CN101965200B - 缀合的因子ⅷ分子 - Google Patents
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Abstract
本发明涉及具有改变的循环半衰期的B-结构域截短的因子VIII分子,所述分子与亲水聚合物共价缀合。本发明此外涉及用于得到这种分子的方法以及这种分子的用途。
Description
发明领域
本发明涉及缀合的凝固因子VIII分子。具体地,本发明涉及具有改变的循环半衰期的缀合的因子VIII分子。
发明背景
A型血友病是由凝固因子VIII(FVIII)活性的缺乏或功能障碍造成的一种遗传性出血障碍。临床表现不是在初级止血-正常形成血凝块-而是由于缺乏次级凝血酶形成所导致的凝块不稳定。通过静脉内注射从血液分离的或重组生产的凝固因子FVIII,治疗该疾病。
流行的治疗推荐正在从传统的在要求时(on-demand)治疗转向预防。内源的FVIII的循环半衰期是12-14小时,因而每周需要进行几次预防性治疗,以使患者得到实际上无症状的生活。对于许多人,尤其是儿童和年轻人,静脉内施用伴有显著的不便和/或疼痛。因而本领域需要新颖的具有因子VIII活性的因子VIII产品,它们优选地在结构上是同质的,优选地是安全的,且优选地具有显著延长的循环半衰期,以减少每周施用因子VIII的次数。此外,本领域需要相对简单的得到和生产这种分子的方法。
本领域已知,为了延长循环半衰期,向因子VIII加入聚乙二醇(PEGylation)。但是,这是得到具有同质结构以及显著提高的循环半衰期的安全产品的障碍。可得到的生产缀合的因子VIII分子的方法经常是费力的,和/或倾向于导致低得率和/或在结构上不同质的产品。在WO2008011633中已经暗示了人工改造的O-联糖基化位点用于得到治疗蛋白的应用,所述治疗蛋白具有延长的治疗蛋白循环半衰期,但是,其中没有公开缀合的因子VIII分子。
发明概述
在第一个方面,本发明涉及具有改变的循环半衰期的B结构域截短的因子VIII分子,所述分子在截短的B结构域中通过O-联寡糖与亲水聚合物共价缀合,其中因子VIII激活导致共价缀合的侧基的去除,且FVIII前体多肽的重链和轻链部分被接头分开,其中所述接头的序列源自FVIII B结构域。
在其它方面,本发明另外涉及用于得到这种分子的方法,这种分子的用途和包含这种分子的药物组合物。
因而,提供了具有改变的循环半衰期的缀合的因子VIII分子,其中缀合的侧基(例如亲水聚合物)在激活后去除。根据本发明的分子优选地在结构上-至少在亲水聚合物在截短的B结构域中的位置这方面-是同质的,且优选地具有有利的安全概况。同样,本文另外提供了相对简单的用于得到这种分子的方法。优选地,根据本发明的激活的因子VIII分子类似于内源激活的因子VIII。
发明详述
定义:
因子VIII分子:FVIII/因子VIII是一种主要由肝细胞产生的大的、复杂的糖蛋白。FVIII由2351个氨基酸组成,包括信号肽,且含有几个根据同源性定义的不同结构域。存在3个A-结构域,1个独特的B-结构域和2个C-结构域。结构域次序可以列成NH2-A1-A2-B-A3-C1-C2-COOH。FVIII在血浆中作为在B-A3边界处分开的两条链进行循环。链通过二价金属离子结合进行连接。A1-A2-B链称作重链(HC),而A3-C1-C2称作轻链(LC)。
内源的因子VIII分子作为一群具有各种大小的B结构域的分子在体内循环。在体内可能发生的是,B结构域的逐渐酶促去除,从而产生一群具有各种大小的B结构域的分子。一般认为,随同凝血酶激活发生在位置740处的切割,由此去除B-结构域的最后一部分。但是,不能排除的是,其中例如在位置740的切割位点已经受损的因子VIII变体可能是有活性的。
本文使用的“因子VIII”或“FVIII”是指一种人血浆糖蛋白,它是内在的凝固途径的一个成员,且是血液凝固所必需的。“天然的FVIII”是在SEQ ID NO.1(氨基酸1-2332)中所示的全长人FVIII分子。B-结构域跨SEQ ID NO 1中的氨基酸741-1648。
SEQ ID NO 1:
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY
根据本发明的因子VIII分子是B结构域截短的因子FVIII分子,其中剩余的结构域对应着SEQ ID NO.1中氨基酸编号1-740和1649-2332所述的序列。因而断定根据本发明的分子是在转化的宿主细胞(优选哺乳动物起源)中生产的重组分子。但是,剩余的结构域(即3个A-结构域和2个C-结构域)可以稍微(例如约1%、2%、3%、4%或5%)不同于在SEQ ID NO 1中所述的氨基酸序列(氨基酸1-740和1649-2332)。具体地,可取的是将氨基酸修饰(取代、缺失等)引入剩余的结构域,例如以修饰因子VIII与各种其它组分(例如vW因子、LPR、各种受体、其它凝固因子、细胞表面等)的结合能力。此外,似乎可取的是根据本发明的因子VIII分子在例如截短的B-结构域中和/或在分子的一个或多个其它结构域中包含其它翻译后修饰。这些其它的翻译后修饰可以是与根据本发明的因子VIII分子缀合的各种分子的形式,例如聚合物、肽化合物、脂肪酸衍生的化合物等。
无论根据本发明的因子VIII分子是否在B结构域之外被修饰、是否具有其它翻译后修饰,它们都具有因子VIII活性,即以在功能上类似于或等同于FVIII的方式在凝固级联中起作用,通过与激活的血小板上的FIXa之间的相互作用诱导FXa的形成,及支持血凝块的形成的能力。通过本领域众所周知的技术,例如凝块分析、内源凝血酶潜力分析等,可以在体外评估活性。根据本发明的因子VIII分子具有的FVIII活性是天然人FVIII的至少约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%和100%或甚至超过100%。
B结构域:因子VIII中的B-结构域跨SEQ ID NO 1中的氨基酸741-1648。B-结构域在几个不同的位点切割,从而产生循环血浆FVIII分子的大异质性。重度糖基化的B-结构域的确切功能是未知的。已知的是,该结构域对于凝固级联中的FVIII活性而言是可有可无的。这种明显的功能缺失得到下述事实的支持,即B结构域缺失的/截短的FVIII似乎具有与全长天然FVIII相同的体内性质。也就是说,有迹象表明,B-结构域可以减少与细胞膜的结合,至少在无血清的条件下。
