TWI425953B - 共軛因子viii分子 - Google Patents

共軛因子viii分子 Download PDF

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TWI425953B
TWI425953B TW098106523A TW98106523A TWI425953B TW I425953 B TWI425953 B TW I425953B TW 098106523 A TW098106523 A TW 098106523A TW 98106523 A TW98106523 A TW 98106523A TW I425953 B TWI425953 B TW I425953B
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Description

共軛因子VIII分子
本發明係關於共軛凝血因子VIII分子。具體而言，本發明係關於具有改良之循環半衰期之共軛因子VIII分子。
A型血友病係由凝血因子VIII(FVIII)活性缺陷或功能障礙而引起之遺傳性出血病症。臨床表現不在於一期止血-血凝塊形成正常發生-而在於由於缺乏二期凝血酶形成導致凝塊不穩定。該疾病係藉由靜脈內注射分離自血液或重組產生之凝血因子FVIII來治療。
當前治療建議正自傳統的按需治療向預防轉移。內源性FVIII之循環半衰期為12-14小時且因此預防性治療需要一週實施數次以使患者獲得實質上無症狀之生活。靜脈內(IV)投與對於許多人(尤其兒童及未成年人)而言導致明顯不便及/或疼痛。因此，業內需要具有因子VIII活性之新穎因子VIII產物，其較佳在結構上係同質的、較佳係安全的且較佳具有顯著延長之循環半衰期以減少因子VIII之每週投與次數。此外，業內亦需要用於獲得及產生該等分子之相對簡單的方法。
業內已知對因子VIII實施聚乙二醇化來延長循環半衰期。然而，獲得具有同質結構以及顯著改良之循環半衰期之安全產物仍為障礙。可用於產生共軛因子VIII分子之方法通常極為費力，及/或往往導致產量較低及/或產物結構不同質。WO2008011633中提出使用人工構造之O-連接糖基化位點來獲得具有延長之治療性蛋白質循環半衰期的治療性蛋白質，然而，其中未揭示共軛因子VIII分子。
 定義：
因子VIII分子： FVIII/因子VIII係主要由肝細胞產生之大的複合糖蛋白。FVIII由2351個胺基酸組成，包括信號肽，且藉由同源性界定含有數種不同結構域。存在三種A結構域、一種B結構域、及兩種C結構域。結構域順序可列示為NH2-A1-A2-B-A3-C1-C2-COOH。FVIII以在B-A3邊界分開之兩條鏈形式在血漿中循環。該等鏈藉由二價金屬離子結合連接。A1-A2-B鏈稱為重鏈(HC)，而A3-C1-C2稱為輕鏈(LC)。
內源性因子VIII分子以具有不同大小B結構域之分子的集合體形式在活體內循環。在活體內很可能發生B結構域之漸進性酶促移除，導致具有不同大小B結構域之分子的集合體。通常認為，位置740處之裂解(藉由其移除B結構域之最後一部分)的發生與凝血酶激活有關。然而，此並不能排除例如位置740處之裂解位點已經損害之因子VIII變體可能具有活性。
本文所用之「因子VIII」或「FVIII」係指為內源性凝血途徑之成員且為凝血所必需之人類血漿糖蛋白。「天然FVIII」係顯示於SEQ ID NO. 1(胺基酸1-2332)中之全長人類FVIII分子。B結構域跨越SEQ ID NO 1中之胺基酸741-1648。
本發明之因子VIII分子係B結構域截短型因子FVIII分子，其中剩餘結構域對應於SEQ ID NO. 1中之1-740號及1649-2332號胺基酸中所示之序列。因此，本發明分子係在轉化宿主細胞(較佳來源於哺乳動物)中產生之重組分子。然而，剩餘結構域(即三種A結構域及兩種C結構域)可與SEQ ID NO 1中所示之胺基酸序列(胺基酸1-740及1649-2332)略有不同，例如約1%、2%、3%、4%或5%。具體而言，在剩餘結構域中引入胺基酸修飾(取代、缺失等)來改良(例如)因子VIII與諸如vW因子、LPR、多種受體、其他凝血因子、細胞表面等多種其他部分的結合能力似乎是可行的。此外，本發明之因子VIII分子在例如截短之B結構域及/或該等分子之一或多種其他結構域中包含其他轉譯後修飾似乎亦是可行的。該等其他轉譯後修飾可呈與本發明之因子VIII分子共軛之多種分子形式，例如聚合化合物、肽化合物、脂肪酸衍生化合物等。
本發明之因子VIII分子無論是否在B結構域外經修飾、是否具有其他轉譯後修飾皆具有因子VIII活性，意味著能夠在凝血級聯反應中以與FVIII功能類似或等效之方式起作用、經由與激活血小板上之FIXa相互作用誘導形成FXa、並支持形成血凝塊。該活性可在活體外藉由業內熟知技術進行評價，例如凝塊分析、內源性凝血酶潛能分析等。本發明之因子VIII分子所具有之FVIII活性佔天然人類FVIII之至少約10%、至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少80%、至少90%、及100%或甚至100%以上。
B結構域： 因子VIII中之B結構域跨越SEQ ID NO 1中之胺基酸741-1648。B結構域在數個不同位點處裂解，使得循環血漿FVIII分子具有較大異質性。高度糖基化B結構域之確切功能尚未可知。吾人已知該結構域對於凝血級聯反應中之FVIII活性不是必要的。以下事實支持此明顯之功能缺乏：B結構域缺失型/截短型FVIII似乎具有與全長天然FVIII所見相同之活體內特性。儘管如此，有跡象表明B結構域可減少與細胞膜之連接，至少在無血清條件下。
B結構域截短型/缺失型因子VIII分子： 所合成之內源性全長FVIII呈單鏈前體分子形式。在分泌之前，前體裂解成重鏈及輕鏈。重組B結構域缺失型FVIII可由兩種不同策略產生。分別合成呈兩條不同多肽鏈形式之不含B結構域之重鏈及輕鏈(雙鏈策略)或合成呈單前體多肽鏈形式之B結構域缺失型FVIII，其以與全長FVIII前體相同之方式裂解成重鏈及輕鏈(單鏈策略)。
在B結構域缺失型FVIII前體多肽中，重鏈及輕鏈部分通常由連接體分開。為使引入B結構域缺失型FVIII中免疫原性抗原決定基之風險最小化，連接體序列較佳衍生自FVIII B結構域。連接體必須包含將B結構域缺失型FVIII前體多肽分成重鏈及輕鏈之蛋白酶識別位點。在全長FVIII之B結構域中，胺基酸1644-1648構成此識別位點。導致B結構域缺失型FVIII激活時連接體移除之凝血酶位點位於重鏈中。因此，連接體之大小及胺基酸序列不可能影響藉由凝血酶激活之其自剩餘FVIII分子之移除。B結構域缺失有利於產生FVIII。儘管如此，B結構域部分可包括於連接體中而不降低生產率。B結構域對生產率之負性效應並未歸因於具有任何特定大小或序列之B結構域。
