CN101981053B - 聚乙二醇化的AβFAB - Google Patents
聚乙二醇化的AβFAB Download PDFInfo
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- CN101981053B CN101981053B CN2008800026216A CN200880002621A CN101981053B CN 101981053 B CN101981053 B CN 101981053B CN 2008800026216 A CN2008800026216 A CN 2008800026216A CN 200880002621 A CN200880002621 A CN 200880002621A CN 101981053 B CN101981053 B CN 101981053B
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Abstract
本发明描述了在预防上和治疗上治疗与Aβ肽活性相关的病症的方法。该方法使用在第13-28位氨基酸之间特异结合人Aβ肽的人源化抗体片段,其中所述抗体片段共价结合聚乙二醇(PEG)分子。
Description
本发明涉及结合淀粉样蛋白β(Aβ)肽且共价结合一个或多个聚乙二醇(PEG)分子的抗体片段。
环状的Aβ肽由前体蛋白质淀粉样蛋白前体蛋白质(APP)切割产生的39-43个氨基酸(大部分为40或42个氨基酸)组成。Aβ从可溶形式向具有高β折叠含量的不可溶形式的转换以及其在脑中作为神经炎斑点和脑血管斑点的沉积似乎与许多病症和疾病(包括阿尔茨海默氏病、唐氏综合症和脑淀粉样血管病(CAA))相关。预防和/或逆转Aβ的沉积可治疗与Aβ肽相关的病症。
影响Aβ沉积的治疗剂包括针对Aβ肽的抗体,例如在WO 2001/62801、WO2004/071408和Tamura,Y.,等,Neurobiol.of Dis.(2005)20:541-545中讨论的人源化抗体和片段。
尽管许多抗体及其衍生物可用于诊断和治疗,但对于特定应用而言常常无法获得理想的抗体药物代谢动力学。旨在治疗与Aβ肽相关的多种病症和疾病的治疗性抗体通常为具有完整Fc区的免疫球蛋白。Fc区负责延长血浆中抗体的半衰期。然而,由于这种延长阻止了有效清除与靶肽结合的抗体,导致抗原抗体复合体在血浆循环中延时存在,因此该延长可能是不利的。随后施用抗体导致血浆中不想要复合体的进一步积累。抗体的Fc部分可能具有某些不必要的效应子作用,因此需要进行修饰以去除这种作用。此外,Fc部分向整体治疗添加了显著的大小,所述治疗常产生与递送路径、递送装置和扩大制备过程相关的问题。
已在体内研究了包括Fab在内的无Fc部分的抗体片段以测定是否此类片段可能为潜在的治疗法。然而,研究提示由于清除率快和半衰期短,所以限制了涉及如Fab的片段的治疗有效性。因此,需要有活性的治疗性抗Aβ肽抗体分子,所述抗体分子具有这样的药物代谢动力学和药物动力学,其允许改善给药方案而避免由血浆中复合体的形成产生和潜在的效应子作用的潜在副作用。
本发明克服了与可能靶向Aβ肽的治疗性抗体或抗体片段相关的许多问题。本发明的化合物包括结合Aβ且共价结合一个或多个聚乙二醇(PEG)分子的抗体片段。这些化合物可在细菌或酵母细胞系统中产生,所述细菌或酵母细胞系统消除了与哺乳动物细胞系中产生抗体相关的多种问题,例如成本问题、纯化问题和污染内源产生的抗原问题。此外,本发明的化合物可经皮下施用并具有理想的药物代谢动力学(PK)和药物动力学(PD)谱,同时保持抗体片段对Aβ亲和力和选择性。
非常无法预测且意外地,申请人还发现共价结合PEG分子至抗体片段的互补决定区(CDR)未改变抗体片段对Aβ的活性、亲和力或选择性。
本发明提供包含抗体片段的分子,所述抗体片段在第13-28位氨基酸之间特异性结合人Aβ肽,其中抗体片段共价结合PEG分子。优选地,抗体片段为Fab片段。
在一个实施方案中,本发明提供包含抗体片段的分子,所述抗体片段具有重链可变区和轻链可变区,其中所述轻链可变区包含具有以下氨基酸序列的CDR区:CFRL1:SSSQSLIYSDGNAYLH(SEQ ID NO:6)、CDRL2:KVSNRFS(SEQ ID NO:7)和CDRL3:TQSTHSPWT(SEQ IDNO:8)且其中所述重链可变区包含具有以下氨基酸序列的CDR区:CDRH1:GYTFSRYSMS(SEQ ID NO:9)、CDRH2:QINIRGCNTYYPDTVKG(SEQ ID NO:10)或QINIRGNNTYYPDTVKG(SEQ ID NO:11)和CDRH3:GDF(SEQ ID NO:12)。优选地,此类分子具有共价结合抗体片段重链可变区或轻链可变区的PEG分子。更优选地,此类分子具有共价结合CDR的PEG分子。甚至更优选地,此类分子具有共价结合CDR内半胱氨酸残基的PEG分子。最优选地,此类分子具有共价结合抗体片段重链可变区CDRH2:QINIRGCNTYYPDTVKG(SEQ ID NO:10)的PEG分子。
在另一实施方案中,本发明提供包含抗体片段的分子,所述抗体片段具有轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:2。优选地,此类分子具有共价结合抗体片段的重链可变区或轻链可变区的PEG分子。更优选地,此类分子具有共价结合抗体片段重链可变区的CDR的PEG分子。甚至更优选地,此类分子具有共价结合抗体片段的重链可变区的CDR内半胱氨酸残基的PEG分子。最优选地,此类分子具有共价结合重链可变区SEQ ID NO:2的第56位氨基酸处的半胱氨酸的PEG分子。
在另一实施方案中,本发明提供包含Fab片段或ScFv片段的分子,其中Fab片段或ScFv片段共价结合PEG分子并具有轻链可变区SEQ IDNO:1和重链可变区SEQ ID NO:2。优选地,此类分子具有共价结合重链可变区SEQ ID NO:2的第56位氨基酸处的半胱氨酸的PEG分子。同样优选地,在此类分子中PEG的分子量约为0.5kD至30kD,更优选为20kD。