B结构域截短的/缺失的因子VIII分子:内源的全长FVIII作为单链前体分子来合成。在分泌之前,前体被切割成重链和轻链。可以从2种不同的策略生产重组的B结构域缺失的FVIII。单独地合成没有B-结构域的重链和轻链,作为2条不同的多肽链(双链策略),或者合成B-结构域缺失的FVIII,作为单条前体多肽链(单链策略),其以与全长FVIII前体相同的方式切割成重链和轻链。
在B结构域缺失的FVIII前体多肽中,重链和轻链部分正常被接头分开。为了使在B结构域缺失的FVIII中引入免疫原性的表位的风险最小化,接头的序列优选地源自FVIII B-结构域。所述接头必须包含蛋白酶的识别位点,其将B结构域缺失的FVIII前体多肽分开成重链和轻链。在全长FVIII的B结构域中,氨基酸1644-1648构成该识别位点。在B结构域缺失的FVIII激活后导致接头去除的凝血酶位点位于重链中。因而,接头的大小和氨基酸序列不太可能影响由凝血酶激活将它从剩余的FVIII分子去除。B结构域的缺失对于FVIII的生产是有利的。尽管如此,在接头中可以包含B结构域部分,而不降低生产力。B结构域对生产力的消极作用尚未归因于B结构域的任意特定大小或序列。
截短的B-结构域可以含有几个O-糖基化位点。优选地,该分子在截短的B-结构域中包含仅1个、或2个、3个或4个O-联寡糖。
优选地,截短的B结构域包含仅1个潜在的O-糖基化位点,且亲水聚合物共价地缀合至该O-糖基化位点。
根据本发明的B-结构域截短的分子中的O-联寡糖可以附着至O-糖基化位点,后者通过重组方式和/或通过截短B-结构域来暴露“隐藏的”O-糖基化位点而人工生成。在两种情况下,可以如下制备这种分子:设计B-结构域截短的因子VIII氨基酸序列,且随后对该氨基酸序列进行预测截短的B-结构域中出现O-糖基化位点的概率的计算机分析。可以在合适的宿主细胞中合成具有相对较高的具备这种糖基化位点的概率的分子,随后分析糖基化模式,再选择在截短的B-结构域中具有O-联糖基化的分子。适用于生产重组因子VIII蛋白的宿主细胞优选地具有哺乳动物起源,以确保该分子被糖基化。在实践本发明时,细胞是哺乳动物细胞,更优选确立的哺乳动物细胞系,包括、但不限于,CHO(例如,ATCC CCL 61)、COS-1(例如,ATCC CRL 1650)、幼仓鼠肾(BHK)和HEK293(例如,ATCC CRL 1573;Graham等人,J.Gen.Virol.36:59-72,1977)细胞系。优选的BHK细胞系是tk-ts13BHK细胞系(Waechter和Baserga,Proc.Natl.Acad.Sci.USA 79:1106-1110,1982),在下文中称作BHK 570细胞。BHK 570细胞系可以从美国典型培养物保藏中心(American Type Culture Collection,12301Parklawn Dr.,Rockville,MD20852)在ATCC登记号CRL 10314下得到。tk-ts13BHK细胞系也可以从ATCC在登记号CRL 1632下得到。优选的CHO细胞系是可从ATCC在登记号CCl61下得到的CHO K1细胞系以及细胞系CHO-DXB11和CHO-DG44。
其它合适的细胞系包括、但不限于,大鼠Hep I(大鼠肝癌;ATCCCRL 1600)、大鼠Hep II(大鼠肝癌;ATCC CRL 1548)、TCMK(ATCCCCL 139)、人肺(ATCC HB 8065)、NCTC 1469(ATCC CCL 9.1)、DUKX细胞(CHO细胞系)(Urlaub和Chasin,Proc.Natl.Acad.Sci.USA77:4216-4220,1980)(DUKX细胞也称作DXB 11细胞)和DG44(CHO细胞系)(Cell,33:405,1983,和Somatic Cell and Molecular Genetics 12:555,1986)。也有用的是3T3细胞、Namalwa细胞、骨髓瘤和骨髓瘤与其它细胞的融合体。在有些实施方案中,细胞可以是突变的或重组的细胞,例如,与它们的来源细胞类型相比,表达在质量上或在数量上不同的酶谱的细胞,所述酶催化蛋白的翻译后修饰(例如,糖基化酶例如糖基转移酶和/或糖苷酶,或加工酶例如前肽)。DUKX细胞(CHO细胞系)是特尤其优选的。
目前优选的细胞是HEK293、COS、中国仓鼠卵巢(CHO)细胞、幼仓鼠肾(BHK)和骨髓瘤细胞,尤其是中国仓鼠卵巢(CHO)细胞。野生型FVIII分子中B结构域的长度是约907个氨基酸。根据本发明的分子中的截短的B结构域的长度可以是约10个氨基酸至约700个氨基酸,例如约12-500个氨基酸、12-400个氨基酸、12-300个氨基酸、12-200个氨基酸、15-100个氨基酸、15-75个氨基酸、15-50个氨基酸、15-45个氨基酸、20-45个氨基酸、20-40个氨基酸或20-30个氨基酸。截短的B-结构域可以包含重链和/或轻链的片段和/或在野生型FVIII分子中不存在的人工引入的序列。术语“B-结构域截短的”和“B-结构域缺失的”可以在本文中互换使用。
改变的循环半衰期:根据本发明的分子具有与野生型因子VIII分子相比改变的循环半衰期,优选增加的循环半衰期。循环半衰期优选地增加了至少10%、优选至少15%、优选至少20%、优选至少25%、优选至少30%、优选至少35%、优选至少40%、优选至少45%、优选至少50%、优选至少55%、优选至少60%、优选至少65%、优选至少70%、优选至少75%、优选至少80%、优选至少85%、优选至少90%、优选至少95%、优选至少100%、更优选至少125%、更优选至少150%、更优选至少175%、更优选至少200%和最优选至少250%或300%。甚至更优选地,这种分子具有与野生型FVIII的循环半衰期相比增加了至少400%、500%、600%或甚至700%的循环半衰期。
亲水聚合物:根据本发明的修饰基团/亲水聚合物优选地是非天然存在的。在一个实例中,“非天然存在的修饰基团”是聚合的修饰基团,其中至少一个聚合部分是非天然存在的。在另一个实例中,非天然存在的修饰基团是修饰的碳水化合物。选择由修饰基团官能化的部位,从而使它不会阻止“修饰的糖”酶促地添加到多肽上。“修饰的糖”也指任意的糖基模仿部分,其被修饰基团官能化,且是天然的或修饰的酶(例如糖基转移酶)的底物。
添加到多肽上的聚合的修饰基团可以改变这种多肽的性质,例如,它的生物利用率、生物活性或它在体内的半衰期。根据本发明的示例性的聚合物包括线性的或分支的水溶性的聚合物,且可以包括一个或多个独立地选择的聚合部分,例如聚(亚烷基二醇)和其衍生物。根据本发明的聚合的修饰基团可以包括水溶性的聚合物,例如聚(乙二醇)和其衍生物(PEG,m-PEG)、聚(丙二醇)和其衍生物(PPG,m-PPG)等。
