at about 220 kDa on non-reducing SDS-PAGE and about 195 kDa on reducing SDS-PAGE.
[0166] In one embodiment, the chimeric polypeptide comprising single-chain factor VIII is purified (or isolated) from the chimeric polypeptide comprising processed factor VIII, and at least about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% of the factor VIII portion of the chimeric polypeptide used herein is single-chain factor VIII. In another embodiment, at least about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII. In other embodiments, about 1% to about 10%, about 5% to about 15%, about 10% to about 20%, about 15% to about 25%, about 20% to about 30%, about 25% to about 35%, or about 30% to about 40% of the Factor VIII portion of the chimeric polypeptides used herein is single-chain Factor VIII. In certain embodiments, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, or about 35% of the Factor VIII portion of the chimeric polypeptides used herein is single-chain Factor VIII. In other embodiments, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the Factor VIII portion of the chimeric polypeptides used herein is single-chain Factor VIII.In some embodiments, the ratio of single-chain factor VIII to processed factor VIII in the chimeric polypeptide is: (a) about 25% single-chain factor VIII and about 75% processed factor VIII, (b) about 20% single-chain factor VIII and about 80% processed factor VIII, (c) about 15% single-chain factor VIII and about 85% processed factor VIII, (d) about 10% single-chain factor VIII and about 90% processed factor VIII, (e) about 5% single-chain factor VIII and about 75% processed factor VIII, (f) about 10% single-chain factor VIII and about 90% processed factor VIII, (g) about 10% single-chain factor VIII and about 90% processed factor VIII, (h) about 10% single-chain factor VIII and about 90% processed factor VIII, (i) about 10% single-chain factor VIII and about 90% processed factor VIII, (j) about 10% single-chain factor VIII and about 90% processed factor VIII, (k) about 10% single-chain factor VIII and about 90% processed factor VIII, (l) about 10% single-chain factor VIII and about 90% processed factor VIII, (m) about 10% single-chain factor VIII and about 90% processed factor VIII, (m) about 10% single-chain factor VIII and about 90% processed factor VIII, (n ... (f) about 1% single-chain factor VIII and about 99% processed factor VIII; (g) about 100% processed factor VIII; (h) about 30% single-chain factor VIII and about 70% processed factor VIII; (i) about 35% single-chain factor VIII and about 65% processed factor VIII; or (j) about 40% single-chain factor VIII and about 60% processed factor VIII. In other embodiments, the ratio of single-chain factor VIII to processed factor VIII in the chimeric polypeptide is: (a) about 30% single-chain factor VIII and about 70% processed factor VIII; (b) about 40% single-chain factor VIII and about 60% processed factor VIII; (c) about 50% single-chain factor VIII and about 50% processed factor VIII; (d) about 60% single-chain factor VIII and about 40% processed factor VIII; (e) about 70% single-chain factor VIII and about 70% processed factor VIII; % single-chain factor VIII and about 30% processed factor VIII, (f) about 80% single-chain factor VIII and about 20% processed factor VIII, (g) about 90% single-chain factor VIII and about 10% processed factor VIII, (h) about 95% single-chain factor VIII and about 5% processed factor VIII, (i) about 99% single-chain factor VIII and about 1% processed factor VIII, or (j) about 100% single-chain factor VIII.
[0167] The factor VIII portion in the chimeric polypeptide used herein has factor VIII activity. Factor VIII activity can be measured by any method known in the art. For example, one of these methods can be a chromogenic assay. The mechanism of the chromogenic assay is based on the principle of the blood coagulation cascade, where activated factor VIII accelerates the conversion of factor X to factor Xa in the presence of activated factor IX, phospholipids, and calcium ions. Factor Xa activity is evaluated by hydrolysis of p-nitroanilide (pNA) substrate specific for factor Xa. The initial rate of release of p-nitroanilide, measured at 405 nM, is directly proportional to factor Xa activity, which in turn is directly proportional to the factor VIII activity in the sample. This chromogenic assay is recommended by the Subcommittee on Factor VIII and Factor IX of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostatsis (ISTH). Since 1994, the chromogenic assay is also the reference method of the European Pharmacopoeia for the assignment of the titer of FVIII concentrates. Thus, in one embodiment, a chimeric polypeptide comprising single-chain factor VIII has a factor VIII activity comparable to a chimeric polypeptide comprising processed factor VIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties), when the factor VIII activity is measured in vitro by a chromogenic assay.
[0168] In another embodiment, a chimeric polypeptide comprising a single-chain factor VIII of the invention has a frequency of factor Xa comparable to a chimeric polypeptide comprising processed factor VIII (e.g., a chimeric polypeptide consisting essentially of, or consisting of, two Fc moieties and processed factor VIII fused to one of the two Fc moieties).
[0169] In order to activate factor X to factor Xa, activated factor IX (factor IXa) hydrolyzes one arginine-isoleucine bond of factor X to form factor Xa in the presence of Ca2+, membrane phospholipids, and cofactor VIII.Therefore, the interaction between factor VIII and factor IX is crucial in the coagulation pathway.In certain embodiments, the chimeric polypeptide comprising single-chain factor VIII can interact with factor IXa at a rate comparable to that of the chimeric polypeptide comprising processed factor VIII (e.g., the chimeric polypeptide consisting essentially of or consisting of two Fc parts and the processed factor VIII fused with one of the two Fc parts).
[0170] In addition, factor VIII is inactive in circulation, but binds to von Willebrand factor. When factor VIII is not bound to vWF, it is rapidly degraded and released from vWF by the action of thrombin. In some embodiments, a chimeric polypeptide comprising single-chain factor VIII binds to von Willebrand factor at a level comparable to that of a chimeric polypeptide comprising processed factor VIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc portions and a processed factor VIII fused to one of the two Fc portions).
[0171] Factor VIII can be inactivated by activated protein C in the presence of calcium and phospholipids. Activated protein C cleaves the heavy chain of factor VIII after arginine 336 in the A1 domain, which disrupts the interaction site of the factor X substrate, and after arginine 562 in the A2 domain, which promotes dissociation of the A2 domain and disrupts the interaction site with factor IXa. This cleavage also splits the A2 domain (43 kDa), generating the A2-N (18 kDa) and A2-C (25 kDa) domains. Thus, activated protein C can catalyze multiple cleavage sites in the heavy chain. In one embodiment, a chimeric polypeptide comprising a single-chain factor VIII is inactivated by activated protein C at a level comparable to a chimeric polypeptide comprising a processed factor VIII (e.g., a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties).
[0172] In other embodiments, the chimeric polypeptides comprising single-chain factor VIII have in vivo factor VIII activity comparable to that of chimeric polypeptides comprising processed factor VIII (e.g., chimeric polypeptides consisting essentially of or consisting of two Fc moieties and processed factor VIII fused to one of the two Fc moieties). In certain embodiments, the chimeric polypeptides comprising single-chain factor VIII can protect hemophilia A mice in a hemophilia A mouse tail vein transection model at a level comparable to that of chimeric polypeptides comprising processed factor VIII (e.g., chimeric polypeptides consisting essentially of or consisting of two Fc moieties and processed factor VIII fused to one of the two Fc moieties).
[0173] The term "comparable" as used herein means that the rate or level compared from using the chimeric polypeptide is equal, substantially equal, or similar to the rate or level of the reference. The term "similar" as used herein means that the rate or level compared has a difference of not more than 10% or not more than 15% from the rate or level of the reference (e.g., the generation rate of FXa by a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and processed factor VIII fused to one of the two Fc moieties). The term "substantially equal" means that the rate or level compared has a difference of not more than 0.01%, 0.5%, or 1% from the rate or level of the reference.
[0174] The invention further includes compositions comprising chimeric polypeptides having Factor VIII activity, where at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a Factor VIII portion and a second portion that is single-chain Factor VIII. In another embodiment, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the chimeric polypeptide in the composition is single-chain Factor VIII. In other embodiments, the second moiety is Fc, XTEN, albumin, a PAS sequence, transferrin or CTP (the 28 amino acid C-terminal peptide of hCG (CTP) having four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin binding polypeptide, an albumin binding small molecule, or a combination of two or more thereof.In yet other embodiments, the compositions of the invention comprise a combination of a chimeric polypeptide comprising processed factor VIII and a chimeric polypeptide comprising single-chain factor VIII, wherein (a) about 30% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 70% of the factor VIII portion of the chimeric polypeptide is processed factor VIII, or (b) about 40% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 70% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (c) about 50% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 50% of the factor VIII portion of the chimeric polypeptide is processed factor VIII; (d) about 60% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 40% of the factor VIII portion of the chimeric polypeptide is processed factor VIII; or (e) about (f) about 80% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 20% of the factor VIII portion of the chimeric polypeptide is processed factor VIII; or (g) about 90% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 10% of the factor VIII portion of the chimeric polypeptide is processed factor VIII. (h) about 95% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 5% of the factor VIII portion of the chimeric polypeptide is processed factor VIII; (i) about 99% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII and about 1% of the factor VIII portion of the chimeric polypeptide is processed factor VIII; or (j) about 100% of the factor VIII portion of the chimeric polypeptide is single-chain factor VIII.