在另一实施方案中,本发明提供包含具有轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:2的抗体片段的分子,其中所述抗体片段在重链可变区SEQ ID NO:2的第56位处共价结合20kD的PEG分子。优选地,在此类分子中PEG分子经马来酰亚胺键进行共价结合。
在另一实施方案中,本发明提供包含在第13-28位氨基酸间特异结合人Aβ肽的抗体片段的分子,其中所述抗体片段共价结合PEG分子并具有轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:3。优选地,此类分子具有共价结合抗体片段绞链区的PEG分子。更优选地,所述PEG经马来酰亚胺键共价结合绞链区。
本发明还包括包含抗体片段的分子,所述抗体片段优选为人源化抗体片段,其中PEG分子共价结合抗体片段,所述抗体片段产生具有允许每周给药方案的药物代谢动力学和药物动力学的活性治疗分子,同时将可能是由血浆中复合体形成造成的潜在副作用最小化且保留或改善抗体片段对Aβ的活性、亲和力和选择性。
本发明还包括治疗、预防或逆转与Aβ肽相关的病症和疾病的方法,所述病症和疾病包括临床前和临床(pre-clinical and clinical)阿尔茨海默氏病、唐氏综合症和临床前和临床脑淀粉样血管病(CAA)认知缺陷、中风、脑溢血和一般性精神衰弱。这些方法包含向受试者施用有效量的此处描述和要求保护的分子。
本发明提供包含在第13-28位氨基酸间特异结合Aβ肽的抗体片段的分子,其中所述抗体片段共价结合PEG分子。我们已发现PEG分子共价连接结合Aβ的抗体片段未负面地改变抗体片段对Aβ的活性、亲和力或选择性。更令人惊奇地,我们发现具有高达20kD分子量的PEG分子共价连接结合Aβ的抗体片段的CDR也未负面地改变抗体片段对Aβ肽的活性、亲和力或选择性。这些抗体片段可经皮下施用且具有用于支持可变通给药方案的治疗用途的改善的PK/PD谱。此外,这些抗体片段可在细菌或酵母细胞系统中产生,所述细菌或酵母细胞系统消除了与在哺乳动物细胞中制备全长抗体相关的多种问题。本发明的聚乙二醇化抗体片段提供了在预防和治疗上预防和治疗人中与Aβ肽相关的病症的可能性。
天然存在的全长抗体为包含四条肽链的免疫球蛋白分子,所述四条肽链为通过二硫键互相连接的两条重(H)链(全长时约为50-70kDa)和两条轻(L)链(全长时约为25kDa)。每一条链的氨基端部分包括主要负责抗原识别的约100-110个或更多个氨基酸的可变区。每一条链的羧基端部分定义主要负责效应子作用的恒定区。
轻链分为κ或λ且特征为具有特定的恒定区。每一条轻链包含N端轻链可变区(此处为“LCVR”)和包含一个结构域CL的轻链恒定区。重链分为γ、μ、α、δ或ε,且分别将抗体的同种型定义为IgG、IgM、IgA、IgD和IgE,并且这些中的几种可进一步分为亚类(同种型)例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。每一种重链类型的特征在于特定的恒定区。每一重链包含N端重链可变区(此处为“HCVR”)和重链恒定区。对IgG、IgD和IgA而言,重链恒定区包含3个结构域(CH1、CH2和CH3);而对IgM和IgE而言,重链恒定区包含4个结构域(CH1、CH2、CH3和CH4)。
HCVR区和LCVR区可进一步细分为高变区,命名为互补决定区(“CDR”),其上散布更保守的命名为框架区(“FR”)的区域。每一个HCVR和LCVR由三个CDR和四个FR组成,自氨基端向羧基端按如下顺序排列:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。此处重链的3个CDR称为“CDRH1、CDRH2和CDRH3”并且轻链的3个CDR称为“CDRL1、CDRL2和CDRL3”。CDR包含与抗原形成特异性相互作用的大部分残基。HCVR和LCVR区内的CDR氨基酸残基的编号及位置与众所周知的Kabat编号规定一致。
每一条轻-重链对的可变区形成抗体的抗原结合位点。如此处所用,“抗原结合部分”或“抗原结合区”或“抗原结合结构域”或“抗原结合位点”可相互取代地指代抗体分子的部分,其包含与抗原相互作用且赋予抗体对抗原的特异性和亲和力的氨基酸残基。该抗体部分包括维持抗原结合残基正常构象所必需的“框架”氨基酸残基。优选地,本发明抗体的框架区为人起源或基本为人起源(至少为80%、85%、90%、95%、96%、97%、98%或99%人起源)且遵循Kabat编号。或者,抗原结合区可来自人序列。
如此处所用,术语“抗体片段”指保留特异结合抗原(例如Aβ)能力的抗体的一个或多个片段。包含在术语抗体的“抗体片段”中的分子的实例包括(i)Fab片段,由VL、VH、CL和CH1结构域组成的单价片段;(ii)F(ab′)2片段,包含在绞链区由二硫键连接的两个Fab片段的二价片段;(iii)由VH和CH1结构域组成的Fd片段;(iv)由抗体单个臂的VL和VH结构域组成的Fv片段,和(v)dAb片段(Ward,等,(1989)Nature341:544-546),其由VH结构域组成。此外,尽管Fv片段的两个结构域VL和VH由不同基因编码,但可使用重组方法通过合成接头(该接头使它们形成单条蛋白质链,其中VL区和VH区配对以形成单价分子)将它们连接起来(称为单链Fv(scFv);参阅,例如Bird等(1988)Science242:423-426:和Huston等(1988)Proc.Natl.Acad.Sci.USA85:5879-5883)。此类单链抗体也旨在包含于术语“抗体片段”中。术语“抗体片段”也包含单链抗体的其他形式,例如双体(diabody)。双体是二价双特异性结合蛋白质,其中VH和VL结构域表达于单条多肽链上,但使用的接头太短以至于无法在同一条链的两个结构域之间形成配对,因此迫使该结构域与另一条链的互补结构域配对并形成两个抗原结合位点(参阅,例如Holliger,P.,等(1993)Proc.Natl.Acad.Sci.USA 90:6444-6448;Poljak,R.J.,等(1994)Structure 2:1121-1123)。