截短之B結構域可含有數個O-糖基化位點。然而，根據一較佳實施例，該分子在截短之B結構域中包含僅一個、或者兩個、三個或四個O-連接寡糖。
根據一較佳實施例，截短之B結構域包含僅一個潛在的O-糖基化位點且親水性聚合物與該O-糖基化位點共價共軛。
本發明B結構域截短型分子中之O-連接寡糖可附接至藉由重組手段及/或藉由截短B結構域暴露「隱藏」O-糖基化位點而人工產生之O-糖基化位點。在兩種情形下，該等分子均可藉由以下來產生：設計B結構域截短型因子VIII胺基酸序列並隨後使該胺基酸序列經受電腦(in silico)分析以預測截短之B結構域中O-糖基化位點之概率。可在適宜宿主細胞中合成以相對高概率具有該等糖基化位點之分子，然後分析糖基化模式並隨後選拔在截短之B結構域中具有O-連接糖基化之分子。用於產生重組因子VIII蛋白質之適宜宿主細胞較佳來源於哺乳動物以確保分子被糖基化。在本發明實施中，細胞係哺乳動物細胞，更佳為已確立之哺乳動物細胞系，包括但不限於CHO(例如，ATCC CCL 61)、COS-1(例如，ATCC CRL 1650)、幼倉鼠腎(BHK)、及HEK293(例如，ATCC CRL 1573；Graham等人，J. Gen. Virol. 36:59-72，1977)細胞系。較佳之BHK細胞系係tk-ts13 BHK細胞系(Waechter及Baserga，Proc. Natl. Acad. Sci. USA 79:1106-1110，1982)，下文中稱為BHK 570細胞。BHK 570細胞系可以ATCC登錄號CRL 10314自美國典型培養物保藏中心(American Type Culture Collection)，12301 Parklawn Dr.，Rockville，MD 20852得到。tk-ts13 BHK細胞系亦可以登錄號CRL 1632自ATCC得到。較佳之CHO細胞系係可以登錄號CCI61自ATCC得到之CHO K1細胞系以及細胞系CHO-DXB11及CHO-DG44。
其他適宜細胞系包括但不限於大鼠Hep I(大鼠肝細胞瘤；ATCC CRL 1600)、大鼠Hep II(大鼠肝細胞瘤；ATCC CRL 1548)、TCMK(ATCC CCL 139)、人類肺(ATCC HB 8065)、NCTC 1469(ATCC CCL 9.1)；DUKX細胞(CHO細胞系)(Urlaub及Chasin，Proc. Natl. Acad. Sci. USA 77:4216-4220，1980)(DUKX細胞亦稱為DXB11細胞)、及DG44(CHO細胞系)(Cell，33:405，1983；及Somatic Cell and Molecular Genetics 12:555，1986)。亦可使用3T3細胞、Namalwa細胞、骨髓瘤及骨髓瘤與其他細胞之融合物。在一些實施例中，細胞可為突變體或重組細胞，例如，與其衍生自之細胞類型相比，表現質量或數量不同之催化蛋白質轉譯後修飾之酶譜系(例如，諸如糖基轉移酶及/或糖苷酶等糖基化酶、或諸如前肽等加工酶)的細胞。DUKX細胞(CHO細胞系)尤其佳。
當前較佳之細胞係HEK293、COS、中國倉鼠卵巢(CHO)細胞、幼倉鼠腎(BHK)及骨髓瘤細胞，中國倉鼠卵巢(CHO)細胞尤佳。
因此，本發明之發明者顯示可藉由截短B結構域來激活因子VIII B結構域中之「隱藏」O-糖基化位點。儘管不欲受限於任何理論，但此現象可歸因於所改變之截短之B構域中該分子之三級結構。由此使得「隱藏」O-糖基化位點在截短之B結構域中可以糖基化。此方法之一個優點係提供具有有利安全性質(例如，就變應原性而言)之重組分子。另一優點可為，其代表獲得B結構域中具有O-連接寡糖之B結構域截短變體之較簡單方法，此乃因B結構域中糖基化位點內在充足，先前證實在重組蛋白質中構造人工O-糖基化位點較為困難。
wt FVIII分子中B結構域之長度為約907個胺基酸。本發明分子中截短之B結構域之長度可自約10個胺基酸至約700個胺基酸不等，例如約12-500個胺基酸、12-400個胺基酸、12-300個胺基酸、12-200個胺基酸、15-100個胺基酸、15-75個胺基酸、15-50個胺基酸、15-45個胺基酸、20-45個胺基酸、20-40個胺基酸、或20-30個胺基酸。該截短之B結構域可包含重鏈片段及/或輕鏈片段及/或未在wt FVIII分子中發現之人工引入序列。術語「B結構域截短」及「B結構域缺失」在本文中可互換使用。
改良之循環半衰期： 與野生型因子VIII分子相比，本發明分子具有改良之循環半衰期，較佳延長之循環半衰期。循環半衰期較佳延長至少10%、較佳至少15%、較佳至少20%、較佳至少25%、較佳至少30%、較佳至少35%、較佳至少40%、較佳至少45%、較佳至少50%、較佳至少55%、較佳至少60%、較佳至少65%、較佳至少70%、較佳至少75%、較佳至少80%、較佳至少85%、較佳至少90%、較佳至少95%、較佳至少100%，更佳至少125%、更佳至少150%、更佳至少175%、更佳至少200%，且最佳至少250%或300%。甚至更佳地，該等分子之循環半衰期相對於野生型FVIII之循環半衰期延長至少400%、500%、600%、或甚至700%。
親水性聚合物： 本發明之修飾基團/親水性聚合物較佳非天然存在。在一個實例中，「非天然存在之修飾基團」係聚合物修飾基團，其中至少一種聚合物部分非天然存在。在另一實例中，非天然存在之修飾基團係經修飾之碳水化合物。選擇修飾基團官能化之位置以不妨礙「修飾糖」以酶促方式添加至多肽上。「修飾糖」亦係指經修飾基團官能化且為天然或修飾酶(例如糖基轉移酶)之受質的任何糖基模擬部分。
添加至多肽之聚合物修飾基團可改變該多肽之特性，例如，其生物可利用性、生物活性或其活體內半衰期。本發明之聚合物實例包括水溶性聚合物，其可為直鏈或具支鏈且可包括一或多種獨立選擇之聚合物部分，例如聚伸烷基二醇及其衍生物。本發明之聚合物修飾基團可包括水溶性聚合物，例如聚乙二醇及其衍生物(PEG、m-PEG)、聚丙二醇及其衍生物(PPG、m-PPG)及諸如此類。
術語「水溶性」係指在水中具有一定可檢測溶解度之部分。檢測及/或定量水溶解度之方法已為業內所熟知。本發明之實例性水溶性聚合物包括肽、糖類、聚醚、聚胺、聚羧酸及諸如此類。肽可具有混合序列且可由單一胺基酸構成，例如，聚離胺酸。多醣實例係聚唾液酸。聚醚實例係聚乙二醇，例如，m-PEG。聚胺實例為聚乙烯亞胺，且聚丙烯酸係代表性聚羧酸。