术语“水溶性的”是指在水中具有一些可检测程度的溶解度的部分。检测和/或定量水溶性的方法是本领域众所周知的。根据本发明的示例性的水溶性的聚合物包括肽、糖、聚(醚)、聚(胺)、聚(羧酸)等。肽可以具有混合的序列,且由单个氨基酸组成,例如,聚(赖氨酸)。示例性的多糖是聚(唾液酸)。示例性的聚(醚)是聚(乙二醇),例如,m-PEG。聚(乙烯亚胺)是示例性的聚胺,聚(丙烯酸)是代表性的聚(羧酸)。
根据本发明的水溶性的聚合物的聚合物主链可以是聚(乙二醇)(即PEG)。与本发明有关的术语PEG包括任意形式的聚(乙二醇),包括烷氧基PEG、双功能的PEG、多臂的PEG、叉状的(forked)PEG、分支的PEG、悬垂的(pendent)PEG(即具有一个或多个从聚合物主链悬垂的官能团的PEG或有关的聚合物)或具有可降解的键的PEG。
聚合物主链可以是线性的或分支的。分支的聚合物主链是本领域普遍已知的。一般地,分支的聚合物具有中心分支核心部分和连接到该中心分支核心的许多线性的聚合物链。PEG通常以分支形式使用,其可以通过将环氧乙烷添加到各种多元醇(例如丙三醇、季戊四醇和山梨糖醇)上来制备。中心分支部分也可以源自几个氨基酸,例如赖氨酸或半胱氨酸。在一个实例中,分支的聚(乙二醇)可以表示为通式R(-PEG-OH)m,其中R代表核心部分,例如丙三醇或季戊四醇,且m代表臂的数目。多臂的PEG分子,例如在本文中整体并入作为参考的美国专利号5,932,462中所述的那些,也可以用作聚合物主链,
图8显示了在本发明的实施方案中使用的代表性分支的PEG聚合物,在本文中称作“SA-丙三醇-PEG”。图8A显示了CMP-SA-丙三醇-PEG或与聚糖或多肽氨基酸连接的SA-丙三醇-PEG的示例性的SA-丙三醇-PEG组分。图8B显示了通过Gal残基连接至聚糖或多肽上的SA-丙三醇-PEG部分。图8C显示了通过Gal-GalNAc残基连接至聚糖或多肽上的SA-丙三醇-PEG部分。图8D显示了通过Gal-GalNAc部分连接至多肽氨基酸上的SA-丙三醇-PEG部分。在各种实施方案中,AA是苏氨酸或丝氨酸。在示例性的实施方案中,通过缺失FVIII多肽的B-结构域,将AA转变成O-联糖基化位点。下文[0032]段落中关于聚合物分子量的讨论,通常也适用于在图8中所示的分支的PEG。在图8中,下标“n”代表任意整数,其提供具有在下面[0032]段落中讨论的所需分子量的线性的(和因而分支的)m-PEG。在各种实施方案中,选择“n”,从而使得线性的m-PEG部分是约20KDa至约40KDa,例如,约20KDa、约30KDa或约40KDa。与这些m-PEG分子量对应的整数等于约400(例如约455)至约900(例如约910)。因此,选择“n”,以提供约40KDa至约80KDa、例如约40KDa、约50KDa、约60KDa、约70KDa或约80KDa的分支的PEG。
许多其它的聚合物也适用于本发明。非肽的和水溶性的聚合物主链在本发明中特别有用。合适的聚合物的实例包括、但不限于,其它聚(亚烷基二醇)例如聚(丙二醇)(″PPG″)、乙二醇和丙二醇的共聚物等,聚(氧乙基化的多元醇),聚(烯醇),聚(乙烯吡咯烷酮),聚(羟丙基甲基丙烯酰胺),聚(α-羟酸),聚(乙烯醇),聚磷腈(polyphosphazene),聚唑啉,聚(N-丙烯酰吗啉),例如在本文中整体并入作为参考的美国专利号5,629,384中所述的那些,以及它们的共聚物、三元共聚物和混合物。
[0032]尽管聚合物主链的每条链的分子量可以变化,但它通常是在从约100Da至约160,000Da、例如从约5,000Da至约100,000Da的范围内。更具体地,根据本发明的每个缀合的亲水聚合物的大小可以从约500Da至约80,000Da变化,例如约1000Da至约80,000Da;约2000Da至约70,000Da;约5000至约70,000Da;约5000至约60,000Da;约10,000至约70,000Da;约20,000至约60,000Da;约30,000至约60,000Da;约30,000至约50,000Da;或约30,000至约40,000Da。应当理解,这些大小代表估计值,而不是精确测量值。根据优选的实施方案,根据本发明的分子缀合亲水聚合物的异质群体,例如大小为例如10,000、40,000或80,000Da±约5000、约4000、约3000、约2000或约1000Da的PEG。
O-联寡糖:通过生产蛋白的细胞,使N-聚糖和O-聚糖附着到蛋白上。随着新生的蛋白从核糖体转运到内质网,细胞的N-糖基化机构识别和糖基化氨基酸链中的N-糖基化信号(N-X-S/T基序)(Kiely等人1976;Glabe等人1980)。
同样地,O-聚糖附着氨基酸链中的特定O-糖基化位点,但是触发O-糖基化的基序比N-糖基化信号的异质性高得多,我们仍然不能预测氨基酸序列中的O-糖基化位点(Julenius等人2004)。人工的O-糖基化位点的构建因而伴有一些不确定性。一般的假设是,天然FVIII分子不含有任何O-糖基化位点,且技术人员因此预期,在实践本发明方面,必须构建至少一个人工的O-糖基化位点,并插入B结构域。
截短的因子VIII B结构域中的O-联寡糖因而可以共价地连接天然存在的O-联糖基化序列或已经通过重组技术人工构建的O-联糖基化序列。
根据本发明的优选的实施方案,O-联寡糖连接至天然存在的O-联糖基化序列,后者在野生型因子VIII分子中不暴露于糖基化,但是作为B结构域截短的结果,变得可进行O-糖基化。其实例显示在实施例和SEQID NO 2中(截短的B-结构域对应着氨基酸742-763)。即使B-结构域在稍微不同的位置被截短,即如果截短的B结构域与SEQ ID NO 2相比稍微更短(例如比SEQ ID NO 2短1、2、3、4或5个氨基酸)或更长(例如长1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45或50个氨基酸),SEQ ID NO 2中的“隐藏的”O-糖基化位点似乎也变成糖基化的。该通过截短B-结构域来激活“隐藏的”O-糖基化位点、而不是生成人工O-糖基化位点的方案,具有生成具有有利的安全概况(即减少的变应原性等)的分子的优点。通过以不同方式截短分子,同样可以激活因子VIII B-结构域中的其它O-糖基化位点。
O-联寡糖的糖-PEG化(Glyco-PEGylation):通过在生物合成的相对早期,添加唾液酸残基,可以修饰和终止O-聚糖的生物合成。某些唾液酸转移酶在核心1GalT作用后能作用于GalNAcα-Ser/Thr或早期O-聚糖核心亚型。术语T抗原与Galβ1-3GalNAcα-Ser/Thr二糖的存在有关。这些结构的产生涉及糖基转移酶之间竞争相同的底物,且因而高尔基体内糖基转移酶的表达水平和亚细胞分布决定了O-聚糖生物合成的结构结果和多样化。如图1所示,仅Galβ1-3GalNAcα-Ser/Thr二糖顺从糖PEG化。