[0175] In certain embodiments, the compositions of the invention have factor VIII activity comparable to a composition comprising processed factor VIII (e.g., a composition comprising a chimeric polypeptide consisting essentially of, or consisting of, two Fc moieties and processed factor VIII fused to one of the two Fc moieties), when the factor VIII activity is measured in vitro by a chromogenic assay.
[0176] In other embodiments, the compositions of the invention have a rate of factor Xa comparable to compositions containing processed factor VIII (e.g., a composition comprising a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties). In yet other embodiments, a composition comprising single-chain factor VIII can interact with factor IXa at a rate comparable to compositions containing processed factor VIII (e.g., a composition comprising a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties). In further embodiments, the single-chain factor VIII in the chimeric polypeptide of the compositions of the invention is inactivated by activated protein C at a level comparable to the processed factor VIII in the chimeric polypeptide of the compositions (e.g., a composition comprising a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties). In certain embodiments, a composition comprising single-chain factor VIII has in vivo factor VIII activity comparable to a composition comprising processed factor VIII (e.g., a composition comprising a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties). In some embodiments, a composition comprising single-chain factor VIII of the invention can protect hemophilia A mice in a hemophilia A mouse tail vein transection model at a level comparable to a composition comprising processed factor VIII (e.g., a composition comprising a chimeric polypeptide consisting essentially of or consisting of two Fc moieties and a processed factor VIII fused to one of the two Fc moieties).
[0177] The present invention further provides methods for treating a bleeding condition in a human subject using the compositions of the present invention. An exemplary method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition / formulation comprising a chimeric polypeptide having factor VIII activity, wherein at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99% of the chimeric polypeptide comprises a factor VIII portion that is single-chain factor VIII and a second portion.
[0178] Bleeding condition can be caused by blood clotting disorder. Blood clotting disorder can also be called coagulopathy. In one example, the blood clotting disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia or von Willebrand's disease (vWD). In another example, the blood clotting disorder that can be treated with the pharmaceutical composition of the present disclosure is hemophilia A.
[0179] In some embodiments, the type of bleeding associated with the bleeding condition is selected from hemarthrosis, muscle bleeding, mouth bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma, gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding into the iliopsoas sheath.
[0180] In other embodiments, the subject suffering from bleeding condition requires treatment for surgery, including, for example, surgical prophylaxis or intraoperative management.In one example, the surgery is selected from minor surgery and major surgery.Exemplary surgical procedures include tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, or joint replacement surgery (e.g., total knee replacement, total hip replacement, etc.), cardiac surgery, and Caesarean section.
[0181] In another embodiment, the subject is simultaneously treated with FIX. The compounds of the present invention can activate FIXa and therefore can be used to pre-activate FIXa polypeptides prior to administration of FIXa to the subject.
[0182] The methods of the invention can be practiced on subjects in need of preventative treatment or on-demand treatment.
[0183] Pharmaceutical compositions comprising at least 30% of single-chain factor VIII may be formulated for any suitable mode of administration, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration.
[0184] The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial, and intraperitoneal injection, and any similar injection or infusion method.The composition can also be, for example, a suspension, emulsion, sustained release formulation, cream, gel, or powder.The composition can be formulated as a suppository with traditional binders and carriers, such as triglycerides.
[0185] In one example, the pharmaceutical formulation is a liquid formulation, for example, a buffered isotonic aqueous solution. In another example, the pharmaceutical composition has a physiological or near-physiological pH. In another example, the aqueous formulation has a physiological or near-physiological osmolality and salinity. It may contain sodium chloride and / or sodium acetate. In some examples, the composition of the present invention is lyophilized.
[0186] Having now described the invention in detail, a further understanding of the invention will be obtained by reference to the following examples, which are included herein for purposes of illustration only and are not intended to limit the scope of the invention. All patents and publications mentioned herein are expressly incorporated by reference. EXAMPLES
[0187] Example 1 Cloning, expression, and purification of rFVIIIFc All molecular biology procedures were performed according to standard techniques. The coding sequence of human FVIII including the native signal sequence (Genbank accession number NM_000132) was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver polyA RNA. Due to the large size of FVIII, the coding sequence was obtained in several sections from separate RT-PCR reactions and assembled by a series of PCR reactions, restriction digestion, and ligation into an intermediate cloning vector containing the B-domain deleted (BDD) FVIII coding region with a fusion of serine 743 (S743) to glutamine 1638 (Q1638), erasing 2682 bp from the B-domain of full-length FVIII. The human IgG1 Fc sequence (e.g., GenBank accession number Y14735) was obtained by PCR from a leukocyte cDNA library, and the final expression cassette was generated in such a way that the BDD FVIII sequence was fused directly to the N-terminus of the Fc sequence (hinge, CH2 and CH3 domains, starting at D221 of the IgG1 sequence, EU numbering) without an intervening linker. For expression of the Fc chain only, a mouse Igκ (kappa) light chain signal sequence was generated using synthetic oligonucleotides and added to the Fc coding sequence using PCR, allowing secretion of the protein product. The FVIIIFc and Fc chain coding sequences were cloned into a dual expression vector, pBudCE4.1 (Invitrogen, Carlsbad, CA).
[0188] HEK293H cells (Invitrogen, Carlsbad, CA) were transfected with the pSYN-FVIII-013 plasmid using Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) and stable cell lines were selected with Zeocin. Cells were grown in serum-free suspension cultures and rFVIIIFc protein was purified from the clarified harvest medium using a four-column purification process that included a combination of anion exchange and hydrophobic interaction columns, followed by a FVIII-specific affinity purification step (McCue J. et al., J. Chromatogr. A., 1216(45):7824-30(2009)).
[0189] Example 2 Biochemical characterization Processed recombinant FVIII-Fc (rFVIIIFc) was synthesized as two polypeptide chains, one consisting of BDD-FVIII (S743-Q1638 fusion, 1438 amino acids) fused to the Fc domain (hinge, CH2 and CH3 domains) of IgG1 (226 amino acids, extending from D221 to G456, EU numbering) and the other consisting of the same Fc region only (226 amino acids), for a total chain length of 1664 amino acids. Cells transfected with FVIIIFc / Fc dual expression plasmid were expected to secrete three products (FVIIIFc dimer, FVIIIFc monomer, and Fc dimer), but only FVIIIFc monomer and Fc dimer were detected in the conditioned medium. Purified FVIIIFc was analyzed by non-reducing and reducing SDS-PAGE analysis (Figure 2A and B). For non-reducing SDS-PAGE, bands migrating at approximately 90 kDa and 130 kDa were observed, consistent with the predicted molecular weights of FVIIIFc heavy chain (HC) and light chain dimeric Fc fusion (LCFc2) (Figure 2A, lane 3). A third band was also detected at approximately 220 kDa, consistent with the predicted molecular weight for single chain FVIIIFc (SC FVIIIFc; HC+LCFc2), in which the arginine residue at position 754 (1648 relative to the full-length sequence) is not cleaved during secretion. For reducing SDS-PAGE analysis, major bands migrating at approximately 25 kDa, 90 kDa, 105 kDa, and 195 kDa were observed, consistent with the predicted molecular weights of single chain Fc, HC, LCFc, and SC FVIIIFc (Figure 2B, lane 3). Cotransfection with human PC5, a member of the proprotein convertase subtilisin / kexin (PCSK)-type proteases, resulted in complete processing of the rFVIIIFc product (Fig. 2A,B, lane 2).
[0190] Densitometric analysis of several batches of rFVIIIFc following SDS-PAGE showed greater than 98% purity of the expected bands, and size exclusion chromatography (SEC) was used to assess the degree of aggregation present, with all batches found to have aggregation levels below 0.5%.