本發明水溶性聚合物之聚合物骨架可為聚乙二醇(即PEG)。與本發明有關之術語PEG包括呈任何形式之聚乙二醇，包括烷氧基PEG、二官能團PEG、多臂PEG、叉狀PEG、具支鏈PEG、有側基的PEG(即具有一或多個側接聚合物骨架之官能團的PEG或有關聚合物)、或其中具有可降解鍵之PEG。
聚合物骨架可為直鏈或具支鏈。具支鏈聚合物骨架已為業內眾所周知。通常，具支鏈聚合物具有中心分支核心部分及複數個與該中心分支核心連接之聚合物直鏈。PEG常以具支鏈形式使用，其可藉由將氧化乙烯添加至諸如甘油、季戊四醇及山梨醇等多元醇中來製備。中心分支部分亦可衍生自諸如離胺酸或半胱胺酸等數種胺基酸。在一個實例中，具支鏈聚乙二醇可以通式R(-PEG-OH)m表示，其中R表示核心部分，例如甘油或季戊四醇，且m表示臂的個數。多臂PEG分子(例如彼等闡述於美國專利第5,932,462號中者，該專利之全部內容以引用方式併入本文中)亦可用作聚合物骨架。
圖8顯示可用於本發明實施例中之代表性具支鏈PEG聚合物，其在本文中稱為「SA-甘油-PEG」。圖8A顯示與聚糖或多肽之胺基酸連接之CMP-SA-甘油-PEG或SA-甘油-PEG的實例性SA-甘油-PEG組份。圖8B顯示透過Gal殘基與聚糖或多肽連接之SA-甘油-PEG部分。圖8C顯示透過Gal-GalNAc殘基與聚糖或多肽連接之SA-甘油-PEG部分。圖8D顯示透過Gal-GalNAc部分與多肽之胺基酸連接之SA-甘油-PEG部分。在多個實施例中，AA係蘇胺酸或絲胺酸。在一實例性實施例中，藉由刪除FVIII多肽之B結構域將AA轉化成O-連接糖基化位點。下文段落中關於聚合物分子量之論述普遍適用於圖8中所顯示之具支鏈PEG。在圖8中，下標「n」表示可提供如段落中所論述之具有期望分子量之直鏈(且因此具支鏈)m-PEG的任何整數。在多個實施例中，「n」經選擇以使直鏈m-PEG部分為約20KDa至約40KDa，例如約20KDa、約30KDa或約40KDa。對應於該等m-PEG分子量之整數對應於約400(例如約455)至約900(例如約910)。因此，「n」經選擇以提供為約40KDa至約80KDa(例如，約40KDa、約50KDa、約60KDa、約70KDa、或約80KDa)之具支鏈PEG。
許多其他聚合物亦適用於本發明。非肽及水溶性之聚合物骨架尤其可用於本發明。適宜聚合物之實例包括但不限於其他聚伸烷基二醇，例如聚丙二醇(「PPG」)、乙二醇與丙二醇及諸如此類之共聚物、聚(氧乙烯化多元醇)、聚(烯醇)、聚(乙烯吡咯啶酮)、聚(羥基丙基甲基丙烯醯胺)、聚([α]-羥酸)、聚(乙烯醇)、聚磷腈、聚噁唑啉、聚(N-丙烯醯基嗎啉)(例如闡述於美國專利第5,629,384號中者，該專利之全部內容以引用方式併入本文中)、以及共聚物、三元共聚物、及其混合物。
儘管聚合物骨架之各鏈的分子量可能有所變化，但通常介於約100Da至約160,000Da範圍內，例如，約5,000Da至約100,000Da。更具體而言，本發明各共軛親水性聚合物之大小可自約500Da至約80,000Da不等，例如約1000Da至約80,000Da；約2000Da至約70,000Da；約5000至約70,000Da；約5000至約60,000Da；約10,000至約70,000Da；約20,000至約60,000Da；約30,000至約60,000Da；約30,000至約50,000Da；或約30,000至約40,000Da。應瞭解，該等大小代表估計值而非準確量測值。根據一較佳實施例，本發明分子與親水性聚合物異質群體共軛，例如，大小為例如10,000、40,000、或80,000Da+/-約5000、約4000、約3000、約2000、或約1000Da之PEG。
O-連接寡糖： N-聚糖及O-聚糖二者均可藉由產生蛋白質之細胞附接至蛋白質上。當初生蛋白自核糖體易位至內質網時，細胞N-糖基化器件識別並糖基化胺基酸鏈中之N-糖基化信號(N-X-S/T基元)(Kiely等人，1976；Glabe等人，1980)。
同樣，可將O-聚糖附接至胺基酸鏈中之特定O-糖基化位點上，但觸發O-糖基化之基元較N-糖基化信號更為異質，且吾人預測胺基酸序列中之O-糖基化位點的能力尚不充分(Julenius等人，2004)。因此，人工O-糖基化位點之構造具有一定不確定性。通常假定天然FVIII分子不含有任何O-糖基化位點，且熟習此項技術者因此預期需要構建至少一個人工O-糖基化位點並插入至與實施本發明有關之B結構域中。
因此，將截短型因子VIII B結構域中之O-連接寡糖共價連接至天然存在之O-連接糖基化序列或藉由重組技術人工構建之O-連接糖基化序列。
根據本發明之一較佳實施例，將O-連接寡糖連接至天然存在之O-連接糖基化序列，此O-連接糖基化序列在野生型因子VIII分子中不經受糖基化，但由於B結構域被截短而變得可經O-糖基化。其實例顯示於實例及SEQ ID NO 2中(截短之B結構域對應於胺基酸742-763)。以下情形似乎可能：即使B結構域在稍微不同的地方被截短，即截短之B結構域與SEQ ID NO 2相比稍微較短(例如較SEQ ID NO 2短1、2、3、4、或5個胺基酸)或較長(例如1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、或50個胺基酸)，SEQ ID NO 2中之「隱藏」O-糖基化位點亦變為經糖基化。該方法藉由截短B結構域來激活「隱藏」O-糖基化位點而非產生人工O-糖基化位點，其具有產生具有有利安全性質(即變應原性降低等)之分子的優點。因子VIII B結構域中之其他O-糖基化位點同樣可藉由以不同方式截短分子來激活。
O-連接寡糖之糖聚乙二醇化： 可改變O-聚糖之生物合成並利用在生物合成之相對早期添加唾液酸殘基來終止。某些唾液酸轉移酶能夠作用於GalNAcα-Ser/Thr、或早期O-聚糖核心亞型(在核心1 GalT作用後)。術語T抗原與Galβ1-3GalNAcα-Ser/Thr二糖之存在有關。該等結構之產生涉及糖基轉移酶之間對相同受質之競爭且因此高爾基體內糖基轉移酶之實際表現程度及亞細胞分佈決定O-聚糖生物合成及多樣化之結構結果。如圖1中所示，僅Galβ1-3GalNAcα-Ser/Thr二糖適於糖聚乙二醇化。
然而，通過用唾液酸酶或核心1 GalT或其組合來處理蛋白質可大大增加該結構之可得量。作為糖聚乙二醇化過程之結果，通過α3鍵結至目標蛋白之Galβ1-3GalNAcα-Ser/Thr二糖將唾液酸PEG添加至天然結構中(圖1)。