但是,通过用唾液酸酶或核心1GalT或它们的组合处理蛋白,可以极大地增强该结构的可利用量。作为糖PEG化过程的结果,通过与靶蛋白的Galβ1-3GalNAcα-Ser/Thr二糖的α3键,唾液酸PEG添加到天然结构上(图1)。
其它亲水聚合物也可以附着到O-联寡糖上。通过O-聚糖将其它亲水聚合物酶促地缀合到FVIII上的基本要求是,如在WO03031464中公开的通过游离氨基使它们与甘氨酰基-唾液酸衍生物偶联的能力。这可以通过本领域技术人员已知的许多种偶联化学来实现。激活的生物相容的聚合物的实例包括聚环氧烷,例如、但不限于,聚乙二醇(PEG)、2-(甲基丙烯酰基氧)乙基磷酸胆碱(mPC)聚合物(如WO03062290所述)、葡聚糖、多聚乙酰神经氨酸或其它基于碳水化合物的聚合物、氨基酸或特定肽序列的聚合物、生物素衍生物、聚乙烯醇(PVA)、聚羧酸酯、聚乙烯吡咯烷酮、乙烯-马来酐共聚物、苯乙烯-苹果酸酐共聚物、聚唑啉、聚丙烯酰吗啉、肝素、清蛋白、纤维素、壳聚糖的水解产物、淀粉例如羟乙基-淀粉和羟丙基-淀粉、糖原、琼脂糖和其衍生物、瓜耳胶、支链淀粉、菊粉、黄原胶、角叉菜聚糖、果胶、藻酸水解产物、其它生物聚合物和它们的任意等价物。
药物组合物:药物组合物在本文中优选地意在包括适合肠胃外施用的包含根据本发明的因子VIIII分子的组合物,例如即可使用的无菌含水组合物或可以在例如水或含水缓冲液中重构的干燥的无菌组合物。根据本发明的组合物可以包含各种药学上可接受的赋形剂、稳定剂等。
这种组合物中的其它成分可以包括润湿剂、乳化剂、抗氧化剂、填充剂、张力调节剂、螯合剂、金属离子、油性载体、蛋白(例如,人血清清蛋白、明胶或蛋白)和两性离子(例如,氨基酸,例如甜菜碱、牛磺酸、精氨酸、甘氨酸、赖氨酸和组氨酸)。这种其它成分当然不应不利地影响本发明的药物制剂的总稳定性。借助于注射器,任选笔样注射器,通过皮下的、肌内的、腹膜内的或静脉内的注射,可以进行肠胃外施用。或者,借助于输注泵,可以进行肠胃外施用。另一个选择是鼻或肺喷雾剂形式的组合物,其可以是用于施用FVIII化合物的溶液或悬浮液。作为另一个选择,含有本发明的FVIII化合物的药物组合物也可以适合于经皮施用(例如通过无针注射或来自贴剂,任选离子电渗疗法贴剂)或跨粘膜的(例如口腔含化的)施用。
在第一个方面,本发明因而涉及具有改变的循环半衰期的B-结构域截短的因子VIII分子,所述分子在截短的B结构域中通过O-联寡糖与亲水聚合物共价缀合,其中因子VIII激活(分子的激活)导致共价缀合的亲水聚合物的去除,且FVIII前体多肽的重链和轻链部分被接头分开,其中所述接头的序列源自FVIII B结构域。
根据一个实施方案,亲水聚合物是PEG。PEG聚合物的大小可以是约10,000至约160,000Da,例如10,000至80,000Da,例如约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。
根据特别优选的实施方案,根据本发明的分子包含如SEQ ID NO 2所述的氨基酸序列。这种分子具有独特的特征,因为激活的FVIII分子与天然的活性FVIII分子相同。该特征似乎在安全性评判方面具有有利的性质。
本发明也涉及包含根据本发明的分子的药物组合物。
本发明此外涉及得到根据本发明的分子的方法,其中所述方法包含,通过截短的B结构域中的O-联寡糖,给B-结构域截短的因子VIII分子缀合亲水聚合物,例如PEG基团。因而断定,本发明也涉及通过这种方法得到的或可以得到的分子。
在另一个方面,本发明涉及治疗血友病的方法,其包含给需要治疗的患者施用治疗有效量的根据本发明的分子。
本文使用的术语“治疗”是指需要治疗的任意人或其它动物受试者的医学疗法。预期所述受试者已经经历医学从业人员的体检,所述人员已经给出推测的或确定的诊断,其可指示所述特定治疗的应用对所述人或其它动物受试者的健康是有益的。根据受试者的健康现状,所述治疗的时间选择和目的可以随个体不同而异。因而,所述治疗可以是预防性的、治标的、针对症状的和/或治愈的。
在另一个方面,本发明涉及根据本发明的分子作为药物的用途,以及根据本发明的分子用于生产治疗血友病的药物的用途。
在最后的方面,本发明涉及工程改造根据本发明的B-结构域截短的因子VIII分子的方法,所述方法包含,(i)截短B-结构域,和任选地对该截短的因子VIII分子的氨基酸序列进行鉴定潜在的O-联糖基化位点的分析,(ii)在合适的宿主细胞中生产该分子,和(iii)选择在截短的B-结构域中具有O-联聚糖的分子。
附图简述
在附图中,缀合基团的大小有时称作“K”,它在本文中意在等同于KDa(千道尔顿)。
图1:O-联寡糖的糖PEG化过程的示意图。该图不代表取得在实施例中得到的产物的可行方式的穷尽列表。
图2:反应混合物在Source 15Q上的离子交换层析(A)。收集的级分的SDS-PAGE,含有分子标记(左边)(B)。
图3:加帽的产物在superdex 200大小排阻层析上的纯化。
图4:使用各种aPTT试剂,O-糖PEG化的rFVIII的凝固活性。(A)显示了凝固活性和生色活性之间的比。(B)显示了比凝固活性。
图5:40K-PEG-[O]-N8在FVIII KO小鼠中的体内作用(闭塞时间)。
图6:显示生产根据本发明的糖PEG化的因子FVIII所包含的过程步骤的流程图。
图7:在实施例中生产的根据本发明的因子VIII分子的示意图。
实施例
实施例1
重组的B结构域截短的O-糖基化的因子VIII的生产
在SEQ ID NO 2中给出了B-结构域缺失的因子VIII分子的氨基酸序列的实例。该多肽也可以称作“N8”。该分子包含21个氨基酸残基的接头序列(SFSQNSRHPSQNPPVLKRHQR,加有下划线的S是在实施例2的含有加入聚乙二醇的O-聚糖的丝氨酸残基)。
根据本发明的因子VIII分子在实施例中可以以各种方式提及,但是所有提及的“因子VIII分子”都表示根据本发明的因子VIII分子,或者在转变成根据本发明的因子VIII分子的过程中的因子VIII分子。
SEQ ID NO 2:
ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRS WYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYS SLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLY
细胞系和培养过程:
使用因子VIII cDNA,构建哺乳动物表达质粒,其编码具有SEQ IDNO 2所述氨基酸序列的B-结构域缺失的因子VIII。该质粒编码包含全长人因子VIII的氨基酸1-740的因子VIII重链和包含全长人因子VIII的氨基酸1649-2332的因子VIII轻链。重链和轻链序列通过具有全长人因子VIII的氨基酸741-750和1638-1648的序列的21氨基酸接头相连。用编码BDD因子VIII的质粒转染中国仓鼠卵巢(CHO)细胞,并用二氢叶酸还原酶系统选择,最终产生在无动物组分的培养基中培养的无性系悬浮生产细胞。
该过程的第一步是,将来自工作细胞库小瓶的细胞小瓶接种进化学成分确知的且无动物组分的生长培养基中。最初,解冻后,在T-瓶中温育细胞。解冻后1或2天,将细胞转移至摇瓶,并通过连续稀释扩大培养体积,以便使细胞密度维持在0.2-3.0×106细胞/ml。下一步是,将摇瓶培养物转移进种子生物反应器。在这里进一步扩大培养体积,然后最终转移至生产生物反应器。相同的化学成分确知的且无动物组分的培养基用于所有接种物增殖步骤。转移至生产生物反应器后,给培养基补充增加产物浓度的组分。在生产生物反应器中,以3天的循环时间,以重复的分批方法培养细胞。在收获时,将80-90%的培养体积转移至收获罐。然后用新鲜的培养基稀释剩余的培养液,以得到最初的细胞密度,且然后开始新的生长期。
通过离心和过滤,澄清收获批,并转移至贮藏罐,然后开始纯化过程。将缓冲液加入贮藏罐中的无细胞的收获物,以稳定化pH。
在生产运行结束前,收集细胞,并冷冻,以造成生产细胞库的结束。测试该细胞库的支原体、无菌性和病毒污染。
纯化:
为了从细胞培养基分离B-结构域缺失的因子VIII,使用4步纯化程序,包括在Capto MMC柱上的浓缩步骤、免疫吸附(immunoabsorbent)层析步骤、阴离子交换层析和最终的凝胶过滤步骤。通常,使用下述程序:以15ml/分钟的流速,用泵将11升无菌过滤的培养基泵到CaptoMMC(GE Healthcare,瑞典)的柱(1.6×12cm)上,后者在缓冲液A中平衡:20mM咪唑,10mM CaCl2,50mM NaCl,0.02%吐温80,pH=7.5。用75ml缓冲液A洗涤柱,然后用含有1.5M NaCl的75ml缓冲液A洗涤。以1ml/分钟的流速,用20mM咪唑、10mM CaCl2、0.02%吐温80、2.5M NaCl、8M乙二醇、pH=7.5洗脱蛋白。收集8ml级分,且测定因子VIII活性(CoA-实验)。合并含有因子VIII的级分,且正常得到约50ml的合并体积。
已经开发了抗因子VIII的单克隆抗体(Kjalke Eur J Biochem 234773)。通过对该抗体进行表位绘图(结果未显示),发现F25识别从氨基酸残基725至740的重链远C-末端序列。基本上如生产商所述,以2.4mg/ml凝胶的密度,将F25抗体偶联至NHS-激活的Sepharose 4FF(GEHealthcare,Bio-Sciences AB,Uppsala,瑞典)。用20mM咪唑、10mMCaCl2、0.02%吐温80、pH=7.3,将来自前述步骤的库稀释10倍,并以0.5ml/分钟的流速,应用于F25Sepharose柱(1.6×9.5cm),后者用20mM咪唑、10mM CaCl2、150mM NaCl、0.02%吐温80、1M丙三醇、pH=7.3平衡过。用平衡缓冲液洗涤柱,直到紫外信号恒定,且然后用20mM咪唑、10mM CaCl2、0.65M NaCl、pH=7.3洗涤,直到紫外信号再次恒定。以1ml/分钟的流速,用20mM咪唑、10mM CaCl2、0.02%吐温80、2.5M NaCl、50%乙二醇、pH=7.3洗脱因子VIII。收集1ml级分,且测定因子VIII活性(CoA-实验)。合并含有因子VIII的级分,且正常得到约25ml的合并体积。
为离子交换步骤制备缓冲液A:20mM咪唑、10mM CaCl2、0.02%吐温80、1M丙三醇、pH=7.3和缓冲液B:20mM咪唑、10mM CaCl2、0.02%吐温80、1M丙三醇、1M NaCl、pH=7.3。以2ml/分钟的流速,用85%缓冲液A/15%缓冲液B平衡Macro-Prep 25Q Support(Bio-RadLaboratories,Hercules,CA,USA)的柱(1×10cm)。用缓冲液A将来自前述步骤的库稀释10倍,且以2ml/分钟的流速泵到柱上。以2ml/分钟的流速,用85%缓冲液A/15%缓冲液B洗涤柱,且以2ml/分钟的流速,以从15%缓冲液B至70%缓冲液B的线性梯度,在120ml范围内洗脱因子VIII。收集2ml级分,且测定因子VIII活性(CoA-实验)。合并含有因子VIII的级分,且正常得到约36ml的合并体积。
将来自前述步骤的库应用到Superdex 200制备级(GE Healthcare,Bio-Sciences AB,Uppsala,瑞典)柱(2.6×60cm),后者用20mM咪唑、10mM CaCl2、0.02%吐温80、1M丙三醇、150mM NaCl、pH=7.3平衡过,并用其在1ml/分钟洗脱。收集3ml级分,且测定因子VIII活性(CoA-实验)。合并含有因子VIII的级分,且正常得到约57ml的合并体积。在-80℃储存含有因子VIII的库。
利用上述的4步纯化程序,得到约15%的总得率,如通过CoA活性和ELISA测量来判断的。
用于生产N8的细胞系是用在pTSV7中的表达质粒#814F8-500稳定转染的重组的中国仓鼠卵巢(CHO)细胞系,所述在pTSV7中的表达质粒#814F8-500由含有插入片段的pTSV7表达载体组成,所述插入片段含有编码F8-500蛋白的cDNA。“N8”在本文中意在对应于具有SEQ IDNO 2所列出的氨基酸序列的蛋白。从N-末端开始,F8-500蛋白(N8)由FVIII信号肽(氨基酸-19至-1)、然后是没有B结构域的FVIII重链(氨基酸1-740)、21氨基酸接头(SFSQNSRHPSQNPPVLKRHQR)和FVIII轻链(野生型人FVIII的氨基酸1649-2332)组成。21氨基酸接头的序列源自FVIII B结构域,且由全长FVIII的氨基酸741-750和1638-1648组成。