[0191] The rFVIIIFc structure was further analyzed by thrombin cleavage, reduction, and analysis by LC / UV and LC / MS. The four factor VIII fragments generated by thrombin (by cleavage at three arginine residues at positions 372, 740, and 795 (795 corresponding to 1689 for the full-length FVIII sequence)) can be detected by UV absorbance, corresponding to the Fc (peak 1), light chain Fc (peak 2), heavy chain to A1 domain (peak 3), and heavy chain to A2 domain (peak 4) protein segments (Figure 2C). The 14 amino acid B domain linker and the approximately 6 kDa a3-related peptide are not detected by UV absorbance due to their small size.
[0192] Analysis of thrombin digestion of rFVIIIFc by HPLC / MS provided detailed information of the four major domains as well as the a3-related peptide of approximately 6 kDa and was compared with REFACTO®, a CHO-derived recombinant BDD-FVIII protein (rBDD FVIII) using the same method. FVIII samples were passed through a Detergent-OUTDTG-100X column (GBioscience, Maryland Heights, MO) for removal of Tween, digested to completion with thrombin, reduced, and analyzed by either RP-HPLC-UV (POROS R1 / 10, Applied Biosystems) or RP-HPLC-MS (Agilent 1200 coupled to an Agilent 6210TOF mass spectrometer) using a gradient of acetonitrile + 0.1% formic acid in water. Peptide sequences were also confirmed using LysC peptide mapping and analyzed by RP-HPLC / MS (Thermo Finnigan LTQ-XL-ETD).
[0193] As expected, the total ion current (TIC) chromatogram of rFVIIIFc (FIG. 2D) appears to be similar to the UV chromatogram (FIG. 2C). Five of the expected six products could be detected by LC / MS, including two forms of the a3 acidic region generated from the processed and single chain isoforms, as well as the thrombin used for digestion. An additional truncated form of the a3 acidic region was also observed, described more fully below. rBDD FVIII yielded a similar TIC chromatogram, but lacked the free Fc chain and had a different mass for the LC compared to rFVIIIFc LC-Fc, consistent with the deletion of the Fc region (data not shown).
[0194] The deconvoluted mass spectra for the A1, LC-Fc, and Fc regions are complex due to the heterogeneity of glycosylation throughout the bulk of the protein, so the identity of all molecular ions has not been established. However, the masses observed for the three major peaks from the Fc region were found to be consistent with the G0, G1, and G2 isotypes found on IgG molecules, which correspond to biantennary oligosaccharides terminating with 0, 1, or 2 galactose residues. The deconvoluted mass spectra of the a3-related peptides and the A2 domain provide the most reliable data, as there is no heterogeneity in post-translational modifications within these regions, allowing the expected masses to be clearly identified.
[0195] After cleavage of R1689, the 6 kDa N-terminal peptide released from the LC is predicted to contain the a3 acidic region (amino acids E1649 to R1689) if derived from the processed isoform, and a 14 amino acid truncated B domain is predicted to be fused to the a3 acidic region if derived from the single chain isoform. Both rFVIIIFc and rBDD FVIII were found to contain a version of the a3 region in proportion to the expected levels based on SDS-PAGE analysis. In addition, both proteins contained a truncated version of the a3 region corresponding to amino acids D1658 to R1689, as reported for other FVIII products, but this was found to be more abundant in rBDD FVIII than in rFVIIIFc.
[0196] The A2 domain contains three potential tyrosine sulfation sites, but no glycosylation sites that could result in complex heterogeneity, and therefore the exact mass of this region can be calculated. In addition to the first expected peak in the deconvoluted mass spectrum of rFVIIIFc, which correlates with the mass of the S373-R740 sequence (Figure 2E), two additional peaks corresponding to known cleavage of the FVIII HC were identified, correlating with the A2 domain truncated at E720 and Y729. These reported truncation forms were also directly observed in the deconvoluted spectrum of rBDD FVIII A2 (Figure 2E). Although both rFVIIIFc and rBDD FVIII A2 contained similar relative amounts of the form truncated at Y729, the rBDD FVIII A2 domain contained significantly higher levels of the form truncated at E720 compared to the same form in rFVIIIFc (Figure 2E).
[0197] The primary sequence of rFVIIIFc was confirmed by peptide mapping by lysyl endopeptidase (Lys-C) digestion followed by detection by ultraviolet and mass spectrometry. Of the 99 theoretical peptides produced from rFVIIIFc, 81 were detected, corresponding to 98% of the total sequence. Post-translational modifications of rFVIIIFc were also characterized by this method. FVIII contains six potential tyrosine sulfation sites corresponding to positions 346, 718, 719, 723, 1664, and 1680. Fully sulfated peptides were found corresponding to these six sites, with a trace of non-sulfated peptide corresponding to position 1680 and no detectable non-sulfated peptides corresponding to other positions, as assessed by integration of the total ion chromatogram in the mass spectrum. BDD FVIII also contains six potential N-glycosylation sites, four of which have been reported to be glycosylated in recombinant FVIII products. Consistent with this, rFVIIIFc was found to have the same four sites that are glycosylated, with N239 and N2118 found to contain high mannose structures, while N41 and N1810 were found to contain more complex carbohydrates similar to those found in rBDD FVIII. N-linked glycosylation of the Fc region was found by LC / MS to be consistent with the G0, G1, and G2 isoforms found in the thrombin map. FVIII has been reported to have O-glycosylation sites at Ser741 and 743 that are partially occupied, and this was also found to be the case for rFVIIIFc.
[0198] The rFVIIIFc polypeptide produced without co-transfected processing enzymes represents 15-25% single chain FVIIIFc (SC FVIIIFc) and differs from processed rFVIIIFc by a single peptide bond between R754 and E755 (R1648 / E1649 for full-length FVIII). This isoform was purified and characterized in all of the biochemical assays described above and found to be comparable to rFVIIIFc, as shown below. The activity of purified single chain FVIIIFc was found to be similar to rFVIIIFc by chromogenic assays, as well as various functional assays described below.
[0199] Measurement of FVIII activity by chromogenic and one-step aPTT assays FVIII activity was measured by FVIII chromogenic assay. The average specific activity from four separate batches of rFVIIIFc was found to be 9762±449 IU / mg by chromogenic assay, which corresponds to 2148±99 IU / nmol. The average specific activity from 14 batches of rFVIIIFc was found to be 8460±699 IU / mg by aPTT assay and 9348±1353 IU / mg by chromogenic assay, which corresponds to 1861±154 and 2057±298 IU / nmol, respectively. The FVIII activity of single-chain FVIII:Fc was also measured by chromogenic assay and compared to fully processed rFVIII:Fc or rFVIII:Fc DS (containing about 25% single-chain rFVIII:Fc). As Table 3A shows, single-chain rFVIIIFc did not show any significant difference in FVIII activity compared to the factor VIII activity of fully processed FVIIIFc or rFVIIIFc DS by chromogenic assay both in the presence and absence of von Willebrand factor (VWF). Table 3B shows that full activity of SCrFVIIIFc was observed in the absence of VWF as measured by a one-stage activated partial thromboplastin time (aPTT) assay. [Table 3A] [Table 3B]
[0200] In a one-stage clotting assay (APTT), SC rFVIIIFc showed a 60% reduction in activity when plasma had normal VWF levels, suggesting a possible role in the activation of SC rFVIIIFc. This observation was further confirmed by spiking human VWF to FVIII / VWF-deficient plasma (Table 3), where the clotting activity of SC rFVIIIFc was reduced to the same level as in congenital FVIII-deficient plasma.
[0201] Xase complex activity FVIII activity was also measured in the Xase complex by incubating activated FIX and thrombin-activated REFACTO® or rFVIIIFc protein on a phospholipid surface in the presence of calcium and monitoring the conversion of FX to FXa, as measured by cleavage of a chromogenic or fluorogenic substrate, thereby determining the rate of FXa generation. This assay was then modified by varying one component of the assay while holding the others constant to examine interactions with each individual component.
[0202] The generation of FXa was determined as a function of different phospholipid concentrations for rFVIIIFc DS, rBDD FVIII, and single-chain rFVIIIFc using synthetic phospholipid vesicles (25% phosphatidylserine / 75% phosphatidylcholine) (Figure 3A). Both proteins were found to have similar activity profiles with peak activity at approximately 156 μM phospholipid.