更进一步,抗体或其抗体片段可为通过抗体或抗体片段与一种或多种其他蛋白质或肽共价或非共价结合形成的更大免疫粘着分子的一部分。此类免疫粘着分子的实例包括使用链霉抗生物素蛋白核心区以产生四聚体scFv分子(Kipriyanov,S.M.,等(1995)Human Antibodies andHybridomas 6:93-101),和使用半胱氨酸残基、标记肽和C端多组氨酸标签以产生二价和生物素化的scFv分子(Kipriyanov,S.M.,等(1994)Mol.Immunol.31:1047-1058)。可使用常规技术如整个抗体的木瓜蛋白酶或胃蛋白酶消化分别从整个抗体中制备抗体片段,例如Fab和F(ab′)2片段。此外,如本领域中众所周知,可使用标准重组DNA技术获得抗体、抗体片段和免疫粘着分子。抗体、抗体片段和免疫粘着分子可为糖基化或非糖基化的且仍落在本发明的范围内。优选地,抗体片段为Fab片段。
术语“人源化抗体”指部分或全部由来自人抗体种系的氨基酸序列或重排序列组成的抗体,其通过改变具有非人CDR的抗体序列获得。可变区的框架区可由对应的人框架区取代。人框架区包括基因组框架区以及包含一个或多个氨基酸取代的那些区域。具体而言,此类取代包括其中由来自非人CDR的天然框架相应位置的氨基酸取代人框架中特定位置的氨基酸的突变。例如,具有小鼠CDR的人源化抗体可包含用相应的小鼠框架氨基酸取代特定的人框架氨基酸的一处或多处取代。其他描述参与人源化可能使用的小鼠抗体的方法的参考是例如Queen等,Proc.Natl.Acad.Sci.USA 88:2869,1991;美国专利号5,693,761;美国专利号4,816,397;美国专利号5,225,539;在Levitt,M.,J.Mol.Biol.168:595-620,1983中描述的计算机程序ABMOD和ENCAD;人源化可基本上按Winter及其同事的方法进行(Jones等,Nature,321:522-525,1986;Riechmann等,Nature,332:323-327,1988;Verhoeyen等,Science,239:1534-1536,1988)。优选地,本发明的抗体为人源化抗体片段。更优选地,本发明的抗体是人源化抗体fab片段。
本发明还包括共价结合一个或多个PEG分子的抗体片段。希望术语“聚乙二醇”和“PEG”可相互取代使用且指代本领域中公知的聚乙二醇或其衍生物(参阅,例如美国专利号:5,445,090;5,900,461;5,932,462;6,436,386;6,448,369;6,437,025;6,448,369;6,495,659;6,515,100和6,514,491)。优选地,PEG共价结合抗体片段的一个或多个赖氨酸或半胱氨酸残基。更优选地,PEG共价结合抗体片段重链可变区中的一个或多个赖氨酸或半胱氨酸残基。甚至更优选地,PEG共价结合抗体片段CDR内的一个或多个赖氨酸或半胱氨酸残基。最优选地,PEG结合所述重链可变区SEQ ID NO:2的第56位氨基酸的半胱氨酸残基。或者,PEG分子可经连接抗体片段绞链区的接头或间隔分子结合到抗体片段上。向绞链区添加接头和间隔分子是本领域众所周知的。此外,PEG可通过本领域公知的技术共价结合到抗体片段的被修饰非天然氨基酸上。
在其一般形式中,“PEG”为具有末端羟基的线性聚合物且具有分子式HO CH2CH2-(CH2CH2O)n-CH2CH2-OH,其中n从约为8至约为4000。端基氢可由保护基团如烷基或链烷醇基团(M-PEG取代。优选地,PEG具有至少一个羟基,更优选地其为末端羟基。优选激活该羟基以与肽反应。使用多种化学修饰来制备具有功能性基团(例如活性碳酸盐、活性酯、醛、tresylate或使用适合于偶联给定靶分子的PEG丙醛)的活性PEG衍生物。活性PEG衍生物随后共价结合多肽药物的反应性基团。有许多用于本发明的PEG形式。本领域中存在大量PEG衍生物且均适合在本发明中使用。共价结合本发明抗体片段的PEG分子不旨在局限于具体类型或大小。PEG分子量优选从约0.5千道尔顿(kD)至约100kD,更优选地从约5kD至约30kD,最优选地从约1kD到约20kD。PEG可为线性的或分支的,且本发明的抗Aβ肽抗体片段可具有1、2、3、4、5或6个与肽结合的PEG分子。最优选为一个PEG分子抗体片段;然而,当每个肽分子存在多于一个PEG分子时,优选为不多于六个。本发明还涵盖PEG分子的两端均适合与两个或多个抗Aβ肽抗体片段分子共同交联。将PEG分子结合到蛋白质、抗体及其片段的方法在本领域是众所周知的。
如此处所用的术语“KD”旨在指特定抗体-抗原相互作用的解离常数。根据方程式:KD=koff/kon(以M测定)计算。如此处所用术语“kon”旨在指结合速率常数,或前者或复合体形成反应的反应比速(specific reactionrate),以单位:M-1sec-1测定。如此处所用术语“koff”旨在指抗体从抗体/抗原复合体上解离的解离速率常数或反应比速,以单位:sec-1测定。
如此处所用的术语“特异结合”指其中特异结合对中的一个成员不显著地结合除它特异结合的一个或多个配偶体之外的其他分子的情况。该术语也适用于例如本发明抗体的抗原结合结构域对于由许多抗原携带的特定表位是特异的,在这种情况下带有抗原结合结构域的特异性抗体将能够结合带有该表位的多种抗原。因此,本发明的分子特异结合Aβ肽,而不特异结合APP。此外,本发明的分子在线性、非线性或包含氨基酸HHQKLVFFAEDVGSNK(13-28)(SEQ ID NO:4)的构象Aβ表位间特异地结合。