用在pTSV7中的814F8-500转染CHO细胞,并用二氢叶酸还原酶系统选择,最终产生在无动物组分的培养基中培养的无性系悬浮生产细胞。如下开始生产运行:解冻工作细胞库小瓶,并增殖细胞,直到转移至生产生物反应器。相同的化学成分确知的且无动物组分的培养基用于所有接种物增殖步骤。转移至生产生物反应器后,给培养基补充增加产物浓度的组分。在生产生物反应器中,以3天的循环时间,以重复的分批方法培养细胞。在收获时,将80-90%的培养体积转移至收获罐。然后用新鲜的培养基稀释剩余的培养液,以得到最初的细胞密度,且然后开始新的生长期。通过离心和过滤,澄清收获批,并转移至贮藏罐,然后开始纯化过程。将缓冲液加入贮藏罐中的无细胞的收获物,以稳定化pH。
实施例2
重组的B结构域截短的且O-糖基化的因子VIII的加入聚乙二醇:使用下述程序,用聚乙二醇(PEG)缀合在实施例1中得到的重组因子VIII分子:
为了有效地进行在实施例1中得到的重组因子VIII分子的糖PEG化,>5mg/ml的FVIII浓度是优选的。由于FVIII在该浓度正常是不溶的,所述筛选选择的缓冲液组合物(参见表1中的一些结果)。
基于这些考虑,发现含有50mM MES、50mM CaCl2、150mMNaCl、20%丙三醇、pH6.0的缓冲液是合适的反应缓冲液。
表1反应条件对FVIII溶解度和聚集的影响的评价
通过在使用分步洗脱的Poros 50HQ柱上的离子交换,在10kDa截止的Sartorius Vivaspin (PES)过滤器上,或在Amicon 10kDa MWCO PES过滤器上,在反应缓冲液中浓缩已经如上所述纯化的重组的FVIII至6-10mg/mL的浓度。通过在反应缓冲液(50mM MES,50mM CaCl2,150mM NaCl,20%丙三醇,0.5mM抗痛素,pH 6.0)中混合因子VIII(BDD)(~4.7mg/mL终浓度)和唾液酸酶(A.urifaciens)(159mU/mL)、CMP-SA-丙三醇-PEG-40kDa(5mol当量)和MBP-ST3Gal1(540mU),开始FVIII的糖PEG化。在32℃温育反应混合物,直到~20-30%的总转变得率。
温育后,用缓冲液A(25mM Tris,5mM CaCl2,20mM NaCl,20%丙三醇,pH7.5)稀释样品,并应用于Source 15Q柱(1cm内径×6cm,4.7mL,1mL/分钟,280nm)上。用缓冲液A洗涤结合的材料,并使用缓冲液B(25mM Tris,5mM CaCl2,1M NaCl,20%丙三醇,pH7.5)的分级梯度进行洗脱。在~25%缓冲液B,从柱洗脱糖PEG化的因子VIII-(O)-SA-丙三醇-PEG-40kDa。图2显示了反应混合物在Source 15Q上的离子交换层析。
为了封闭在唾液酸酶处理过程中已经暴露于N-聚糖上的游离的半乳糖部分,在反应缓冲液50mM MES、20mM CaCl2、150mM NaCl、10mM MnCl2、20%丙三醇、pH6.0中,将因子VIII-SA-丙三醇-PEG-40kDa(1.0mg/mL终浓度)的合并级分与CMP-SA(2,000mol当量)和MBP-SBD-ST3Gal3(400mU/mL)相混合,且在32℃温育11小时。
以0.25mL/分钟的流速,通过用50mM MES、50mM CaCl2、150mMNaCl、10%丙三醇、pH6.0平衡的Superdex 200柱(10cm内径×300mm;280nm)上的凝胶过滤,从CMP-SA和ST3GalIII分离得到的加帽的糖PEG化的因子VIII-SA-丙三醇-PEG-40kDa。产物因子VIII-SA-丙三醇-PEG-40kDa在38分钟洗脱。图3显示了使用superdex 200大小排阻层析纯化加帽的产物。收集峰级分,等分试样,并进行后续分析。
加帽程序的目的是减少缀合的因子VIII分子的体内清除。
实施例3
O-聚糖加入聚乙二醇的rFVIII在生色FVIII活性测定中的活性:如下使用Coatest SP试剂(Chromogenix),在生色FVIII测定中评价在实施例2中得到的O-糖PEG化的rFVIII的活性:在Coatest测定缓冲液(50mM Tris,150mM NaCl,1%BSA,pH7.3,具有防腐剂)中稀释rFVIII样品和校准物(calibrator)(来自NIBSC的第7国际FVIII标准品)。将50μl样品、标准品和缓冲液负对照一式两份地加入96-孔微量滴定板(Nunc)。以5∶1∶3(体积∶体积∶体积)混合来自Coatest SP试剂盒的因子IXa/因子X试剂、磷脂试剂和CaCl2,且将75μl加入孔中。在室温温育15分钟后,加入50μl因子Xa底物S-2765/凝血酶抑制剂I-2581混合物,且在室温温育反应物10分钟,然后加入25μl 1M柠檬酸pH3。在Spectramax微量滴定板读数器(Molecular Devices)上测量在415nm的吸光度,在620nm的吸光度用作参考波长。从所有样品减去负对照的值,且通过相对于FVIII浓度绘制的吸光度值线性回归,制备校准曲线。如下计算比活性:通过将样品的活性除以由尺寸排阻HPLC通过积分HPLC层析谱中的轻链峰测得的蛋白浓度,即不包括PEG-部分。表2中的数据证实,O-糖PEG化的rFVIII化合物保持比生色活性,这意味着因子VIII活性看起来在加入聚乙二醇的的变体中保持。
表2.具有不同PEG基团大小的O-糖PEG化的rFVIII的比生色活性
数据是在括号中指出的独立测定数的平均值和标准差。
实施例4
在FVIII凝固活性测定中O-聚糖加入聚乙二醇的rFVIII的活性
在FVIII凝固测定中,进一步评价了O-糖PEG化的rFVIII的活性。将rFVIII样品在HBS/BSA(20mM hepes,150mM NaCl,pH7.4,含有1%BSA)中稀释至约10U/ml,随后在含有VWF(Dade Behring)的FVIII-缺乏的血浆中稀释10-倍。随后将样品和校准的血浆标准品(来自Instrumentation Laboratory的HemosIL校准血浆)在HBS/BSA中稀释至4个(样品)或6个(校准物)不同的浓度。使用单因子程序,在ACL9000仪器(Instrumentation laboratory)上测量凝固时间,其中将样品/标准品与等体积的含有VWF(Dade Behring)、钙和aPTT试剂的FVIII-缺乏的血浆相混合,并测量凝固时间。使用下述试剂:Synthasil(HemosIL,Instrumentation Laboratory),Actin FS(激活的PTT试剂,Dade Behring)Stago(PTT-A,Stago),和dAPPTin(TC,Technoclone)。基于凝固时间相对于校准物浓度的半对数图,计算样品的活性。