[0203] The rate of FXa generation was then determined as a function of the different FX concentrations, and the Km and Vmax were calculated (Figure 3B). The activity profiles for rFVIIIFc DS, rBDD FVIII, and single-chain rFVIIIFc were found to be similar, with similar Km and Vmax (Table 4). Finally, the rate of FXa generation was determined as a function of the different FIX concentrations (Figure 3C). The activity profiles appeared to be similar, with similar Kd and Vmax (Table 4). Similar results were obtained using platelets as the phospholipid source (unpublished data, June 2009). [Table 4] [Table 5]
[0204] Inactivation by APC Upon activation, FVIII is inactivated by cleavage by activated protein C (APC) and dissociation of the A2 domain. Both rFVIIIFc and rBDD FVIII were activated by thrombin and then incubated with APC for different times and activities determined in an FXa generation assay (Figure 4). In the absence of thrombin activation, FXa generation was barely detectable and increased significantly with thrombin digestion. Treatment with APC for 90 minutes resulted in a significant decrease in the FXa generation rate, similar to that of the non-activated sample, and these results were similar for rFVIIIFc DS, rBDD FVIII, and single-chain rFVIIIFc.
[0205] Affinity for vWF The interaction of FVIII with von Willebrand factor (vWF) was measured by real-time biomolecular interaction analysis (BIAcore) based on surface plasmon resonance (SPR) technology to determine the binding kinetics of rFVIIIFc and rBDD FVIII to vWF (Table 6). The kinetic parameters of Ka (on-rate) and Kd (off-rate) as well as affinity KD (Kd / Ka) were determined for each FVIII interaction under identical conditions. Both rFVIIIFc and rBDD FVIII were found to have low nM binding affinity (KD) to vWF, 1.64±0.37 and 0.846±0.181 nM, respectively. The proteins had similar off-rates with a two-fold difference in on-rate resulting in a two-fold difference in affinity. [Table 6]
[0206] As shown in Table 6, the affinity of rFVIII:Fc DS or single-chain rFVIIIFc with vWF was found to be in the low nM range, nearly two-fold higher than that of BDD FVIII alone. At physiological concentrations, this would result in a slight decrease in the percentage of rFVIIIFc (processed or single-chain) complexed with vWF compared to free FVIII, but in vivo studies have shown that despite this slightly lower affinity, the half-life of rFVIIIFc is significantly extended over full-length or BDD FVIII, so this does not appear to impair the half-life of this molecule. Free rFVIIIFc is more effectively recycled via the FcRn pathway, so this may contribute to the longer extension of the half-life.
[0207] Affinity for and thrombin-mediated release from vWF Recombinant B-domain deleted factor VIII Fc (rFVIIIFc) was expressed in HEK293 cells. During biosynthesis in HEK293 cells, the majority of rFVIIIFc is processed by limited proteolysis to generate FVIII heavy chain (HC) and FVIII light chain (LC) to which the Fc portion is attached. Spontaneous dissociation of HC and LC during storage in plasma and in FVIII drug formulations is believed to contribute to the loss of FVIII activity. The remaining portion of the biosynthesized rFVIIIFc that is not processed forms a single-chain isoform of rFVIIIFc (SC rFVIIIFc), which may provide increased manufacturability and stability compared to processed rFVIIIFc.
[0208] This example describes an assay comparing the interaction of rFVIIIFc with vWF to the interaction of SC rFVIIIFc with von Willebrand factor (vWF). The interaction with vWF was measured by real-time biomolecular interaction analysis (BIAcore) based on surface plasmon resonance (SPR) technology to determine the kinetic binding kinetics of rFVIIIFc and rBDD FVIII to vWF (Table 11 and Figure 16A). Human plasma-derived vWF, free of FVIII, was immobilized by amine coupling on the biosensor surface at a level low enough to prevent mass transport limitations. rFVIIIFc and SC rFVIIIFc were injected sequentially in single-cycle kinetic mode at concentrations ranging from 0.13 to 5.0 nM. The sensorgram data were fitted to a 1:1 interaction model.
[0209] The kinetic parameters of Ka (on-rate) and Kd (off-rate) and affinity KD (Kd / Ka) were determined for each FVIII interaction under identical conditions. Both rFVIIIFc and rBDD FVIII were found to have low nM binding affinity (KD) for vWF, 0.34±0.1 and 0.31±0.1 nM, respectively. Both isoforms also had similar on-rate and off-rate. [Table 11]
[0210] Next, thrombin-mediated release of rFVIIIFc, SC rFVIIIFc, and B-domain deleted FVIII lacking the Fc portion (rBDD FVIII) was measured at both 25 and 37 °C. Human vWF was immobilized by amine coupling at similar levels in three flow cells on the biosensor surface. The remaining flow cells served as blanks for reference purposes. rFVIIIFc and SC rFVIIIFc proteins were captured by vWF and slowly dissociated, and the concentrations of rFVIIIFc and SC rFVIIIFc were adjusted to obtain equimolar capture levels by the end of the dissociation phase. Human α-thrombin solutions were prepared by two-fold serial dilutions and applied at concentrations ranging from 0.005 to 20 U / mL, resulting in the proteolytic release of FVIIIa species from vWF. Thrombin-mediated release of activated rFVIIIFc, SC rFVIIIFc, and rBDD FVIII from vWF was monitored in real time using an SPR-based optical biosensor (Figure 16B). After subtraction of blank reference values (Figure 16C), the release rate as a function of α-thrombin concentration was determined (Figure 16D). The half-maximal effective concentration (EC) of thrombin for SC rFVIIIFc was 3.9±0.3 U / mL compared to 3.9±0.3 U / mL for rFVIIIFc at 25°C. 50 ) was 12±1 U / mL and 15±1 U / mL for SC rFVIIIFc compared to 4.8±0.2 U / mL for rFVIIIFc at 37° C. (FIG. 16E). rFVIIIFc had similar thrombin EC50 values compared to rBDD FVIII, with values of 3.9±0.3 U / mL and 3.3±0.3 U / mL, respectively, at 25° C. and 4.8±0.2 U / mL and 4.0±0.2 U / mL, respectively, at 37° C. 50 SC rFVIIIFc had an EC value of thrombin approximately 3-fold higher than rFVIIIFc. 50The attenuation of thrombin-mediated release from vWF may underlie the hemospecific decrease in specific activity for SC rFVIIIFc observed in the aPTT assay in the presence of vWF (Table 3B).
[0211] Example 3 This phase I / IIa open-label, crossover, dose-escalation, multicenter, first-in-human study was designed to evaluate the safety, tolerability, and pharmacokinetics of a single dose of rFVIIIFc in subjects with severe (defined as <1 IU / dL [1%] endogenous factor VIII [FVIII]) hemophilia A. A total of approximately 12 previously treated patients were enrolled and received rFVIIIFc at 25 or 65 IU / kg. After screening (scheduled within 28 days prior to the first dose of the reference comparator ADVATE® [rFVIII]) and a minimum of 4 days (96 hours) of no FVIII treatment prior to the first injection, approximately 6 subjects received a single 25 IU / kg dose of ADVATE®, followed by a 3-day (72-hour) pharmacokinetic (PK) profile, then a crossover study, followed by a single, open-label dose of 25 IU / kg of rFVIIIFc for 7-day (168-hour) PK profiling. The first three subjects were dosed sequentially. For the first three subjects dosed with 25 IU / kg rFVIIIFc, each subject underwent inhibitor evaluation 14 days (336 hours) after injection of rFVIIIFc. Dosing of the next subject (only for the first three subjects) was performed upon completion of the inhibitor study. After the third subject completed the 14-day inhibitor evaluation, the remaining three subjects began enrollment at 25 IU / kg and six subjects at 65 IU / kg, sequentially within each dose group, separated by at least one day.
[0212] Approximately 6 different subjects were recruited for the 65 IU / kg cohort one week after the last subject received a 25 IU / kg dose of rFVIIIFc. Each subject in the 65 IU / kg cohort received a single 65 IU / kg dose of ADVATE®, followed by 4-day (96-hour) PK profiling, then a crossover study, followed by a single, open-label 65 IU / kg dose of rFVIIIFc for 10-day (240-hour) profiling. If a bleeding episode occurred before the first injection of rFVIIIFc in any cohort, the subject's pre-test FVIII product was used for treatment, and at least a 4-day interval had to elapse before receiving the first injection of rFVIIIFc for PK profiling.