亦可將其他親水性聚合物附接至O-連接寡糖上。以酶促方式經由O-聚糖使其他親水性聚合物與FVIII共軛之基本要求係能夠如WO03031464中所揭示經由游離胺基使其偶聯至甘胺醯基-唾液酸衍生物。此可通過彼等熟習此項技術者所習知之多種偶聯化學法來達成。具活性生物相容聚合物之實例包括聚氧化烯烴，例如但不限於聚乙二醇(PEG)、2-(甲基丙烯醯氧基)乙基磷醯膽鹼(mPC)聚合物(如WO03062290中所述)、右旋糖苷、多聚乙醯神經胺酸或其他基於碳水化合物之聚合物、胺基酸或特定肽序列之聚合物、生物素衍生物、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯啶酮、聚乙烯-共-馬來酸酐、聚苯乙烯-共-蘋果酸酐、聚噁唑啉、聚丙烯醯基嗎啉、肝素、白蛋白、纖維素、殼聚糖水解產物、諸如羥基乙基澱粉及羥基丙基澱粉等澱粉、糖原、瓊脂糖及其衍生物、瓜爾豆膠、支鏈澱粉、菊糖、黃原膠、角叉菜膠、果膠、海藻酸水解產物、其他生物聚合物及其任何等效物。
醫藥組合物： 醫藥組合物在本文中較佳意欲涵蓋包含本發明因子VIIII分子之適於非經腸投與的組合物，例如即用型無菌水性組合物或可於例如水或水性緩衝液中重構之乾燥無菌組合物。本發明組合物可包含多種醫藥上可接受之賦形劑、穩定劑等。
該等組合物中之額外成份可包括潤濕劑、乳化劑、抗氧化劑、增積劑、張力調節劑、螯合劑、金屬離子、油質媒劑、蛋白質(例如，人類血清白蛋白、明膠或蛋白質)及兩性離子(例如，胺基酸，例如甜菜鹼、牛磺酸、精胺酸、甘胺酸、離胺酸及組胺酸)。當然，該等額外成份不應不利地影響本發明醫藥調配物之總體穩定性。非經腸投與可藉由藉助注射器(視情況鋼筆型注射器)皮下、肌內、腹膜內或靜脈內注射來實施。或者，非經腸投與可藉助輸注幫浦來實施。另一選擇係組合物可為適於呈鼻或肺噴霧形式投與FVIII化合物之溶液或懸浮液。作為又一選擇，含有本發明FVIII化合物之醫藥組合物亦可適於經皮投與(例如藉由無針注射或貼片，視情況為離子導入貼片)、或經黏膜(例如含服)投與。
因此，在第一態樣中，本發明係關於具有改良之循環半衰期的B結構域截短型因子VIII分子，該分子經由截短之B結構域中之O-連接寡糖與親水性聚合物共價共軛，其中因子VIII激活(分子激活)導致共價共軛之親水性聚合物移除。
根據一個實施例，親水性聚合物係PEG。PEG聚合物之大小可自10,000至約160,000Da不等；例如10,000至80,000Da，例如約10,000；15,000；20,000；25,000；30,000；35,000；40,000；45,000；50,000；55,000；60,000；65,000；70,000；75,000；或80,000Da。較佳地，將O-連接寡糖附接至藉由截短B結構域而非藉由插入未在wt FVIII分子中發現之人工O-糖基化位點而產生之O-糖基化位點上。
根據一尤佳實施例，本發明分子包含如SEQ ID NO 2中所示之胺基酸序列。該等分子具有激活之FVIII分子與天然活性FVIII分子相同的特徵。該特徵在安全評價中似乎具有有利特性。
本發明亦係關於包含本發明分子之醫藥組合物。
本發明進一步係關於獲得本發明分子之方法，其中該方法包含使B結構域截短型因子VIII分子與諸如PEG基團等親水性聚合物經由截短之B結構域中之O-連接寡糖共軛。因此，本發明亦係關於藉由該等方法獲得或可藉由該等方法獲得之分子。
在另一態樣中，本發明係關於治療血友病性疾病之方法，其包含向有需要之患者投與治療有效量之本發明分子。
本文所用之術語「治療」係指對有需要之任何人類或其他動物個體實施醫學治療。期望該個體已由從業醫師實施身體檢查，該從業醫師給出嘗試性或確定性診斷，該診斷指出使用該特定治療有益於該人類或其他動物個體之健康。該治療之時間選擇及目的可根據個體體質現狀因人而異。因此，該治療可為預防治療、姑息治療、對症治療及/或治癒性治療。
在再一態樣中，本發明係關於本發明分子作為藥劑之用途以及本發明分子用以製造用於治療血友病之藥劑的用途。
在最後態樣中，本發明係關於構造本發明之B結構域截短型因子VIII分子之方法，該方法包含：(i)截短B結構域並視情況對該截短型因子VIII分子之胺基酸序列實施分析以識別潛在的O-連接糖基化位點；(ii)在適宜宿主細胞中產生該分子；及(iii)選拔在截短之B結構域中具有O-連接聚糖之分子。
實例 實例1 產生重組B結構域截短型O-糖基化因子VIII
在SEQ ID NO 2中給出B結構域缺失型因子VIII分子之胺基酸序列的實例。此多肽亦可稱為「N8」。該分子包含21胺基酸殘基連接體序列(SFSQNSRHPS QNPPVLKRHQR-加以下劃線之S 係絲胺酸殘基，其具有在實例2中經聚乙二醇化之O-聚糖)。
本發明之因子VIII分子在實例中可以多種方式提及-但因子VIII分子之所有提及皆係指本發明之因子VIII分子、或另一選擇為在轉化成本發明因子VIII分子過程中之因子VIII分子。
細胞系及培養方法：
使用因子VIII cDNA來構建編碼具有如SEQ ID NO 2中所示胺基酸序列之B結構域缺失型因子VIII的哺乳動物表現質粒。該質粒編碼包含全長人類因子VIII之胺基酸1-740的因子VIII重鏈及包含全長人類因子VIII之胺基酸1649-2332的因子VIII輕鏈。藉由21胺基酸連接體將該等重鏈及輕鏈序列與全長人類因子VIII之胺基酸741-750及1638-1648序列連接。將中國倉鼠卵巢(CHO)細胞用BDD因子VIII編碼質粒轉染並利用二氫葉酸還原酶系統進行選拔，最終產生在無動物組份培養基中培養之純系懸浮液生產細胞。
此方法之第一步驟係接種細胞小瓶，自工作細胞庫小瓶至化學限定及無動物組份生長培養基中。解凍後，先將細胞在T-燒瓶中進行培育。解凍後一天或兩天，將細胞轉移至搖瓶中，並藉由連續稀釋擴展培養物體積以使細胞密度保持在0.2-3.0×106 個細胞/ml之間。下一步驟為將搖瓶培養物轉移至種子生物反應器中。此時再次擴展培養物體積，隨後最終轉移至生產生物反應器中。所有接種物擴展步驟皆使用相同的化學限定及無動物組份培養基。在轉移至生產生物反應器中之後，向培養基中補充能夠增大產物濃度之組份。在生產生物反應器中，以重複分批方法以三天為週期培養細胞。收穫時，將80-90%培養物體積轉移至收穫槽中。隨後用新鮮培養基稀釋剩餘培養液以達到初始細胞密度，並隨後開始新的生長期。
藉由離心及過濾來淨化收穫批料並轉移至儲存槽中，隨後開始純化過程。將緩衝液添加至儲存槽中之游離細胞收穫物中以穩定pH值。
到生產運行結束時，收集細胞並冷凍以終了生產細胞庫。測試該細胞庫之黴漿菌、無菌度及病毒污染。純化：
使用包括以下之四步驟純化程序，自細胞培養基分離B結構域缺失型因子VIII：在Capto MMC管柱上之濃縮步驟、免疫吸附層析步驟、陰離子交換層析及最後的凝膠過濾步驟。通常使用以下程序：將11公升無菌過濾培養基抽吸至Capto MMC管柱(GE Healthcare,Sweden)(1.6×12cm)上，該管柱已經過15ml/min流速之緩衝液A(20mM咪唑、10mM CaCl2 、50mM NaCl、0.02% Tween 80，pH=7.5)平衡。將管柱用75ml緩衝液A洗滌，隨後用含有1.5M NaCl之75ml緩衝液A洗滌。用20mM咪唑、10mM CaCl2 、0.02% Tween 80、2.5M NaCl、8M乙二醇(pH=7.5)以1ml/min流速來溶離蛋白質。收集8ml溶離份並分析其因子VIII活性(CoA-測試)。集中含有因子VIII之溶離份且通常獲得約50ml之集合體積。