依赖于PEG大小和使用的aPTT试剂,O-糖PEG化的rFVIII化合物(对照,分别是10、40和80kDA PEG)的凝固活性(图4)降低至各种程度。使用Synthasil或dAPPTin作为aPTT试剂,导致凝固活性随着PEG-大小逐渐降低。利用Stago的aPTT试剂,对于评价的所有3种O-糖PEG化的N8化合物,观察到降低了50%的比凝固活性。当使用ActinFS作为aPTT试剂时,维持约10,000IU/mg的比凝固活性。数据表明,aPTT测定受到PEG部分的存在的影响,但是,使用选择的aPTT试剂例如Actin FS,rFVIII的比凝固活性在O-糖PEG化后不受损害。
实施例5:
rFVIII的O-联加入聚乙二醇对辅因子活性和FVIII激活速率的作用激活的FVIII向FIXa-FVIIIa复合物中的掺入增强FIXa-催化的FX激活的催化效率5个数量级(van Dieijen等人(1981)J Biol Chem256:3433),且FIXa-FVIIIa复合物装配和FX激活动力学的特征是FVIIIa分子的功能完整性的灵敏度量。通过在有磷脂和凝血酶-激活的rFVIII或PEG-rFVIII存在下测定FIXa-催化的FX激活的动力学参数,表征凝血酶-激活的rFVIII或PEG-rFVIII的辅因子活性。使用FVIIIa活性测定(FIXa-辅因子活性测定),分别针对固定浓度(0.1nM)的rFVIIIa或FIXa进行FIXa和FVIIIa的倒数滴定(reciprocal titration),以得到FIXa对rFVIIIa的表观亲和力(K1/2FIXa)和功能性的FVIIIa浓度。从针对固定浓度的FIXa-FVIIIa复合物的FX滴定,得到FX激活的米氏常数(km)和转化数(kcat)。
如下进行FIXa-辅因子活性测定:通过将rFVIII(通常0.7nM,1U/mL)与5nM人α-凝血酶在37℃温育精确的30秒,为每个实验新鲜制备凝血酶-激活的rFVIII和PEG-rFVIII变体。随后,通过将上面的激活反应物二次取样进制备的FIXa、磷脂载体(来自Rossix瑞典]的磷脂TGT)、蛭素、Pefabloc Xa和CaCl2的混合物中,定量FX激活的速率;通过加入FX,开始FX激活,且允许在37℃进行30秒或60秒。通过将FX激活反应物稀释进冰冷的含有EDTA的缓冲液中,终止激活。使用FXa特异性的生色底物,通过在ELISA读数器中读出在405nM的吸光度,定量FXa的浓度。使用纯化的FXa制备的参考曲线,用于将吸光度转变成FXa浓度。从激活的rFVIII或PEG-rFVIII变体装配成的FIXa-rFVIIIa复合物的转化数,用于将FX激活速率转变成rFVIIIa浓度。
通过定量含有0.7nM rFVIII或PEG-rFVIII和0.13nM人α-凝血酶的混合物中rFVIIIa的起始(0-3分钟)形成,测量凝血酶-催化的rFVIII激活的速率。FVIIIa的形成在时间上是线性的。将FVIIIa激活的速率表达为每摩尔最初存在的rFVIII每分钟形成的摩尔rFVIIIa(v/[rFVIII]0)。
rFVIII的O-联糖PEG化没有影响凝血酶-催化的rFVIII激活的速率或在有激活的rFVIII存在下FIXa-催化的FX激活的km或kcat(参见表3)。此外,O-联糖PEG化没有影响rFVIIIa-FIXa相互作用的表观Kd(K1/2FIXa)。
图4显示了使用各种aPTT试剂O-糖PEG化的rFVIII的凝固活性。数据表示为凝固活性和生色活性之间的比(A)或比凝固活性(B)。显示了来自3个独立实验的值的平均值和标准差。
表3:rFVIII激活的速率和FIXa对FX激活的动力学常数
数据是3-6次测量的平均值和标准差。
实施例6
糖PEG化的B-结构域缺失的(BDD)-FVIII在FVIII KO 小鼠和vWFKO小鼠中的药物代谢动力学
在给FVIII KO小鼠静脉内施用280IU/kg后,研究用各种PEG大小糖PEG化的BDD-FVIII的药物代谢动力学。
研究了下述化合物: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和N-聚糖0129-0000-1008-1A)、BDD-FVIII-80K PEG(N-聚糖,0129-0000-1012,O-聚糖0129-0000-1009)。
动物研究的设计:
基于C57Bl/6背景下外显子16KO,在Taconic M&B繁殖因子VIII敲除的(FVIII KO)小鼠。采用重约25g且年龄在19-26周范围内的雄性和雌性(约1∶1)的混合物。小鼠不完全回交。在该小鼠品系中没有检测到FVIII。
在尾静脉中给小鼠单次静脉内注射280IU/kg上面列出的化合物。如果在静脉周给小鼠给药,则小鼠与另一只小鼠互换。给药后,使用未包被的毛细管玻璃管,从给药前至给药后64小时,收集眶丛血样。从每只小鼠采集3个样品,且在每个时点收集2、3或4个样品。在柠檬酸钠(9∶1)中稳定化血液,并在FVIII COA SP缓冲液(1∶4)中稀释,然后在4000g离心5分钟。在干冰上冷冻从稀释的血液得到的血浆,在借助于FVIII生色活性和/或FVIII抗原分析进行定量分析之前,在-80℃保存。
定量血浆分析:
使用来自Coatest SP试剂盒(Chromogenix)的试剂,测定FVIII生色活性。稀释的血浆样品、在Coatest SP-缓冲液中的校准物(ILS校准血浆)和缓冲液负对照(50μl)一式两份地加入96-孔微量滴定板(Nunc)。以5∶1∶3(体积∶体积∶体积),混合来自Coatest SP试剂盒的因子IXa/因子X试剂、磷脂试剂和CaCl2,且将75μl混合物加入孔中。在室温温育15分钟后,加入50μl因子Xa底物S-2765/凝血酶抑制剂I-2581混合物,且在室温温育反应物10分钟,然后加入25μl 2%柠檬酸。在Spectramax微量滴定板读数器(Molecular Devices)上,测量在405nm的吸光度。从通过稀释校准的国际血浆标准品(ILS)制备的校准曲线,计算血浆样品中的FVIII活性。
FVIII抗原测定是可从Diagnostica Stago(Asserachrom VIII:CAg)商业得到的ELISA试剂盒,其中使用2种针对人FVIII的轻链的单克隆抗体。在试剂盒提供的coatest SP稀释缓冲液中稀释校准物(化合物的稀释物)或血浆样品至少50-倍,应用于预包被的孔,且根据生产商的说明书进行ELISA。用于报告药物代谢动力学研究的值基于从化合物本身制备的标准曲线。
药物代谢动力学参数估计:
使用ILS作为校准物(基于生色活性的数据),使用化合物本身作为校准物(基于ELISA的数据),通过数据的无房室法(NCA),进行药物代谢动力学分析。从数据估计下述参数:Cmax(静脉内施用后的最大浓度,这是在第一个取样时点)、Tmax(静脉内施用后的最大浓度的时间,这是第一个时点)、AUC0-∞(从时间0至无限的曲线下面积)、T1/2,(末端半衰期)、CL(清除率)和Vss(在稳态时的分布体积)。使用WinNonlin Proversion 4.1进行所有计算。
给FVIII KO小鼠静脉内注射280IU/Kg BDD-FVIII、BDD-FVIII-10KDa PEG、BDD-FVIII-40KDa PEG、BDD-FVIII-2x40KDaPEG和BDD-FVIII-80KDa PEG后,在7.8h(BDD-FVIII)至15-16h的范围内,半衰期随着渐增的PEG大小而增加(表4),这对应着2-倍增加。类似地,清除率降低,且MRT随着渐增的PEG大小而增加(表4)。
表4:基于生色活性对静脉内施用给FVIII KO小鼠后用不同大小的PEG糖PEG化的FVIII的药物代谢动力学参数估计(BDD:B-结构域缺失的)
结论:
给FVIII KO小鼠静脉内施用280IU/kg后,与BDD-FVIII相比,BDD-FVIII的糖PEG化使T1/2增加至1.3-2.1倍。随着PEG基团的大小在10KDa至80KDa PEG的范围内增加,观察到渐增的T1/2。
实施例7
在FeCl3诱导的A型血友病小鼠损伤模型中40K-PEG-[O]-N8与Advate相比延长的止血作用
在FeCl3诱导的血友病A(F8-KO)小鼠损伤模型中,研究了40K-PEG-[O]-N8与重组FVIII(Advate)相比的作用持续时间。麻醉小鼠,且将其放置在加热垫(37℃)上以维持体温。暴露出颈动脉,且将通过超声测量血流量的流量探测器(0.5PSB Nanoprobe)放置在动脉周围。通过将在10%FeCl3溶液中简短浸渍的滤纸(2×5mm)应用在暴露的颈动脉周围,诱导损伤(一种铁介导的化学氧化)。3分钟后取下滤纸。然后用0.9%NaCl洗涤动脉3次,且最后应用Surgilube (一种声耦合器),以置换流量探测器中的空气,并确保血流量的最佳测量。取下FeCl3饱和的滤纸后,记录血流量(ml/分钟)25分钟,且通过测量从取下FeCl3饱和的滤纸到血流量为0ml/分钟的时间(按分钟计算),测定闭塞时间。如果25分钟后没有发生闭塞,则将闭塞时间记录为25分钟,尽管在观察时间段期间没有发生闭塞。用Advate(280U/kg)、40K-PEG-[O]-N8(280U/kg)或载体处理F8-KO小鼠(n=6-10)。在给药后5分钟(急性作用)或24、48、60和72小时,用FeCl3诱导损伤。取下FeCl3后,记录血流量(ml/分钟)25分钟,且随后测定闭塞时间。
在载体治疗的F8-KO小鼠中没有发生闭塞,然而在给药后5分钟(急性作用)在用40KDa-PEG-[O]-N8和Advate治疗的所有小鼠中发生闭塞,平均闭塞时间分别是4.3±0.4分钟和5.2±0.7分钟。在40KDa-PEG-[O]-N8治疗的F8-KO小鼠中,在给药后72小时,平均闭塞时间增加至13.8±3.4分钟。相反,Advate治疗的F8-KO小鼠在24和48小时后分别具有13.0±3.4分钟和15.9±2.9分钟的闭塞时间。重要的是,在施用Advate后60和72小时,没有观察到闭塞。在用40KDa-PEG-[O]-N8治疗的所有小鼠中,在给药后24小时观察到闭塞,然而仅67%的用Advate治疗的小鼠闭塞。72小时后,在63%的用40KDa-PEG-[O]-N8治疗的小鼠中仍然观察到闭塞,然而在施用Advate后60和72小时没有观察到闭塞。
40KDa-PEG-[O]-N8在F8-KO小鼠中延长的作用
在给药280IU/kg 40KDa-PEG-[O]-N8、280IU/kg Advate或载体后5分钟(急性作用)、24、48、60和72小时,用FeCl3诱导损伤。取下FeCl3后,记录血流量(ml/分钟)25分钟,且随后测定闭塞时间。在给药后60和72小时,在施用Advate的小鼠中不发生闭塞。显示了每组6-10只小鼠的平均值和SEM。使用Kruskal-Wallis检验,其包括Dunn氏后检验(post test),对比不同组之间的闭塞时间。*:p<0.05;**:p<0.01。
总之,在FeCl3诱导的F8-KO小鼠损伤模型中,与Advate相比,40KDa-PEG-[O]-N8的止血作用显著延长。
Claims (13)
1.具有改变的循环半衰期的B-结构域截短的因子VIII分子,所述分子在截短的B结构域中通过O-联寡糖与亲水聚合物共价缀合,其中所述截短的B结构域的长度为10-700个氨基酸,且其中(i)因子VIII激活导致共价缀合的亲水聚合物的去除;和(ii)FVIII前体多肽的重链和轻链部分被接头分开,其中所述接头的序列源自FVIII B结构域。
2.根据权利要求1的分子,其中所述B结构域的长度为20-30个氨基酸。
3.根据权利要求1-2中任一项的分子,其中所述亲水聚合物是PEG。
4.根据权利要求3的分子,其中所述PEG的大小是10,000至160,000Da。
5.根据权利要求4的分子,其中所述PEG的大小是40,000Da。
6.根据权利要求1-2中任一项的分子,其包含如SEQ ID NO 2所述的氨基酸序列。
7.药物组合物,其包含根据权利要求1-6中任一项的分子。
8.制备根据权利要求1-6中任一项的分子的方法,其中所述方法包含,通过截短的B结构域中的O-联寡糖,给B-结构域截短的因子VIII分子缀合亲水聚合物,其中所述截短的B结构域的长度为10-700个氨基酸。
9.通过根据权利要求8的方法可得到的分子。
10.根据权利要求1-6和9中任一项的分子在生产治疗血友病的药物中的用途。
11.根据权利要求1-6和9中任一项的分子或根据权利要求7的药物组合物在生产用于治疗血友病的皮下药物中的用途。
12.根据权利要求1-6和9中任一项的分子或根据权利要求7的药物组合物在生产用于治疗血友病的静脉内药物中的用途。
13.工程改造根据权利要求1-6或9中任一项的B-结构域截短的因子VIII分子的方法,所述方法包含,(i)截短B-结构域,和任选地对该截短的因子VIII分子的氨基酸序列进行鉴定潜在的O-联糖基化位点的分析,(ii)在合适的宿主细胞中生产该分子,和(iii)选择在截短的B-结构域中具有O-联聚糖的分子,其中所述截短的B结构域的长度为10-700个氨基酸。
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