[0213] All subjects were followed for safety evaluation periods of 14 days (336 hours) and 28 days after administration of 25 IU / kg or 65 IU / kg of rFVIIIFc. All subjects underwent pharmacokinetic sampling pre- and post-dose administration, along with blood samples for analysis of FVIII activity at designated time points.
[0214] Pharmacokinetic data from Phase I / IIa clinical trials showed the following results for FVIIIFc: FVIIIFc had significantly increased systemic exposure (AUC INF ), a 50% increase in clearance (Cl), and a 50-70% increase in elimination half-life and MRT. In addition, FVIIIFc showed increased C168, TBLP1, TBLP3, and TBLP5 values compared to ADVATE®. AUC INF Area under the concentration-time curve from zero to infinity Beta HL Emission layer half-life;t 1 / 2β Also called C168 Estimated FVIIIFc activity above baseline approximately 168 hours after dose administration Cl clearance MRT Average residence time TBLP1 Model-predicted time after dose administration when FVIIIFc activity declined to approximately 1 IU / dL above baseline TBLP3 Model-predicted time after dose administration when FVIIIFc activity would decrease to approximately 3 IU / dL above baseline TBLP5 Model-predicted time after dose administration when FVIIIFc activity declined to approximately 5 IU / dL above baseline
[0215] Example 4 A recombinant B-domain deleted factor VIII-Fc (rFVIIIFc) fusion protein was generated as an approach to extend the half-life of FVIII. The pharmacokinetics (PK) of rFVIIIFc was compared to rFVIII in hemophilia A mice. The terminal half-life was found to be two-fold longer for rFVIIIFc compared to rFVIII. To confirm that the underlying mechanism for the extension of the half-life is due to protection of rFVIIIFc by FcRn, PK was evaluated in FcRn knockout and human FcRn transgenic mice. A single intravenous dose (125 IU / kg) was administered and plasma concentrations were measured using a chromogenic activity assay. Cmax was similar between rFVIIIFc and rFVIII (XYNTHA®) in both mouse strains. However, the half-life for rFVIIIFc was comparable to that of rFVIII in FcRn knockout mice, whereas the half-life for rFVIIIFc was extended approximately 2-fold longer than rFVIII in hFcRn transgenic mice. These results confirm that FcRn mediates or is involved in the extended half-life of rFVIIIFc compared to rFVIII. Because whole blood hemostasis as measured by rotational thromboelastometry (ROTEM®) has been shown to correlate with the potency of clotting factors in hemophilic mouse bleeding models as well as in clinical applications, we sought to assess the ex vivo potency of rFVIIIFc in hemophilia A mice using ROTEM®. Hemophilia A mice were administered a single intravenous dose of 50 IU / kg of rFVIIIFc, XYNTHA® (FVIII), or ADVATE® (FVIII). Five minutes after dose administration, clot formation was similar with respect to clotting time (CT), clot formation time (CFT), and alpha angle. However, rFVIIIFc showed significant improvement in CT at 72 and 96 hours after dose administration, and CFT and alpha angle were also improved at 96 hours compared to both XYNTHA® (FVIII) and ADVATE® (FVIII), consistent with PK prolongation of rFVIIIFc.Thus, the construction of an Fc fusion of FVIII produces a molecule with an increased half-life and a defined mechanism of action that has the potential to provide long-term protection from bleeding.
[0216] Example 5 This example shows the final analysis results for FVIII activity from 16 patients treated with 25 and 65 IU / kg FVIII products, see Example 3.
[0217] In this example, rFVIIIFc is a recombinant fusion protein consisting of a single molecule of recombinant B-domain deleted human FVIII (BDD-rFVIII) fused to the dimeric Fc domain of human IgG1, with no intervening linker sequence. This protein construct is also referred to herein as rFVIIIFc heterodimer hybrid protein, FVIIIFc monomeric Fc fusion protein, FVIIIFc monomeric hybrid, monomeric FVIIIIFc hybrid, and FVIIIFc monomer-dimer. See Example 1, Figure 1, and Table 2A.
[0218] Preclinical studies with rFVIIIFc have shown an approximately two-fold extension of the half-life of rFVIII activity compared to commercial rFVIII products. The rationale for this study was to evaluate the safety and tolerability of single-dose administration of rFVIIIFc in a frozen liquid formulation and to provide data on PK in severe hemophilia A subjects. For this study, 16 evaluable subjects were available for PK evaluation. Single doses of two doses of both rFVIIIFc and ADVATE® at nominal doses of 25 (n=6) and 65 IU / kg / body weight (n=10) were infused intravenously over approximately 10 minutes. Blood samples for plasma PK evaluation were obtained pre-infusion and up to 10 days after dose administration. The PK of FVIII activity for both ADVATE® and rFVIIIFc was characterized in this study using model-dependent methods.
[0219] the purpose The primary objective of this study was to evaluate the safety and tolerability of a single dose of two doses of rFVIIIFc (25 and 65 IU / kg) in previously treated patients (PTPs) aged 12 years or older with severe hemophilia A.
[0220] A secondary objective was to determine pharmacokinetic (PK) parameters as determined by pharmacodynamic (PD) activity of FVIII over time following a single dose of 25 or 65 IU / kg rFVIIIFc compared to ADVATE® in one-stage clotting and chromogenic assays.
[0221] Study Design (See Example 3) Blood samples were collected for FVIII activity PK assessment at the screening visit (within 28 days prior to administration of a dose of ADVATE®); pre-injection (injection of ADVATE®) on day 0 and 10 and 30 minutes and 1, 3, 6, and 9 hours post-injection; on day 1, 24 hours post-injection of ADVATE®; on day 2, 48 hours post-injection of ADVATE®; on day 3, 72 hours post-injection of ADVATE®; and on day 4, 96 hours post-injection of high-dose ADVATE® (Cohort B only).
[0222] Blood samples were collected for FVIII activity PK assessment immediately prior to administration of rFVIIIFc, on the day of rFVIIIFc injection at 10 and 30 min and 1, 3, 6, and 9 h after rFVIIIFc injection; on day 1 at 24 h after rFVIIIFc injection; on days 2-5 at 48, 72, 96, and 120 h after rFVIIIFc injection; on day 7 at 168 h after rFVIIIFc injection; and on days 8, 9, and 10 at 192, 216, and 240 h after high-dose rFVIIIFc injection (cohort B only). FVIII activity was also measured at the final study visit at 672 h after rFVIIIFc injection (day 28 after rFVIIIFc injection). Pharmacokinetic Modeling and Calculations Abbreviation TBLP1 = model predicted time after dose administration when FVIII activity declines to approximately 1 IU / dL above baseline. TBLP3 = model predicted time after dose administration when FVIII activity declines to approximately 3 IU / dL above baseline. KV_M = Cmax_M / practical dose (IU / kg) KV_OB=Cmax_OB / practical dose (IU / kg) IVR_M=100×Cmax_M×plasma volume(dL) / total volume(IU);plasma volume(mL)=(23.7×body length(cm))+(9.0×body weight(kg))-1709. IVR_OB=100×Cmax_OB×plasma volume(dL) / total volume(IU);plasma volume(mL)=(23.7×body length(cm))+(9.0×body weight(kg))-1709.
[0223] Results Single-dose pharmacokinetics (one-stage assay) The observed FVIII activity increased acutely after a brief IV infusion of ADVATE® or rFVIIIFc, with mean (±standard deviation) model-predicted Cmax values of 56.6±4.74 and 121±28.2 IU / dL for ADVATE® and 55.6±8.18 and 108±16.9 IU / dL for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. All patients treated with ADVATE® and rFVIIIFc had a dose-dependent increase in FVIII activity. Cmax and AUC INF The increases observed in both were slightly less than dose proportional over the dose range evaluated.
[0224] After the end of the infusion, the decline in observed FVIII activity showed a monoexponential decay profile until baseline levels were reached. The rate of decline in FVIII activity was lower for rFVIIIFc than for ADVATE®, with mean (±standard deviation) model-predicted elimination half-life values of 11.9±2.98 and 10.4±3.03 hours for ADVATE® and 18.0±3.88 and 18.4±6.99 hours for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. Elimination half-life values appeared to be dose-independent across the dose range evaluated for both FVIII products.
[0225] Total systemic FVIII exposure (AUC INF (assessed by ) were approximately 48% and 61% greater following rFVIIIFc administration than ADVATE® at the 25 and 65 IU / kg dose levels, respectively. Mean (± standard deviation) model predicted AUC INF Values were 974±259 and 1810±606 hours for ADVATE® for the 25 and 65 IU / kg dose groups, respectively. * IU / dL and for rFVIIIFc 1440 ± 316 and 2910 ± 1320 h * IU / dL.