参考本发明的分子,术语“活性”包括但不限于表位/抗原亲和力和特异性、在体内或体外中和或拮抗Aβ肽活性的能力、IC50、抗体的体内稳定性和抗体的免疫特性。本领域中抗体识别的其他可识别的生物学特性或特征包括例如交叉反应性、(即通常与靶肽的非人同源物、或与其他蛋白质或组织的交叉反应性)、和在哺乳动物细胞中保持蛋白质高表达水平的能力。可使用包括但不限于ELISA、竞争性ELISA、Biacore或KinExA表面等离子共振分析、不受限的体外或体内中和试验、受体结合、细胞因子或生长因子产生和/或分泌、信号转导和对于来自包括人、灵长类或任何其他来源的不同来源的组织切片的免疫组织化学在内的领域认可的技术观察、测定或评估前文提到的特性或特征。
此处可互相取代使用的术语“个体”、“受试者”和“患者”指哺乳动物,优选为人。在特定实施方案中,受试者特征还在于患有将从Aβ肽活性下降中受益的疾病或障碍或病症。
如此处所用,表述“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互相取代使用并包括单独的细胞或细胞培养物,其为本发明任何分离的多核苷酸或包含编码HCVR、LCVR或本发明单克隆抗体的序列的任何一个或多个重组载体的接受者。宿主细胞包括单个宿主细胞的后代。由于自然的、意外的或有目的性的突变和/或改变,后代可能在形态或总DNA补体上与原始亲本细胞不完全一致。宿主细胞包括用表达本发明的抗体片段或其轻链或重链的重组载体或多核苷酸转化的、转导的或感染的细胞。包含本发明重组载体的宿主细胞无论是否稳定整合入宿主染色体,也可称为“重组宿主细胞”。用于产生本发明宿主细胞的优选细胞为CHO细胞(例如ATCC CRL-9096)、NS0细胞、SP2/0细胞、COS细胞(ATCC例如,CRL-1650、CRL-1651)和HeLa(ATCC CCL-2)。本发明中使用的另外的宿主细胞包括植物细胞、酵母细胞、其他哺乳动物细胞和原核细胞。更优选地,本发明中使用的细胞为酵母或原核细胞。
术语“涉及Aβ肽的病症或疾病”或“与具有Aβ活性的疾病相关的病症”意指包括所有与以下相关的病症、病症和疾病:1)脑中β淀粉样蛋白斑的发展,2)Aβ异常形式的合成,3)Aβ独特毒性形式的形成,或4)异常的Aβ合成速率、降解速率或清除速率。已知或推测如阿尔茨海默氏病、唐氏综合症、脑淀粉样血管病、某些血管性痴呆和轻度认知缺损的病症和疾病与Aβ具有这样的关系。
本发明提供包含在第13-28位氨基酸间特异结合Aβ肽的抗体片段的分子,其中所述抗体片段共价结合到PEG分子上。该抗体片段优选为人源化抗体片段,如Fab片段和/或scFv片段。最优选地,该抗体片段为Fab片段。本发明的分子特异结合Aβ肽允许所述分子待用于治疗与Aβ肽相关的疾病和病症,即从抑制Aβ肽的生物活性中受益的病症、疾病或病症。
在本发明的一个实施方案中,抗体片段具有重链可变区和轻链可变区,其中轻链可变区包含具有以下氨基酸序列的CDR区:CDRL1:SSSQSLIYSDGNAYLH(SEQ ID NO:6)、CDRL2:KVSNRFS(SEQ IDNO:7)和CDRL3:TQSTHSPWT(SEQ ID NO:8),和/或其中重链可变区包含具有以下氨基酸序列的CDR区:CDRH1:GYTFSRYSMS(SEQ IDNO:9)、CDRH2:QINIRGCNTYYPDTVKG(SEQ ID NO:10)或QINIRGNNTYYPDTVKG(SEQ ID NO:11),和CDRH3:GDF(SEQ IDNO:12)。优选地,本发明抗体片段的六个CDR共同存在。包含本发明CDR的组合物通常为抗体重链或轻链序列或其基本部分,其中CDR位于与Kabat编号一致的位置上。在框架区内提供由以下化学式呈现的作为连续序列的每条重链和轻链的三个CDR区:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。当以有序状态与CDR排列为连续序列时,重链或轻链FR1、FR2、FR3和FR4结合形成抗体片段的完整框架区。优选地,本发明抗体的框架区为人起源或基本为人起源(即超过约80、82、85、87、90、92、95、97%)。
优选地,本发明的抗体片段包含LCVR和HCVR,
所述LCVR包含以下序列的肽:
DIVMTQTPLSLSVTPGQPASISCSSSQSLIYSDGNAYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCTQSTHSPWTFGGGTKVEIK(SEQ ID NO:1),
所述HCVR包含具有选自以下序列的序列的肽:
EVQLVESGGGLVKPGGSLRLSCAASGYTFSRYSMSWVRQAPGKGLEWVGQINIRGCNTYYPDTVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGDFWGQGTLVTVSS (SEQ ID NO:2)
EVQLVESGGGLVKPGGSLRLSCAASGYTFSRYSMSWVRQAPGKGLEWVGQINIRGNNTYYPDTVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGDFWGQGTLVTVSS(SEQ ID NO:3);
或者,抗体片段包含LCVR(包含具有由SEQ ID NO:1组成的序列的肽)和HCVR(包含具有选自SEQ ID NO:2或SEQ ID NO:3的序列的肽),其中所述HCVR和LCVR共同存在于抗体片段中。本领域技术人员将理解本发明的抗体片段不局限于HCVR和LCVR的特异性序列,而且还包括这些序列的变体,所述变体当在本发明的分子中存在时,其保持或改善抗原结合能力和亲本抗体的至少一种其他功能特性,例如表位特异性、与亲本抗体竞争结合Aβ肽的能力、结合Aβ肽的IC50和/或KD或koff值。