已研發出對抗因子VIII之單株抗體(Kjalke Eur J Biochem 234 773)。已由抗原決定基之圖譜分析(結果未顯示)發現，此抗體F25能從胺基酸殘基725至740中識別出重鏈的之遠C-端序列。基本上如製造商所述將此F25抗體以2.4mg/ml凝膠之密度偶聯至NHS激活瓊脂糖4FF(GE Healthcare,Bio-Sciences AB,Uppsala,Sweden)上。將來自先前步驟之集合液用20mM咪唑、10mM CaCl2 、0.02% Tween 80(pH=7.3)稀釋10倍並施加至F25瓊脂糖管柱(1.6×9.5cm)上，該管柱已用0.5ml/min流速之20mM咪唑、10mM CaCl2 、150mM NaCl、0.02% Tween 80、1M甘油(pH=7.3)平衡。用平衡緩衝液洗滌管柱，直至UV信號穩定，並隨後用20mM咪唑、10mM CaCl2 、0.65M NaCl(pH=7.3)洗滌管柱，直至UV信號再次穩定。用20mM咪唑、10mM CaCl2 、0.02% Tween 80、2.5M NaCl、50%乙二醇(pH=7.3)以1ml/min流速來溶離因子VIII。收集1ml溶離份並分析其因子VIII活性(CoA-測試)。集中含有因子VIII之溶離份且通常獲得約25ml之集合體積。
製備供離子交換步驟用之緩衝液A(20mM咪唑、10mM CaCl2 、0.02% Tween 80、1M甘油，pH=7.3)及緩衝液B(20mM咪唑、10mM CaCl2 、0.02% Tween 80、1M甘油、1M NaCl，pH=7.3)。用85%緩衝液A/15%緩衝液B以2ml/min流速平衡Macro-Prep 25Q Support管柱(1×10cm)(Bio-Rad Laboratories,Hercules,CA,USA)。將來自先前步驟之集合液用緩衝液A稀釋10倍，並以2ml/min流速抽吸至管柱上。用85%緩衝液A/15%緩衝液B以2ml/min流速洗滌管柱並用自15%緩衝液B至70%緩衝液B之線性梯度，以2ml/min流速經120ml來溶離因子VIII。收集2ml溶離份並分析其因子VIII活性(CoA-測試)。集中含有因子VIII之溶離份且通常獲得約36ml之集合液體積。
將來自先前步驟之集合液施加至Superdex 200製備級(GE Healthcare,Bio-Sciences AB,Uppsala,Sweden)管柱(2.6×60cm)中，用20mM咪唑、10mM CaCl2 、0.02% Tween 80、1M甘油、150mM NaCl(pH=7.3)以1ml/min平衡及溶離該管柱。收集3ml溶離份並分析其因子VIII活性(CoA-測試)。集中含有因子VIII之溶離份且通常獲得約57ml之集合液體積。將含有因子VIII之集合液於-80℃下儲存。
藉由CoA活性及ELISA量測所判斷，使用上述四步驟純化程序獲得約15%之總產率。
用於製造N8之細胞系係重組中國倉鼠卵巢(CHO)細胞系，其經過由pTSV7表現載體與含有編碼F8-500蛋白質之cDNA的插入物組成之pTSV7中的表現質粒#814 F8-500穩定轉染。「N8」在本文中意欲對應於具有如SEQ ID NO 2中所列示之胺基酸序列的蛋白質。自N-端開始，F8-500蛋白質(N8)由FVIII信號肽(胺基酸-19至-1)、繼之無B結構域之FVIII重鏈(胺基酸1-740)、21胺基酸連接體(SFSQNSRHPSQNPPVLKRHQR)、及FVIII輕鏈(野生型人類FVIII之胺基酸1649-2332)組成。21胺基酸連接體序列係衍生自FVIII B結構域且由全長FVIII之胺基酸741-750及1638-1648組成。
用pTSV7中之814 F8-500轉染CHO細胞並利用二氫葉酸還原酶系統進行選拔，最終產生在無動物組份培養基中培養之純系懸浮液生產細胞。藉由解凍工作細胞庫小瓶並在轉移至生產生物反應器中之前擴展細胞來開始生產運行。所有接種物擴展步驟皆使用相同的化學限定及無動物組份培養基。在轉移至生產生物反應器中之後，向培養基中補充能夠增大產物濃度之組份。在生產生物反應器中，以重複分批方法以三天為週期培養細胞。收穫時，將80-90%培養物體積轉移至收穫槽中。隨後用新鮮培養基稀釋剩餘培養液以達到初始細胞密度，並隨後開始新的生長期。藉由離心及過濾來淨化收穫批料並將其轉移至儲存槽中，隨後開始純化適程。將緩衝液添加至儲存槽中之游離細胞收穫物中以穩定pH值。
實例2 重組B結構域截短型及O-糖基化之因子VIII之聚乙二醇化：
利用以下程序使實例1中獲得之重組因子VIII分子與聚乙二醇(PEG)共軛。
為使實例1中獲得之重組因子VIII分子有效地糖聚乙二醇化，大於5mg/ml之FVIII濃度較佳。由於FVIII在此濃度下通常不溶解，故對所選緩衝液組合物實施篩選(一些該等結果參見表1)。
基於該等考慮，吾人發現含有50mM MES、50mM CaCl2 、150mM NaCl、20%甘油(pH 6.0)之緩衝液係適宜反應緩衝液。
將經如上所述純化之重組FVIII在反應緩衝液中藉由在Poros 50 HQ管柱上利用分步溶離、在Sartorius Vivaspin(PES)過濾器上(以10kDa為截斷值)或在Amicon 10kDa MWCO PES過濾器上實施離子交換來濃縮至6-10mg/mL濃度。藉由將因子VIII(BDD)(最終約4.7mg/mL)與唾液酸酶(產脲節桿菌(A. ureafaciens))(159mU/mL)、CMP-SA-甘油-PEG-40kDa(5mol.eq.)及MBP-ST3Gal1(540mU)混合於反應緩衝液(50mM MES、50mM CaCl2 、150mM NaCl、20%甘油、0.5mM抗蛋白酶素，pH 6.0)中來開始對FVIII實施糖聚乙二醇化。在32℃下培育反應混合物，直至總轉化產率為約20-30%。
培育後，將樣品用緩衝液A(25mM Tris、5mM CaCl2 、20mM NaCl、20%甘油，pH 7.5)稀釋並施加至Source 15Q管柱(1cm內徑x 6cm，4.7mL，1mL/min，280nm)上。將結合物質用緩衝液A洗滌並使用分步梯度用緩衝液B(25mM Tris、5mM CaCl2 、1M NaCl、20%甘油，pH 7.5)實施溶離。在約25%緩衝液B下自管柱中溶離出經糖聚乙二醇化之因子VIII-(O)-SA-甘油-PEG-40kDa。圖2顯示反應混合物在Source 15Q上之離子交換層析。