[0226] Similar to the elimination half-life, the MRT was prolonged for rFVIIIFc compared to ADVATE®. Mean (± standard deviation) model predicted MRT values were 17.1±4.29 and 14.9±4.38 hours for ADVATE® and 25.9±5.60 and 26.5±10.1 hours for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. MRT values appeared to be dose-independent across the dose range evaluated for both FVIII products.
[0227] In addition, the first PK parameter values for CL and V were determined. The CL values for rFVIIIFc accounted for only about 66% of those observed for ADVATE® at equivalent doses. The mean (±standard deviation) model predicted CL values were 2.70±0.729 and 4.08±1.69 mL / hr / kg for ADVATE® and 1.80±0.409 and 2.69±1.25 mL / hr / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. V values were comparable between ADVATE® and rFVIIIFc, with mean (±standard deviation) model-predicted V values of 43.9±4.27 and 56.1±13.4 mL / kg for ADVATE® and 45.3±7.23 and 61.6±10.6 mL / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. A slight increase in mean CL and V values was observed with increasing doses of ADVATE® and rFVIIIFc, but the increase in standard deviation at the 65 IU / kg dose coupled with limited dose levels confounded the assessment of dose-dependence of these parameters. For example, the CV% geometric mean CL values for the rFVIIIFc treatment groups increased from 23.0% (25 IU / kg) to 48.6% (65 IU / kg).
[0228] In addition to the primary PK parameters, secondary PK parameters (e.g., K values, IVR, etc.) were determined to assess FVIII duration of effect. Evidence of PK differences was also observed with rFVIIIFc, which showed increased TBLP1 and TBLP3 values compared to ADVATE® at equivalent doses. IVR and K values for ADVATE® and rFVIIIFc appeared to be comparable. Slight increases in TBLP1 and TBLP3 values were observed with increasing doses of ADVATE® and rFVIIIFc. In contrast, slight decreases in mean IVR and K values were noted with increasing doses of ADVATE® and rFVIIIFc. As previously mentioned, assessment of dose dependency of these parameters is confounded by limited dose levels.
[0229] The mean (±standard deviation) observed TBLP1 was 2.88±0.733 and 2.93±0.848 IU / dL per IU / kg for ADVATE® and 4.28±0.873 and 5.16±2.02 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The mean (±standard deviation) observed TBLP3 was 2.06±0.527 and 2.26±0.666 IU / dL per IU / kg for ADVATE® and 3.09±0.623 and 3.93±1.59 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively.
[0230] Mean IVR and K values calculated using observed Cmax values (subtracting baseline and residual drug in the model) were generally greater than those determined using model-predicted Cmax values, consistent with the slightly lower estimates of peak activity observed using the one-compartment model. Mean (±standard deviation) observed K values were 2.57±0.198 and 2.13±0.598 IU / dL per IU / kg for ADVATE® and 2.46±0.330 and 1.85±0.332 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The mean (±standard deviation) observed IVR values were 94.1±15.6 and 85.8±16.5% for ADVATE® and 89.5±11.9 and 74.8±6.72% for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively.
[0231] Single-dose pharmacokinetics (chromogenic assay) Observed FVIII activity increased acutely after brief IV infusion of ADVATE® or rFVIIIFc, with mean (±SD) model-predicted Cmax values of 70.2±9.60 and 157±38.6 IU / dL for ADVATE® and 70.3±10.0 and 158±34.7 IU / dL for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively.
[0232] All patients treated with ADVATE® and rFVIIIFc had a dose-dependent increase in FVIII activity. Cmax and AUC INF The increases observed in both were slightly less than dose proportional over the dose range evaluated.
[0233] After the end of the infusion, the decline in observed FVIII activity showed a monoexponential decay profile until baseline levels were reached. The rate of decline in FVIII activity was lower for rFVIIIFc than for ADVATE®, with mean (±standard deviation) model-predicted elimination half-life values of 10.7±1.98 and 10.3±3.27 hours for ADVATE® and 16.2±2.92 and 19.0±7.94 hours for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. Elimination half-life values appeared to be dose-independent across the dose range evaluated for both FVIII products.
[0234] Total systemic FVIII exposure (AUC INF (assessed by ) were approximately 53% and 84% greater following rFVIIIFc administration than ADVATE® at the 25 and 65 IU / kg dose levels, respectively. Mean (± standard deviation) model predicted AUC INF Values were 1080±236 and 2320±784 hours for the 25 and 65 IU / kg dose groups, respectively, and for ADVATE®. * IU / dL and for rFVIIIFc, 1650 ± 408 and 4280 ± 1860 h * IU / dL.
[0235] Similar to the elimination half-life, the MRT was prolonged for rFVIIIFc compared to ADVATE®. Mean (±standard deviation) model-predicted MRT values were 15.3±2.86 and 14.8±4.72 hours for ADVATE® and 23.4±4.22 and 27.3±11.4 hours for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. MRT values appeared to be dose-independent across the dose range evaluated for both FVIII products.
[0236] In addition, the first PK parameter values for CL and V were determined. The CL values for rFVIIIFc accounted for only about 58-66% of those observed for ADVATE® at equivalent doses. The mean (± standard deviation) model predicted CL values were 2.39±0.527 and 3.21±1.40 mL / hr / kg for ADVATE® and 1.57±0.349 and 1.86±0.970 mL / hr / kg for rFVIIIFc for the 25 and 65 IU / KG dose groups, respectively. V values were comparable between ADVATE® and rFVIIIFc, with mean (±standard deviation) model-predicted V values of 35.8±5.52 and 43.6±11.2 mL / kg for ADVATE® and 35.9±6.65 and 42.7±8.91 for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. Increases in mean CL and V values were observed with escalating doses of ADVATE® and rFVIIIFc, but the increased standard deviation at 65 IU / kg coupled with limited dose levels confounded assessment of dose-dependence of these parameters.
[0237] In addition to the primary PK parameters, secondary PK parameters (e.g., K values, IVR, etc.) were determined to assess FVIII duration of effect. Evidence of PK differences was also observed with rFVIIIFc, indicating increased TBLP1 and TBLP3 values compared to ADVATE® at equivalent doses. IVR and K values for ADVATE® and rFVIIIFc appeared to be comparable.
[0238] A slight increase in TBLP1 and TBLP3 values was observed with increasing doses of ADVATE® and rFVIIIFc. In contrast, a slight decrease in mean IVR and K values was observed with increasing doses of ADVATE® and rFVIIIFc. As mentioned above, the assessment of the dose-dependence of these parameters is confounded by the limited dose levels.
[0239] The mean (±SD) observed TBLP1 was 2.70±0.511 and 3.09±0.978 IU / dL per IU / kg for ADVATE® and 4.06±0.798 and 5.66±2.38 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The mean (±SD) observed TBLP3 was 1.98±0.377 and 2.39±0.718 IU / dL per IU / kg for ADVATE® and 3.04±0.598 and 4.44±1.84 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively.
[0240] Mean IVR and K values calculated using observed Cmax values (subtracting baseline and residual drug in the model) were generally greater than those determined using model-predicted Cmax values, consistent with the slightly lower estimates of peak activity observed using the one-compartment model. Mean (±standard deviation) observed K values were 3.08±0.429 and 2.85±0.721 IU / dL per IU / kg for ADVATE® and 3.12±0.451 and 2.92±0.985 IU / dL per IU / kg for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively. The mean (±standard deviation) observed IVR values were 112±14.5 and 116±26.9% for ADVATE® and 113±16.3 and 117±33.6% for rFVIIIFc for the 25 and 65 IU / kg dose groups, respectively.
[0241] conclusion All patients treated with ADVATE® and rFVIIIFc had comparable dose-dependent increases in Cmax and AUCINF across the dose range evaluated. Peak plasma levels of ADVATE® and rFVIIIFc activity were generally observed within 1 hour of the end of the infusion and remained detectable for several days after dose administration. After the end of the infusion, the decrease in baseline-corrected FVIII activity showed a monoexponential decay profile until reaching baseline levels for both products. Parameter values for elimination half-life and MRT appeared to be dose-dependent across the dose range evaluated for both FVIII products. Although a slight increase in mean CL and V values was observed with increasing doses of ADVATE® and rFVIIIFc, the increased intersubject variability at 65 IU / kg coupled with the limited dose levels confounded the assessment of dose-dependency of these parameters.