在本发明的另一实施方案中,所有可变区或部分可变区受限于由SEQID NO:1显示的特殊LCVR序列和在SEQ ID NO:2或SEQ ID NO:3中显示的HCVR,并且进一步的特征在于其在体内或体外拮抗或中和至少一种Aβ肽活性。本发明抗体进一步的特征在于其特异结合人Aβ肽但不结合人APP,所述抗体中所有可变区或部分可变区受限于由此处的LCVR SEQID NO:1,和HCVR SEQ ID NO:2或SEQ ID NO:3中显示的特殊序列。
在本发明的一个方面中,PEG(或其衍生物)共价结合抗体片段的一个或多个赖氨酸、半胱氨酸或非天然修饰的氨基酸残基。优选地,PEG分子共价结合抗体片段的重链可变区或轻链可变区。更优选地,此类分子具有共价结合到抗体片段重链可变区的CDR上的PEG分子。最优选地,此类分子具有共价结合重链可变区SEQ ID NO:2的第56位氨基酸处的半胱氨酸的PEG分子。或者,PEG分子可经针对抗体片段绞链区的接头或间隔分子结合到抗Aβ肽Fab抗体片段上。
在本发明的另一方面中,PEG分子共价结合本发明抗体的一个或多个改造的赖氨酸、半胱氨酸或非天然修饰的氨基酸残基,以取代抗体分子上存在的糖基化而不显著影响抗体片段对Aβ的亲和力和选择性。优选地,PEG分子在抗体片段的重链可变区或轻链可变区上取代糖基化信号。更优选地,PEG分子在抗体片段重链可变区的CDR上取代糖基化信号。最优选地,PEG分子在重链可变区SEQ ID NO:2的第56位处取代糖基化信号。
在本发明中共价结合到抗体上的PEG分子不旨在局限于特定的类型或大小。PEG分子量优选从约0.5kD至约100kD,更优选地从约0.5kD至约30kD,最优选地从约1kD到约20kD。或者PEG分子量可选自约0.5kD、约1kD、约5kD、约10kD和约20kD。PEG可为线性的或分支的,且本发明的聚乙二醇化的抗Aβ肽抗体可具多于一个结合到肽上的PEG分子。优选地,每一聚乙二醇化的抗Aβ肽抗体具有一个PEG分子。
更优选地,本发明的抗体分子包含具有轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:2的抗体片段,其中所述抗体片段在重链可变区SEQ ID NO:2的第56位处共价结合到20kD的PEG分子上。
本发明抗体结合的抗原性Aβ肽表位是包含氨基酸HHQKLVFFAEDVGSNK(SEQ ID NO:4)的线性、非线性或构象表位。结合所述表位的抗体与它们结合的APP相比特异且优先结合Aβ肽。本发明的单克隆抗体结合Aβ肽比它结合人APP高出至少2、5、10、20、30、40、50、60、70、80、90或100倍(例如更高亲和力或更高特异性);更优选地比它结合APP高出至少150、200、250、300、350、400、450、500、550或600倍,甚至更优选地它在高于如ELISA试验、竞争性ELISA试验或Biacore或KinExA试验中的KD值测定的背景水平的水平上不结合APP。
本发明的抗体片段结合氨基酸HQKLVFFAEDVGSNK(SEQ ID NO:5)间的表位比不包含氨基酸HQKLVFFAEDVGSNK(SEQ ID NO:5)的表位高出至少2、5、10、20、30、40、50、60、70、80、90或100倍(例如更高亲和力或更高特异性)。更优选地,比不包含氨基酸HQKLVFFAEDVGSNK(SEQ ID NO:5)的表位高出至少150、200、250、300、350、400、450、500、550或600倍,甚至更优选地它在高于如ELISA试验、竞争性ELISA试验测定的背景水平的水平上,或Biacore或KinExA试验中的KD值不结合不包含氨基酸HQKLVFFAEDVGSNK(SEQ ID NO:5)的表位。
在优选的实施方案中,本发明提供抗体片段,所述抗体片段对Aβ肽具有强亲和力,即结合Aβ肽或其包含序列HQKLVFFAEDVGSNK(SEQID NO:5)的部分[即与HQKLVFFAEDVGSNK多肽接触的抗体],具有通过KinExA方法测定的对人Aβ肽低于约200pM、100pM、50pM、40pM或30pM,优选地为低于20pM的结合亲和力(KD)。或者,对人Aβ肽的结合亲和力(KD)介于0.1pM-200pM之间。可按下文中实施例或本领域可得的其他方法中描述的进行测定抗体亲和力。
本发明抗体的给药途径可为口服、肠胃外、通过吸入或局部。优选地,本发明抗体可整合入适合肠胃外给药的药物组合物中。如此处所用的术语肠胃外包括静脉内、肌内、皮下、直肠、阴道、腹膜内施用。优选通过静脉内或腹膜内或皮下注射的外周全身给药。更优选地,本发明抗体的给药途径是经皮下注射。用于此类注射的合适载体在本领域是显而易见的。
本发明的抗体片段在血浆中较相应的抗Aβ肽全长抗体具有更短的半衰期,且较相应的抗Aβ肽全长抗体能更快地被从血浆中清除。或者,本发明的抗体较相应的非共价结合PEG分子的抗Aβ肽Fab片段具有更长的血浆半衰期,且较相应的非共价结合PEG分子的抗Aβ肽Fab片段能更慢地被从血浆中清除(实施例1、2和3)。参考如此处所用的抗体的术语“相应的”指具有相同LCVR和HCVR的抗体。例如,参考具有SEQ ID NO:1的LCVR和由SEQ ID NO:2组成的HCVR的抗体Fab片段的相应全长抗体将具有相同的SEQ ID NO:1的LCVR和由SEQ ID NO:2组成的HCVR,及完整的Fc结构域。
在另一方面,本发明涉及当表达时编码抗体的重组多核苷酸包含SEQID NO:1的LCVR和由SEQ ID NO:2组成的HCVR。由于密码子简并性,其他多核苷酸序列可容易被取代成这些序列。本发明特别优选的多核苷酸编码当表达时包含SEQ ID NO:6-8的轻链CDR,和SEQ ID NO:9、10或11和12的重链CDR,或SEQ ID NO:1-SEQ ID NO:3的任何可变区的抗体。编码SEQ ID NO:1的LCVR和SEQ ID NO:2的HCVR的多核苷酸序列的实例分别示于SEQ ID NO:13(LCVR)和SEQ ID NO:14(HCVR)中。