為封端唾液酸酶處理期間暴露於N-聚糖之游離半乳糖部分，將因子VIII-SA-甘油-PEG-40kDa(最終為1.0mg/mL)之集合溶離份與CMP-SA(2,000mol eq)及MBP-SBD-ST3Gal3(400mU/mL)混合於反應緩衝液(50mM MES、20mM CaCl2 、150mM NaCl、10mM MnCl2 、20%甘油，pH 6.0)中並在32℃下培育11小時。
在Superdex 200管柱(10cm內徑×300mm；280nm)上藉由凝膠過濾將所得封端之經糖聚乙二醇化因子VIII-SA-甘油-PEG-40kDa與CMP-SA及ST3GalIII分開，該管柱已用50mM MES、50mM CaCl2 、150mM NaCl、10%甘油(pH 6.0)以0.25mL/min流速平衡。產物因子VIII-SA-甘油-PEG-40kDa在38min時溶離出來。圖3顯示使用Superdex 200尺寸排除層析對封端產物實施純化。收集峰溶離份，等分並隨後實施分析。
封端程序之目的係降低共軛因子VIII分子之活體內清除率。
實例3 在產色FVIII活性分析中O-聚糖聚乙二醇化rFVIII之活性：
在產色FVIII分析中使用Coatest SP試劑(Chromogenix)如下對實例2中獲得之O-糖聚乙二醇化rFVIII的活性實施評價：將rFVIII樣品及校準物(來自NIBSC之第7國際FVIII標準物)稀釋於Coatest分析緩衝液(50mM Tris、150mM NaCl、1% BSA，pH 7.3，含有防腐劑)中。將50μl樣品、標準物及緩衝液陰性對照一式兩份添加至96孔微量滴定板(Nunc)中。將來自Coatest SP套組之因子IXa/因子X試劑、磷脂試劑及CaCl2 以5:1:3(vol:vol:vol)混合並將75μl此物質添加至孔中。在於室溫下培育15min後，添加50μl因子Xa受質S-2765/凝血酶抑制劑I-2581混合物並在室溫下將反應物培育10min，隨後添加25μl 1M檸檬酸(pH 3)。在Spectramax微量滴定板讀數器(Molecular Devices)上使用620nm作為參考波長量測415nm處之吸光度。減去所有樣品之陰性對照值並藉由吸光度值與FVIII濃度之圖形之線性回歸繪製校準曲線。比活性係藉由將樣品活性除以蛋白質濃度來計算，該蛋白質濃度係藉由尺寸排除HPLC並積分HPLC層析譜中之輕鏈峰(即不包括PEG部分)來測定。表2中之數據顯示，O-糖聚乙二醇化rFVIII化合物之比產色活性得以保持，意味著聚乙二醇化變體中似乎保留因子VIII活性。
表2.具有不同PEG基團大小之O-糖聚乙二醇化rFVIII的比產色活性
圓括號中所注明之數據係單獨測定數值之平均偏差及標準偏差。
實例4 在FVIII凝固活性分析中O-聚糖聚乙二醇化rFVIII之活性
在FVIII凝固分析中進一步評價O-糖聚乙二醇化rFVIII之活性。將rFVIII樣品稀釋於HBS/BSA(20mM hepes、150mM NaCl，pH 7.4，含有1% BSA)中至約10U/ml，隨後10倍稀釋於含有VWF(Dade Behring)之FVIII缺乏血漿中。隨後將樣品及校準用血漿標準物(HemosIL校準血漿，來自Instrumentation Laboratory)稀釋於HBS/BSA中以產生四種(樣品)或六種(校準物)不同濃度。在ACL9000儀器(Instrumentation laboratory)上使用單因子程式來量測凝固時間，其中將樣品/標準物與等體積的含有VWF(Dade Behring)、鈣及aPTT試劑之FVIII缺乏血漿混合，並量測凝固時間。使用以下試劑：Synthasil(HemosIL,Instrumentation Laboratory)、肌動蛋白FS(激活部分促凝血酶原激酶時間(Activated PTT)試劑，Dade Behring)、Stago(STAPTT-A,Stago)、及dAPPTin(DAPPTINTC,Technoclone)。樣品活性係基於凝固時間對校準物濃度之半對數圖來計算。
端視PEG大小及所用aPTT試劑，O-糖聚乙二醇化rFVIII化合物(分別為對照、10、40及80kDA PEG)之凝固活性(圖4)有不同程度的降低。使用Synthasil或dAPPTin作為aPTT試劑導致凝固活性隨PEG大小逐漸降低。使用Stago's aPTT試劑，對於所評價之所有三種O-糖聚乙二醇化N8化合物均觀察到比凝固活性降低50%。當使用肌動蛋白FS作為aPTT試劑時，比凝固活性保持在10,000IU/mg左右。數據表明，aPTT分析受存在之PEG部分影響，然而，使用選擇之aPTT試劑(例如肌動蛋白FS)O-糖聚乙二醇化時rFVIII之比凝固活性並未減弱。
實例5：
rFVIII之O-連接聚乙二醇化對輔因子活性及FVIII激活速率的影響
將激活之FVIII納入至FIXa-FVIIIa複合物中會使FIXa催化之FX激活的催化效率提高五個數量級(van Dieijen等人，(1981)J Biol Chem 256:3433)且FIXa-FVIIIa複合物組裝及FX激活動力學之表徵係FVIIIa分子之功能完整性之靈敏量度。藉由在磷脂及凝血酶激活之rFVIII或PEG-rFVIII存在下測定FIXa催化之FX激活的動力學參數來表徵凝血酶激活之rFVIII或PEG-rFVIII的輔因子活性。利用FVIIIa活性分析(FIXa-輔因子活性分析)，對固定濃度(0.1nM)之rFVIIIa或FIXa分別實施FIXa及FVIIIa之相互滴定以獲得FIXa對於rFVIIIa之表觀親和常數()及功能FVIIIa濃度。FX激活之米氏常數(Michaelis constant)(km )及轉換數(kcat )係自針對固定濃度之FIXa-FVIIIa複合物滴定FX來獲得。
如下實施FIXa-輔因子活性分析：藉由在37℃下將rFVIII(通常為0.7nM，1U/mL)與5nM人類α-凝血酶一起培育恰好30秒來製備用於每一測試之新鮮的凝血酶激活之rFVIII及PEG-rFVIII變體。隨後，藉由將上文激活反應物二次取樣至FIXa、磷脂囊泡(磷脂TGT，來自Rossix[,Sweden])、水蛭素、廣譜蛋白酶抑制劑(Pefabloc)Xa及CaCl2 之製備好的混合物中來定量FX激活速率；藉由添加FX並容許在37℃下進行30秒或60秒來開始FX激活。藉由將FX激活反應物稀釋至含有EDTA之冰冷緩衝液中來終止激活。使用FXa特異性產色受質藉由在ELISA讀數器中讀取405nM處之吸光度來定量FXa濃度。利用使用經純化FXa製得之參考曲線將吸光度轉化成FXa濃度。使用自激活之rFVIII或PEG-rFVIII變體組裝之FIXa-rFVIIIa複合物的轉換數將FX激活速率轉化成rFVIIIa濃度。