[0242] Comparison of rFVIIIFc and ADVATE® activity PK demonstrated an increase in systemic exposure of approximately 48-61% (single-stage assay) or 53-84% (chromogenic assay) for rFVIIIFc compared to ADVATE® at comparable doses, an approximately 30-40% reduction in clearance, and an approximately 50-80% increase in both elimination half-life and MRT. Evidence of PK differences was also observed with rFVIIIFc, which demonstrated increased TBLP1 and TBLP3 values compared to ADVATE® at equivalent doses. IVR and K values for ADVATE® and rFVIIIFc appeared to be comparable.
[0243] PK parameters derived from the chromogenic assay results were generally consistent with those from the one-stage assay, except that the chromogenic assay yielded higher estimates of exposure parameters (e.g., Cmax, AUCINF, etc.).
[0244] With the observed improvements in PK, rFVIIIFc may provide long-term protection from bleeding and may allow for less frequent injections for individuals with hemophilia A.
[0245] Example 6 Based on the interim PK analysis from the first-in-human study of rFVIII:Fc (Example 3), the A-LONG study was designed. A-LONG is an open-label, multicenter evaluation of the safety, pharmacokinetics, and efficacy of recombinant factor VIII Fc fusion (FVIII:Fc) in preventing and treating bleeding in previously treated subjects with severe hemophilia A (defined as <1 IU / dL [<1%] endogenous FVIII).
[0246] Approximately 106 subjects will be enrolled in one of three regimens: a goal-directed prophylaxis regimen (arm 1), a weekly dosing regimen (arm 2), and an on-demand regimen (arm 3).
[0247] Arm 1: Tailored Prophylaxis Regimen Arm 1 includes the full group and the PK subgroup. Approximately 66 subjects will be enrolled. The initial regimen will be 25 IU / kg on the first day, followed by 50 IU / kg twice weekly on the fourth day of that week. Subjects will receive rFVIIIFc on this weekly prophylaxis regimen until PK results for rFVIIIFc are available. Based on these results, a tailored prophylaxis regimen will be established for each individual, with doses and intervals determined to maintain a trough level of 1-3% FVIII activity. Each subject will then receive their individual tailored prophylaxis regimen throughout the study.
[0248] Subjects will be monitored throughout the study and ongoing dose and interval adjustments will be made. Adjustments will only be considered if a subject experiences unacceptable bleeding episodes, defined as 2 or more spontaneous bleeding episodes over a 2-month period, in which case adjustments will target trough levels of 3-5%.
[0249] Arm 2: Weekly dosing regimen Approximately 20 subjects will be enrolled / randomized to undergo abbreviated PK profiling of rFVIIIFc as follows: at least a 96 hour washout; administration of a single dose of 65 IU / kg of rFVIIIFc; and convenience sampling starting with rFVIIIFc on day 0, including pre-injection and at 10 (± 2) minutes, 3 hours (± 15 minutes), 72 (± 2) hours [day 3], and 96 (± 2) hours [day 4] from the start of injection. After abbreviated PK profiling, subjects will then receive a flat dose of 65 IU / kg rFVIIIFc every 7 days for at least 28 weeks and up to 52 weeks.
[0250] Arm 3: On-demand regimen A minimum of 10 major surgeries in at least 5 subjects will be evaluated in this study. Major surgery is defined as any surgical procedure (elective or emergency) involving general anesthesia and / or respiratory support that penetrates or exposes a major body cavity or results in substantial impairment of physical or physiological function (e.g., laparotomy, thoracotomy, craniotomy, joint replacement, and limb amputation).
[0251] For prophylaxis during surgery, subjects are treated with 20-50 IU / kg rFVIIIFc every 12-24 hours. Prior to surgery, the physician reviews the subject's PK profile of rFVIIIFc and evaluates the factor VIII replacement dosing regimen typically required for the type of planned surgery as well as the subject's clinical condition. Recommendations for appropriate dosing of rFVIIIFc during the surgical procedure, including any rehabilitation time, take these factors into account.
[0252] The primary objectives of this study are (a) to evaluate the safety and tolerability of rFVIIIFc administered as a prophylactic, on-demand, and surgical treatment regimen, and (b) to evaluate the efficacy of rFVIIIFc administered as a prophylactic, on-demand, and surgical treatment regimen. The secondary objectives of this study are (a) to characterize the PK profile of rFVIIIFc and compare the PK of FVIIIFc to that of the current marketed product, ADVATE®, (b) to evaluate individual responses to FVIIIFc, and (c) to evaluate FVIIIFc consumption. Main purpose To evaluate the safety and tolerability of rFVIIIFc administered as prophylactic, weekly, on-demand, and surgical treatment regimens To evaluate the efficacy of rFVIIIFc administered as tailored prophylactic, on-demand, and surgical treatment regimens Secondary Objectives To characterize the PK profile of rFVIIIFc and compare the PK of rFVIIIFc with that of the current marketed product, ADVATE® To evaluate individual responses to rFVIIIFc Characterize the range of doses and schedules required to adequately prevent bleeding in prophylactic regimens, maintain homeostasis in surgical settings, or treat bleeding episodes in on-demand, weekly treatment, or prophylactic settings. To assess rFVIIIFc consumption (e.g., total annual rFVIIIFc consumption per subject)
[0253] Example 7 Clinical ROTEM® Evaluation In the study in Example 7, in addition to measuring plasma FVIII activity by the one-stage activated partial thromboplastin time (aPTT) assay, whole blood rotational thromboelastometry (ROTEM®) was also examined to evaluate the improvement in global hemostasis with rFVIIIFc and ADVATE® in two subjects, specifically, one in the low-dose cohort and one in the high-dose cohort.
[0254] rFVIIIFc and ADVATE® appear to be equally active in clot formation when added to a subject's blood prior to rFVIIIFc treatment. Clotting times (CT) were linear with respect to dose of rFVIIIFc and ADVATE® in the range of approximately 1% to 100% of normal, and the dose response was comparable between rFVIIIFc and ADVATE® in the same subjects.
[0255] After ADVATE® and subsequent rFVIIIFc dose administration, citrated whole blood was sampled at various time points, and clot formation after recalcification was monitored by ROTEM®. Despite variable baseline CT due to residual FVIII levels before ADVATE® or rFVIIIFc dose administration, both products effectively corrected CT to comparable levels 30 minutes after injection. In addition, the improvement in CT was well sustained at the time of injection of 25 IU / kg rFVIIIFc and 3 hours after injection in subjects dosed at this low dose compared to ADVATE®. However, the differential improvement of rFVIIIFc vs. ADVATE® was less readily detectable at the 65 IU / kg dose.
[0256] Example 8 Hemophilia A mice were used for the tail clip study. Mice were first anesthetized and then injected with either 4.6 μg / kg, 1.38 μg / kg, or 0.46 μg / kg of processed rFVIIIFc (drug substance containing approximately 75%-85% processed rFVIIIFc) and purified single-chain rFVIIIFc. After injection, the tail was clipped from the tip and immediately placed in a tube to collect blood. The percentage of protection in survival was measured for processed rFVIIIFc (drug substance) and single-chain FVIIIFc, as shown in Table 7 and Figure 7. [Table 7]
[0257] As shown in Table 7 and FIG. 7, protection in survival by single-chain rFVIIIFc is comparable to that of processed rFVIIIFc (DS).
[0258] In vitro coagulation activity by ROTEM The clotting titer of rFVIIIFc was further examined by whole blood rotational thromboelastometry (ROTEM) across a range of concentrations and compared with both rBDD FVIII (Xyntha) and recombinant full-length FVIII (rflFVIII; Advate). For in vitro ROTEM, rFVIII protein was added in triplicate to citrated pooled blood collected from the vena cava of 5–6 male hemophilia A mice to final concentrations of 0, 0.1, 1, 10, and 100% of normal plasma FVIII levels. Clotting was initiated by addition of CaCl2 (NATEM) and clotting time (CT), clot formation time (CFT), alpha angle, and maximum clot firmness (MCF) were recorded in a ROTEM system (Pentapharm GmbH, Munich, Germany). Clotting times (CT), clot formation times (CFT), and alpha angles for the three proteins added to the blood of hemophilia A mice at increasing doses from 0.1 to 100% of normal FVIII levels are shown in Figure 14. Over the wide range of 0.1 to 100% of normal, CT and CFT are comparable among rFVIIIFc, rBDD FVIII, and rflFVIII. The alpha angle is the only significant difference between rFVIIIFc and rBDD FVIII at 10% (p<0.05).