多核苷酸通常还将包括有效连接到人源化免疫球蛋白编码序列上的表达控制多核苷酸,其包含天然结合的或异源的启动子区。优选地,表达控制序列将为在能够转化或转染真核细胞的载体中的真核启动子系统,但也可使用用于原核细胞的控制序列。一旦将载体整合进合适的宿主细胞系中,宿主细胞在适合表达核苷酸序列的条件下繁殖,并且如期望的,可接着收集和纯化轻链、重链、轻/重链二聚体或完整抗体、结合片段或其他免疫球蛋白形式。
可使用多种众所周知的技术从多种不同多核苷酸(基因组寡核苷酸或cDNA、RNA、合成寡核苷酸等)和组分(例如V、J、D和C区)形成能够最终表达所期望抗体或抗体片段的本发明的核酸序列。将合适的基因组序列和合成序列连接起来是制备的常用方法,但也可利用cDNA序列。
可根据众所周知的方法从多种人细胞中分离人恒定区DNA序列,但优选从不凋亡的B细胞中分离。可从本领域众所周知的多种来源中获得用于多核苷酸序列的合适的来源细胞和用于免疫球蛋白表达和分泌的宿主细胞。
除此处特别描述的人源化抗体或抗体片段外,可利用对本领域技术人员熟知的多种重组DNA技术容易地设计并制备其他“基本同源的”修饰后抗体。例如,框架区可通过几个氨基酸替代、末端和中部添加和缺失等而在一级结构水平上与天然序列不同。此外,多种不同的人框架区可单独或组合用作与本发明人源化抗体的基础。通常,可通过多种众所周知的技术如定向诱变来容易地完成基因修饰。
如前文陈述,当序列已有效连接到(即定位以确保功能)表达控制序列之后多核苷酸将在宿主中进行表达。这些表达载体通常作为游离基因或作为宿主染色体DNA的整合部分在宿主细胞中复制。通常,表达载体将包含选择标记,例如四环素或新霉素以允许检测那些转化有所期望DNA序列的宿主细胞。这些细胞的表达载体可包括表达控制序列,如复制起始区、启动子、增强子和必需的加工信息位点例如核糖体结合位点、RNA剪切位点、多腺苷酸化位点和转录终止序列。优选的表达控制序列是来自免疫球蛋白基因、SV40、腺病毒、牛乳头状瘤病毒、巨细胞病毒等的启动子。
可通过众所周知的方法(其根据细胞类型而有所不同)将包含目的多核苷酸序列(例如重链和轻链编码序列和表达控制序列)的载体转入宿主细胞。可使用本领域周知的技术将多种宿主用于表达本发明的抗体。优选的细胞系包括COS、CHO、SP2/0、NS0(可从如公共保藏处ATCC,美国典型培养物保藏中心Manassas,VA获得)和酵母细胞系。优选地,本发明的宿主细胞包含一种或多种包含本发明核酸分子的载体或构建体。本发明的宿主细胞是已引入本发明载体的细胞,所述载体包含编码本发明的LCVR的多核苷酸和/或编码本发明的HCVR的多核苷酸。本发明也提供已引入本发明的两个载体的宿主细胞;一个载体包含编码本发明抗体的LCVR的多核苷酸,一个载体包含编码存在于本发明抗体中的HCVR的多核苷酸,并且每一载体有效连接启动子序列。细胞类型包括哺乳动物细胞、细菌细胞、植物细胞和酵母细胞。优选地,细胞为CHO细胞、COS细胞、SP2/0细胞、NS0细胞、酵母细胞或任何优选细胞类型的衍生物或后代。
本发明的完整抗体、其二聚体、各个轻链和重链、或其他免疫球蛋白形式一旦表达,可根据本领域的标准方法对其进行纯化,所述方法包括硫酸铵沉淀、离子交换、亲和、反相、疏水性相互作用柱层析、凝胶电泳等。对药物使用而言,优选基本上至少约90%、92%、94%或96%同质性的基本纯免疫球蛋白,且最优选98%至99%或更高同质性的基本纯免疫球蛋白。肽一旦被部分纯化或纯化达到所期望的同质性,就可如此处所示在治疗上或预防上使用。
导致认知缺陷、中风、脑溢血和一般性精神衰弱的许多症状似乎与含Aβ肽的脑中神经炎和脑血管斑相关。这些病症为临床前和临床阿尔茨海默氏病、唐氏综合症、和临床前和临床脑淀粉样血管病(CAA)。淀粉样蛋白斑形成自Aβ肽。这些肽通常以与脂蛋白的复合形式在血液和脑脊液(CSF)中循环。循环形式的Aβ肽由从常见前体蛋白质,常命名为APP的淀粉样前体蛋白质剪切获得的39-43个氨基酸(主要为40或42个氨基酸)组成。可溶性AP的一些形式自身具有神经毒性且可决定神经变性和/或认知衰退的严重性(McLean,C.A.,等,Ann.Neturol.(1999)46:860-866;Lambert,M.P.,等(1998)95:6448-6453;Naslund,J.,J.Am.Med.Assoc.(2000)283:1571)。
因此,包含本发明分子的药物组合物可用于治疗或预防这样的病症,其中Aβ肽的存在引起或促进不期望的病理学效果或Aβ肽活性的降低在哺乳动物优选人中具有治疗益处,所述病症包括但不限于临床或临床前阿尔茨海默氏病、唐氏综合症、临床或临床前淀粉样蛋白血管病(CAA)、前驱阿尔茨海默氏病、轻度认知缺损(MCI)和认知缺陷、中风、脑溢血和一般性精神衰弱,所述病症似乎与含Aβ肽的脑中神经炎和脑血管斑相关。此处涵盖本发明的分子用于治疗或预防至少一种上述病症(其中Aβ肽活性是有害的或其从生物活性Aβ肽水平降低中受益)的用途。此外,涵盖本发明分子用于制备治疗至少一种上述病症的药物中的用途。
如此处所用,术语“治疗(treatment)”、“治疗(treating)”等指获得所期望的药理学和/或生理学效果。所述效果可以是对疾病和/或归属于疾病发展的不利作用的部分治疗或完全治疗。如此处所用,“治疗(treatment)”包括尤其是向人施用化合物,且包括:(a)抑制疾病,即停滞其发展;或(b)缓解疾病,即导致疾病或病症的复原或缓和其症状或并发症。可调整给药方案来提供最佳的期望应答(例如,治疗或预防应答)。例如,可快速浓注施用,可在一段时间内施用几份分次剂量或根据治疗情况的紧急程度所示按比例减少或增加剂量。