凝血酶催化之rFVIII激活速率係藉由在含有0.7nM rFVIII或PEG-rFVIII及0.13nM人類α-凝血酶之混合物中定量rFVIIIa之初始(0-3min)形成來量測。FVIIIa之形成與時間成線性關係。將FVIIIa激活速率表示為每分鐘每莫耳最初存在之rFVIII所形成rFVIIIa之莫耳數(v/[rFVIII]0 )。
rFVIII之O-連接糖聚乙二醇化不影響於激活之rFVIII存在下凝血酶催化之rFVIII激活速率或FIXa催化之FX激活的km 或kcat (參見表3)。此外，O-連接糖聚乙二醇化不影響rFVIIIa-FIXa相互作用之表觀Kd ()。
圖4顯示使用多種aPTT試劑之O-糖聚乙二醇化rFVIII的凝固活性。數據以凝固活性與產色活性之比(A)或以比凝固活性(B)顯示。顯示來自三項獨立實驗之值的平均值及標準偏差。
數據係3-6次量測之平均值及標準偏差。
實例6
糖聚乙二醇化B結構域缺失型(BDD)-FVIII在FVIII KO小鼠及vWF KO小鼠中之藥物代謝動力學
在靜脈內投與280IU/kg至FVIII KO小鼠中之後對具有多種PEG大小之糖聚乙二醇化BDD-FVIII的藥物代謝動力學實施研究。
研究以下化合物：BDD-FVIII、BDD-FVIII-10K PEG(O-聚糖，0129-0000-1005)、BDD-FVIII-40K PEG(O-聚糖，0129-0000-1003)、BDD-FVIII-2x40K PEG(O及N-聚糖0129-0000-1008-1A)、BDD-FVIII-80K PEG(N-聚糖，0129-0000-1012，O-聚糖0129-0000-1009)。動物研究設計：
基於C57Bl/6外顯子16基因剔除之背景，在Taconic M&B飼養因子VIII基因剔除(FVIII KO)小鼠。以約1:1之比例使用重量大約為25g及19-26週齡之雄性與雌性小鼠。小鼠未完全回交。在該小鼠品系中未檢測到FVIII。
在尾靜脈中給小鼠單次靜脈內注射280IU/kg上文所列示化合物。若小鼠係經靜脈外圍投藥，則用另一隻小鼠替換該小鼠。投藥後，自投藥前直至投藥後64小時使用未經塗敷之玻璃毛細管採集眼血管叢血液樣品。自每隻小鼠取三份樣品，且在每一時間點採集2、3或4份樣品。將血液在檸檬酸鈉(9:1)中穩定化並稀釋於FVIII COA SP緩衝液(1:4)中，隨後在4000g下離心5分鐘。將自經稀釋血液獲得之血漿在乾冰中於保持-80℃下冷凍，隨後藉助FVIII產色活性及/或FVIII抗原分析實施定量分析。
定量血漿分析：
藉由使用來自Coatest SP套組(Chromogenix)之試劑來測定FVIII產色活性。將經稀釋血漿樣品、存於Coatest SP緩衝液中之校準物(ILS校準血漿)、及緩衝液陰性對照(50μl)一式兩份添加至96孔微量滴定板(Nunc)中。將來自Coatest SP套組之因子IXa/因子X試劑、磷脂試劑及CaCl2 以5:1:3(vol:vol:vol)混合並將75μl此物質添加至孔中。在於室溫下培育15min後，添加50μl因子Xa受質S-2765/凝血酶抑制劑I-2581混合物並在室溫下將反應物培育10min，隨後添加25μl 2%檸檬酸。在Spectramax微量滴定板讀數器(Molecular Devices)上量測405nm處之吸光度。自藉由校準用國際血漿標準物(ILS)稀釋物製得之校準曲線來計算血漿樣品中之FVIII活性。
FVIII抗原分析係自Diagnostica Stago(Asserachrom VIII:CAg)購得之ELISA套組，其使用兩種針對人類FVIII之輕鏈的單株抗體。在藉由該套組提供之coatest SP稀釋緩衝液中將校準物(化合物之稀釋物)或血漿樣品稀釋至少50倍，將其施加至預塗覆孔中並按照製造商說明書實施ELISA。用於報告藥物代謝動力學研究之值係基於自化合物本身製得之標準曲線。
藥物代謝動力學參數估計：
藥物代謝動力學分析係藉由數據之非分域方法(NCA)使用ILS作為校準物(數據基於產色活性)、使用化合物本身作為校準物(數據基於ELISA)來實施。自數據估計以下參數：Cmax(在於第一取樣時間點靜脈內投與後之最大濃度)、Tmax(在於第一時間點靜脈內投與後之最大濃度時間)、AUC0-∞(自0時間至無窮之曲線下面積)、(終末半衰期)、CL(清除率)及Vss(穩態分佈容積)。所有計算皆係使用WinNonlin Pro 4.1版本實施。
在靜脈內注射280IU/Kg BDD-FVIII、BDD-FVIII-10KDa PEG、BDD-FVIII-40KDa PEG、BDD-FVIII-2x40KDa PEG及BDD-FVIII-80KDa PEG至FVIII KO小鼠中後，隨PEG大小增加半衰期延長至7.8h(BDD-FVIII)至15-16h範圍內(表4)，此對應於2倍增加。類似地，隨PEG大小增加，清除率降低且MRT增加(表4)。
表4：在靜脈內投與至FVIII KO小鼠中後基於產色活性具有不同大小PEG之糖聚乙二醇化FVIII的藥物代謝動力學參數估計(BDD：B結構域缺失)。
結論：
在靜脈內投與280IU/kg至FVIII KO小鼠中之後，與BDD-FVIII相比，BDD-FVIII之糖聚乙二醇化使延長1.3-2.1倍。隨PEG基團大小在10KDa至80KDa PEG範圍內增加，吾人觀察到延長。
實例7 在FeCl3 誘導之損傷模型中於A型血友病小鼠中與艾菲特(Advate)相比40K-PEG-[O]-N8之延長止血效果
在FeCl3 誘導之損傷模型中於A型血友病(F8-KO)小鼠中研究40K-PEG-[O]-N8相對於重組FVIII(艾菲特)之作用持續時間。
將小鼠麻醉並置於加熱墊(37℃)上以保持體溫。暴露頸動脈並藉由超音將量測血液流量之流量探針(0.5PSB奈米探針)置於動脈周圍。藉由將短暫浸於10% FeCl3 溶液中之濾紙(2×5mm)放置於暴露之頸動脈周圍來誘導損傷(鐵調介之化學氧化)。3min後移除濾紙。隨後將動脈用0.9% NaCl洗滌三次並最後施加消毒用外科潤滑劑(Surgilube)(聲音耦合劑)以排除流量探針中之空氣並確保血液流量之量測最佳化。在移除FeCl3 飽和濾紙後記錄血液流量(ml/min)達25min並藉由量測自移除FeCl3 飽和濾紙直至血液流量為0ml/min之時間(min)來測定阻塞時間。若在25min後未發生阻塞，則阻塞時間報告為25min，即使在觀察期間未發生阻塞。用艾菲特(280U/kg)、40K-PEG-[O]-N8(280U/kg)、或媒劑對F8-KO小鼠(n=6-10)實施治療。在投藥後5min(急性效應)或24、48、60、及72小時用FeCl3 誘導損傷。在移除FeCl3 後記錄血液流量(ml/min)達25min，並隨後測定阻塞時間。