[0259] Coagulation activity by ex vivo ROTEM The pharmacokinetics of rFVIIIFc was compared to rBDD FVIII and rflFVIII after a single intravenous injection in hemophilia A mice, as measured by ROTEM. For ex vivo ROTEM, male hemophilia A mice were intravenously injected with a single dose of 50 IU / kg of rFVIIIFc, ADVATE®, and XYNTHA®, and five mice were sacrificed at each time point (5 min, 24, 48, 72, and 96 h after dose administration). Individual citrated whole blood collected from the vena cava was immediately analyzed by NATEM on the ROTEM system, and parameters were measured as described above. CT, CFT, and alpha angle were determined for samples collected 5 min to 96 h after dose administration and are shown in Figure 15. All yielding similar CT, CFT, and alpha angle at 5 min are equally effective (Figure 15A-C). However, rFVIIIFc showed significantly improved (p<0.05) CT at 72 and 96 hours, CFT and alpha angle at 96 hours compared to rBDD FVIII and rflFVIII (FIGS. 15A-C).
[0260] Example 9 Recombinant factor VIIIFc (rFVIIIFc) consists of B-domain deleted (BDD) rFVIII genetically fused to the Fc domain of human immunoglobulin G1 (IgG1). Prior to secretion from HEK293 cells, the majority of rFVIIIFc is processed into FVIII heavy chain (HC) and light chain (LC+Fc). In circulation, rFVIIIFc is complexed with von Willebrand factor (VWF) and released upon activation in a manner indistinguishable from native FVIII. Spontaneous separation of HC and LC is believed to contribute to the loss of FVIII activity in plasma and during storage of FVIII drug formulations. Here we describe a single-chain unprocessed isoform of rFVIIIFc (SC rFVIIIFc) that may offer superior manufacturability and increased stability compared to native FVIII.
[0261] SC rFVIIIFc was purified from rFVIIIFc containing a fraction of unprocessed isoforms. Compared to rFVIIIFc, SC rFVIIIFc showed equivalent chromogenic activity by one-stage (aPTT) assay, but approximately 60% reduced activity (Table 3A-B). Thrombin generation assay (TGA) was performed using an auto-calibrated thrombogram (by Thrombinoscope®). In the thrombin generation assay (TGA), SC rFVIIIFc also showed reduced thrombin availability (Figure 13A) and peak thrombin (Figure 13B) compared to rFVIIIFc. However, as shown in Table 3B, full activation of SC rFVIIIFc by aPTT was observed in the absence of vWF, suggesting that release from vWF may be delayed due to covalent binding of the a3 acidic region to HC after Arg1680 cleavage in SC rFVIIIFc, compared to a3 release and dissociation from fully processed FVIII. The delayed dissociation from vWF may explain the reduced activity observed in the aPTT assay and TGA, whereas full activity was observed in the two-step chromogenic assay. In the presence of vWF, the reduced rate of activation was confirmed in a modified chromogenic substrate assay with limited thrombin as the activator of FVIII.
[0262] The in vivo function of SC rFVIIIFc was evaluated in the hemophilia A mouse tail vein transection (TVT) model. Hemophilia A male mice were treated with the indicated doses of either the drug formulation of rFVIIIFc or SC rFVIIIFc 48 hours prior to TVT. Tail rebleeding and survival were monitored hourly up to 12 hours after TVT, with a final observation at 24 hours after TVT. SC rFVIIIFc and rFVIIIFc demonstrated equivalent in vivo efficacy in this model, with ED50 of 1.17 μg / kg and 1.23 μg / kg, respectively, when TVT was performed 48 hours after injection (Figure 7(A)). Comparable survival curves (p≧0.65) (FIG. 7(B)) and rebleeding rates (FIG. 7(C)) at 24 hours after TVT in hemophilia A mice were observed for SC rFVIIIFc and rFVIIIFc at each tested dose level, indicating that SC rFVIIIFc was as effective as rFVIIIFc, despite a lower apparent aPTT activity. Thus, the delayed in vitro activation of SC rFVIIIFc in the presence of vWF does not appear to have a significant effect on in vivo efficacy. Hence, SC rFVIIIFc represents a novel and effective isoform of rFVIIIFc that may have clinical applications. Further studies will be required to demonstrate increased product stability in this Fc fusion protein.
[0263] Example 10 Current factor VIII (FVIII) products have a half-life (t...
Claims
1. 1. A pharmaceutical composition comprising: (i) a chimeric polypeptide comprising a factor VIII (FVIII) portion and a second portion; and (ii) at least one pharma- ceutically acceptable excipient; Including, (1) 15% to 40% of the FVIII portion of the chimeric polypeptide comprises single-chain FVIII; (2) 60% to 85% of the FVIII portion of the chimeric polypeptide comprises processed FVIII; the single-chain FVIII comprises a FVIII heavy chain and a FVIII light chain on a single polypeptide chain, and the processed FVIII comprises a FVIII heavy chain and a FVIII light chain on two polypeptide chains; the single-chain FVIII portion comprises an amino acid sequence that is at least 95% identical to amino acids 20-1457 of SEQ ID NO:2; The second portion comprises an amino acid sequence that is at least 95% identical to amino acids 21-247 of SEQ ID NO:
4.
2. (a) 15% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 85% of the FVIII of the chimeric polypeptide comprises processed FVIII; (b) 20% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 80% of the FVIII of the chimeric polypeptide comprises processed FVIII; or (c) 25% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 75% of the FVIII of the chimeric polypeptide comprises processed FVIII; or (d) 30% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 70% of the FVIII of the chimeric polypeptide comprises processed FVIII; or (e) 35% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 65% of the FVIII of the chimeric polypeptide comprises processed FVIII; or (f) 40% of the FVIII of the chimeric polypeptide comprises single-chain FVIII and 60% of the FVIII of the chimeric polypeptide comprises processed FVIII.
2. The pharmaceutical composition of claim 1.
3. The pharmaceutical composition of claim 1 or 2, wherein the chimeric polypeptide is a rFVIIIFc monomer-dimer hybrid.
4. The pharmaceutical composition of any one of claims 1 to 3, wherein the single-chain FVIII comprises an amino acid sequence identical to amino acids 20 to 1457 of SEQ ID NO:
2.
5. The pharmaceutical composition of any one of claims 1 to 4, wherein the rFVIIIFc monomer-dimer hybrid is produced by human embryonic kidney 293 (HEK293) cells.
6. 6. The pharmaceutical composition of claim 5, wherein the second portion comprises an amino acid sequence identical to amino acids 21-247 of SEQ ID NO:
4.
7. A pharmaceutical composition according to any one of claims 1 to 6, for subcutaneous or intravenous injection into humans.
8. 8. A pharmaceutical composition according to any one of claims 1 to 7 for use in reducing bleeding in a subject with hemophilia A.
9. 9. The pharmaceutical composition for use according to claim 8, for use in the prevention, reduction or treatment of bleeding episodes in said subject, prophylactic treatment of bleeding episodes in said subject, on-demand treatment of bleeding episodes in said subject.
10. 10. The pharmaceutical composition for use according to claim 9, wherein the bleeding episode is associated with hemarthrosis, muscle bleeding, mouth bleeding, bleeding, bleeding into muscle, oral bleeding, trauma, head trauma (head trauma), gastrointestinal bleeding, intracranial bleeding, intraperitoneal bleeding, intrathoracic bleeding, fracture, central nervous system bleeding, retropharyngeal bleeding, retroperitoneal bleeding, or bleeding into the iliopsoas sheath.
11. The pharmaceutical composition for use according to claim 9, wherein the subject is in need of surgery.
12. 12. The pharmaceutical composition of claim 11, wherein the surgical procedure is minor surgery, major surgery, tooth extraction, tonsillectomy, inguinal herniotomy, synovectomy, total knee arthroplasty, craniotomy, osteosynthesis, trauma surgery, intracranial surgery, intraperitoneal surgery, intrathoracic surgery, emergency surgery or joint replacement surgery.
13. A pharmaceutical composition according to any one of claims 1 to 7 or a pharmaceutical composition for use according to any one of claims 8 to 12, which is lyophilized.
Citation Information
Patent Citations
Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof
WO2011069164A2