可将本发明分子掺入适合于向受试者施用的药物组合物中。本发明分子可按一次剂量或多剂量单独施用或与可药用载体、稀释剂和/或赋形剂组合施用。设计用于施用的药物组合物以适合选定的施用方式,并在适当时使用可药用稀释剂、载体和/或赋形剂如分散剂、缓冲液、表面活性剂、防腐剂、增溶剂、等渗剂、稳定剂等(例如见此处的实施例14)。按照例如Remington,The Science and Practice of Pharmacy,第19版,Gennaro编著,Mack Publishing Co.,Easton,PA 1995中的常规技术设计所述组合物,所述文献提供了从业者公知的制备技术一览表。
可使用包括口服、静脉内、腹膜内、皮下、肺部、透皮、肌内、鼻内、口腔、舌下或栓剂施用在内的标准施用技术向具有如此处所述病理学风险或出现如此处所述病理学的受试者施用包含本发明分子的药物组合物。优选地,可通过皮下施用向具有如此处所述病理学风险或出现如此处所述病理学的受试者施用本发明的分子。
本发明的药物组合物优选为“治疗有效量”或“预防有效量”的本发明分子。“治疗有效量”指在剂量和必需的时间周期上获得所期望治疗结果的有效量。分子的治疗有效量可根据如个体的疾病病症、年龄、性别和体重等因素以及分子在个体中诱导所期望应答的能力而有所变化。治疗有效量还是其中治疗有益作用大于分子的任何毒害作用或不利作用的量。“预防有效量”指在剂量和必需的时间周期上获得所期望的预防结果的有效量。通常,由于在受试者患病之前或早期使用预防剂量,预防有效量将少于治疗有效量。
治疗有效量或预防有效量是必需给予受试者治疗益处的活性物质的至少最小剂量但低于中毒剂量。换言之,治疗有效量的本发明分子在哺乳动物优选为人中降低Aβ肽活性(例如结合Aβ肽)的量,其中Aβ肽的存在引起或促进不想要的病理效应或Aβ肽的降低在哺乳动物优选人中引起有益的疗效。
本发明分子的给药途径可以是口服、肠胃外、通过吸入、或局部给药。优选地,可将本发明的抗体掺入到适合肠胃外给药的药物组合物中。如此处所用的术语肠胃外包括静脉内、肌内、皮下、直肠、阴道、或腹膜内施用。优选通过静脉内或腹膜内或皮下注射的外周全身给药。最优选皮下注射。用于此类注射的合适载体在本领域中是显而易见的。
药物组合物通常必须为无菌且在生产和所提供的容器(例如包括密封的管或注射器)中贮存的条件下是稳定的。因此,药物组合物可在制成后进行无菌过滤,或另在微生物(方面)上制成可接受的。静脉输注的典型组合物可以具有多达250-1000ml液体的体积(例如无菌的林格氏溶液、生理盐水、葡萄糖溶液和Hank’s溶液)和治疗有效剂量(例如1至100mg/ml或更多)的治疗剂,以递送下文列出的典型剂量。剂量可基于疾病的类型和严重性而变化。如在医学领域众所周知的,用于任何一名受试者的剂量取决于许多因素,所述因素包括患者的大小、体表面积、年龄、待施用的特定化合物、性别、给药时间和途径、一般健康和同时施用的其他药物。例如,典型剂量可在0.001至1000mg范围内;然而,特别是考虑到上文提到的因素时,可考虑低于或高于该示例性范围的剂量。每日肠胃外剂量方案可以为每天从约0.1mg/kg至约100mg/kg总体重,优选为从约0.3mg/kg至约10mg/kg且更优选为从约1mg/kg至1mg/kg,甚至更优选为从约0.5至10mg/kg体重。可通过周期评估来监测进程。对于在几天或更长时间的重复施用,根据情况重复治疗直至产生期望的疾病症状的抑制。然而,其他剂量方案也是有用的,并不由此未排除在外。根据从业人员希望获得的药物代谢动力学衰减模式(pattern of pharmacokineticdecay),可通过分子的单次快速浓注施用、多次浓注施用或连续输注施用来递送所期望的剂量。
本发明分子的这些建议用量受许多治疗上的考虑。选择合适剂量和时间安排上的关键因素是所获得的结果。在该上下文中考虑的因素包括治疗的具体病症、治疗的具体哺乳动物、个别患者的临床情况、病症的原因、抗体递送位点、抗体的具体类型、施用方法、施用时间安排和医疗从业人员公知的其他因素。
本发明的治疗剂可冷冻或冻干用于贮存并且在使用之前在合适的无菌载体中重构。冻干和重构可能会导致抗体活性不同程度的损失。可能需要调整剂量来补偿。
以下实施例旨在说明而非不限制本发明。下文的实施例使用命名为“266”(m266)的鼠类单克隆抗体(其最初通过用人Aβ肽第13-28位残基组成的肽免疫制备)和命名为266(m266-Fab)的鼠类单克隆抗体的Fab片段。确认抗体与该肽可以进行免疫反应。前面已经描述过m266的制备。为将PEG分子共价结合到m266-Fab上,可在重链可变的CDR2(N56C)中引入半胱氨酸残基将Fab突变且以下文显示的方式将其聚乙二醇化(实施例4)。如此处实施例描述在鼠类系统中进行的实验,鼠类单克隆抗体的使用是令人满意的。然而,在旨在用于人的本发明的治疗方法中,优选本发明抗体的人源化形式或其片段。在下文的实施例中涉及的1A1-Fab是包含SEQ ID NO:1的LCVR和SEQ ID NO:2的HCVR的人源化抗体Fab片段。
实施例1
PDAPP小鼠中m266-Fab PEG皮下PK/PD研究
使用幼小的(3个月大)转基因PDAPP小鼠以研究抗体和抗体-Aβ复合体的药物代谢动力学/药物动力学血浆反应。研究了几种抗体,包括小鼠Fab(m266-Fab)、m266-Fab+5KD PEG、m266-Fab+10KD PEG、m266-Fab+20KD PEG和完整的全长m266IgG抗体。用1mg/kg的抗体通过皮下注射PDAPP+/-小鼠,随后在以下时间点分离血浆:服药后1、4、8、24、48、96、168和240小时。由于这部分的快速翻转,所以在额外早的时间点对接受m266-Fab抗体的动物进行分析。用于m266-Fab的时间点如下:服药后1、4、8、12、16、24和48小时。在每个时间点对每种抗体分析了总共5只动物。通过用连接到1CC注射器上的23号针头进行心脏穿刺获得全血,所述1CC注射器先前已经用0.5M EDTA冲洗过。在分离过程中将血样在冰上进行温育,并随后在4℃,冷冻微量离心机中以14,000转/分钟离心15分钟。将所得血浆样品等分并储存在-80℃。