在經媒劑治療之F8-KO小鼠中未發生阻塞，而在經40KDa-PEG-[O]-N8及艾菲特治療之所有小鼠中皆於投藥後5min(急性效應)發生阻塞，平均阻塞時間分別為4.3±0.4min及5.2±0.7min。在經40KDa-PEG-[O]-N8治療之F8-KO小鼠中，在投藥後72小時平均阻塞時間延長至13.8±3.4min。相比之下，經艾菲特治療之F8-KO小鼠在24及48小時後分別具有13.0±3.4min及15.9±2.9min之阻塞時間。重要的是，在投與艾菲特60及72小時未觀察到阻塞。在經40KDa-PEG-[O]-N8治療之所有小鼠中，在投藥後24小時觀察到阻塞，而經艾菲特治療之小鼠僅67%發生阻塞。在72小時後，在經40KDa-PEG-[O]-N8治療之小鼠中63%仍然可以看到阻塞，而在投與艾菲特後60及72小時未觀察到阻塞。
40KDa-PEG-[O]-N8於F8-KO小鼠中之延長效果。
在投藥280IU/kg 40KDa-PEG-[O]-N8、280IU/kg艾菲特、或媒劑後5min(急性效應)、24、48、60、及72小時用FeCl3 誘導損傷。在移除FeCl3 後記錄血液流量(ml/min)達25min，並隨後測定阻塞時間。在投藥後60及72小時，在投藥艾菲特之小鼠中未發生阻塞。顯示每一群組6-10隻小鼠之平均值及SEM。利用Kruskal-Wallis檢驗(包括Dunn事後檢驗(Dunn's post test))來比較不同組間之阻塞時間。*：p<0.05；**：p<0.01。
總之，在FeCl3 誘導之損傷模型中於F8-KO小鼠中與艾菲特相比40KDa-PEG-[O]-N8之止血效果顯著延長。
在第一態樣中，本發明係關於具有改良之循環半衰期的B結構域截短型因子VIII分子，該分子經由截短之B結構域中之O-連接寡糖與親水性聚合物共價共軛，其中因子VIII激活導致共價共軛之側基移除。
在其他態樣中，本發明進一步係關於獲得該等分子之方法、該等分子之用途及包含該等分子之醫藥組合物。
因此，本文提供具有改良之循環半衰期之共軛因子VIII分子，其中共軛側基(例如親水性聚合物)在激活後移除。本發明分子較佳具有同質結構-至少對於親水性聚合物在截短之B結構域中的位置而言-且較佳具有有利的安全性質。另外，本文進一步提供獲得該等分子之相對簡單方法。較佳地，本發明之激活之因子VIII分子與內源性激活之因子VIII類似。
在圖中，共軛基團之大小有時稱為「K」，在本文中其與KDa(千道爾頓)具有相同含義。
圖1： O-連接寡糖之糖聚乙二醇化過程的示意圖。此圖並不代表達成實例中所獲得產物之可能方式的詳盡無遺列示。
圖2： 反應混合物在Source 15Q上之離子交換層析(A)。所收集溶離份之分子標記(左)的SDS-PAGE(B)。
圖3： 在superdex 200尺寸排除層析上對封端產物實施純化。
圖4： 使用多種aPTT試劑之O-糖聚乙二醇化rFVIII的凝固活性。(A)顯示凝固活性與產色活性之比。(B)顯示比凝固活性。
圖5： 40K-PEG-[O]-N8在FVIII KO小鼠中之活體內效果(阻塞時間)。
圖6： 顯示根據本發明產生糖聚乙二醇化因子FVIII所涉及製程步驟之流程圖。
圖7： 實例中產生之本發明之因子VIII分子的示意圖。
圖8： 顯示可用於本發明實施例中之代表性具支鏈PEG聚合物，其在本文中稱為「SA-甘油-PEG」。
(無元件符號說明)

Claims (15)

  1. 一種具有改良之循環半衰期的B結構域截短型因子VIII分子，該分子經由該截短之B結構域中之O-連接寡糖與親水性聚合物共價共軛，其中因子VIII之激活導致該共價共軛親水性聚合物之移除，其中該截短之B結構域的長度為10-700個胺基酸。
  2. 如請求項1之分子，其中該O-連接寡糖附接至藉由截短該B結構域而產生之O-糖基化位點上。
  3. 如請求項1之分子，其中該親水性聚合物係PEG。
  4. 如請求項3之分子，其中該PEG之大小係約10,000至約160,000Da。
  5. 如請求項4之分子，其中該PEG之大小係約40,000Da。
  6. 如請求項1至5中任一項之分子，其包含如SEQ ID NO 2中所示之胺基酸序列。
  7. 一種具有改良之循環半衰期的B結構域截短型因子VIII分子，該分子經由該截短之B結構域中之O-連接寡糖與親水性聚合物共價共軛，其中：(i)因子VIII之激活導致該共價共軛親水性聚合物之移除；以及(ii)因子VIII前體多肽之重鏈及輕鏈部分藉由連接體分開，其中連接體序列係衍生自因子VIII B結構域。
  8. 如請求項7之分子，其中該B結構域的長度為20-30個胺基酸。
  9. 一種具有改良之循環半衰期的因子VIII分子，其包含由 SEQ ID NO：2之胺基酸741-760所組成之截短型B結構域，其中該因子VIII分子經由對應於SEQ ID NO：2位置750之絲胺酸殘基上的O-連接寡糖與親水性聚合物共價共軛，其中親水性聚合物的大小係約40,000Da。
  10. 如請求項9之分子，其係由包含下列步驟之方法製得：a)以編碼請求項9的因子VIII分子之載體轉染哺乳動物宿主細胞；b)在適合於宿主細胞中表現因子VIII分子的條件下培養步驟a)之宿主細胞；c)自步驟b)之宿主細胞培養物收穫因子VIII分子；及d)經由對應於SEQ ID NO：2位置750之絲胺酸殘基上的O-連接寡糖將因子VIII分子與親水性聚合物共價共軛，其中親水性聚合物的大小係約40,000Da。
  11. 一種醫藥組合物，其包含如請求項1至10中任一項之分子。
  12. 一種製備如請求項1至10中任一項之分子的方法，其中該方法包括使B結構域截短型因子VIII分子與親水性聚合物經由該截短之B結構域中之O-連接寡糖共軛。
  13. 一種可藉由如請求項12之方法獲得之分子。
  14. 一種如請求項1至10中任一項之分子的用途，其係用以製造用於治療血友病之藥劑。
  15. 一種構成如請求項1至10中任一項之B結構域截短型因子VIII分子的方法，該方法包括(i)截短該B結構域並視情況分析該截短型因子VIII分子之胺基酸序列，以識別潛 在的O-連接糖基化位點；(ii)在適宜宿主細胞中產生該分子；及(iii)選拔在該截短之B結構域中具有O-連接聚糖之分子。
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