A.用于Fab PK分析的方法学
使用抗原捕获ELISA测定血浆Fab的浓度。简言之,在4℃用Aβ-BSA缀合物过夜包被平板或在37℃包被1小时,然后用Pierce酪蛋白缓冲液进行封闭。向平板中加入标准样品、对照样品和研究样品,然后在室温下温育1小时。山羊抗小鼠HRP用于检测并用OPD底物对比色反应进行显色。平板在A493的吸光度处进行读数,参考A700。从标准曲线测定来自血浆样品的免疫反应性的浓度,使用4/5-参数算法从小鼠血浆中m266 Fab的已知量制备所述标准曲线。m266 Fab的测定范围是0.05到0.5μg/mL。聚乙二醇化的Fab的范围是0.075到0.8μg/mL。
从标准曲线测定来自血浆样品的免疫反应性的浓度,使用4/5-参数算法从小鼠血浆中m266 Fab的已知量制备所述标准曲线。266 Fab的测定范围是0.05到0.5μg/mL。聚乙二醇化Fab的范围是0.075到0.8μg/mL。结果清晰地证实,添加PEG分子并增加PEG分子的大小提高了聚乙二醇化Fab相比于非聚乙二醇化m266-Fab(8小时后为350ng/ml)在血浆中的停滞(对20K聚乙二醇化m266-Fab而言,8小时后为2545ng/ml)。
B.m266AβELISA测定
为了测定在不存在或存在治疗抗体(全长或Fab片段)的情况下血浆Aβ的量,发展并使用了ELISA测定。在这些测定法中测定的Aβ肽是全长Aβ1-40或Aβ1-42。用PBS中10μg/ml(每孔100μl)的C端捕获抗体(对Aβ40平板而言为m2G3或对Aβ42平板而言为m21F12)在4℃过夜包被96孔Immulon 4HBX 96孔ELISA板(ThermoLabsystems)。在过夜温育过程中将测试平板密封以防止蒸发。第二天,去除孔内溶液并用Labsystems 96-孔板洗涤器用PBS(每孔400μl)将孔洗涤3次。加入封闭缓冲液(360μl的1%牛奶-PBS)并在37℃温育平板1小时。通过稀释血浆将样品制备成样品稀释液,以产生以下:20%血浆、0.5M胍、5mM Tris pH 8.0、0.5X蛋白酶抑制剂混合物、25μg/ml m266和PBS。因存在高水平的Aβ肽,所以在某些时间点上可能需要减少用于测定的血浆体积,并且在这些情况下,用大鼠血浆调整剩余的血浆体积(最终百分比体积维持在20%)。在标准稀释液(20%大鼠血浆、0.5M胍、5mM Tris pH 8.0和完全无EDTA的0.5X蛋白酶抑制剂混合物(Roche Diagnostics)、25μg/ml m266和PBS)中产生浓度在250pg/ml到3.9pg/ml之间变化的Aβ标准物。需要在样品和标准稀释液中掺入25μg/ml的完整m266,以中和测定中不同水平的中心结构域抗体可能产生的任何负干扰。封闭后,用PBS洗涤平板4次。一式三份来装载样品和标准物(每孔100μl),封闭平板并在4℃温育过夜。第二天早晨,用PBS-T(PBS+0.05%Tween-20)洗涤平板4次,并在室温下将孔与生物素化的第二抗体m3D6(在0.5%BSA/PBS-T中稀释的每孔100μl)温育2小时。用PBS-T洗涤平板4次后,它们与链霉抗生物素蛋白-多聚HRP(在0.5%BSA/PBS-T中为1∶5000)在室温下温育1.5小时。用PBS-T洗涤平板4次并每孔加入100μl的TMB(Sigma)底物。在15、30和60分钟,650nm处监测比色反应的进行。
表1.药物动力学结果:用于Aβ40(pg/ml)的平均血浆浓度
时间(h) | m266Fab | m266Fab+5KDPEG | m266Fab+10KDPEG | m266Fab+20KDPEG | 完整m266 |
1481218244896168240 | 246.7498.8576.7530.9344.1181.2 | 253.5693.2997.8914.7200.179.162.6450.14 | 178.4898.110111728789.6104.876.4998.24 | 196.3816.6125929662642329.3143.3101.2 | 223.711101852691985571079299236114 |
除了可在PEG大小基础上进行调整的更灵活的给药方案外,该结果证实聚乙二醇化Fab抗原复合体不像完整抗体(完整的m266)一样可在血浆循环中延时积累。完整抗体延长了血浆中抗体的半衰期并导致抗原:抗体复合体在血浆循环中延时存在(>240小时)。另一方面,天然Fab(m266Fab)具有限制它们用于治疗的快清除率和短半衰期(<24小时)。相反,如表1中证实,所述聚乙二醇化Fab提供具有允许改善给药方案的药物代谢动力学和药物动力学的抗体分子。
实施例2
PDAPP小鼠中1A1-Fab PEG皮下PK/PD研究
在幼小的(3个月大)转基因PDAPP小鼠中进行研究,以研究抗体和抗体-Aβ复合体的药物代谢动力学/药物动力学血浆反应。研究了几种抗体,包括人源化的1A1-Fab、1A1-Fab+5KD PEG、1A1-Fab+10KD PEG和1A1-Fab+20KD PEG。用1mg/kg的抗体通过皮下注射PDAPP+/-小鼠,随后根据抗体注射组在不同的时间点分离血浆。以下时间点用于不同抗体:
服药后1、4、8、12、24和48小时对1A1-Fab放血
服药后1、4、8、24、48、96和168小时对1A1-Fab+5KD PEG放血
服药后1、4、8、24、48、96和168小时对1A1-Fab+10KD PEG放血
服药后1、8、24、48、96、168和240小时对1A1-Fab+20KD PEG放血
在每个时间点上对每种抗体分析总共5只动物。将所得血浆样品等分并储存在-80℃。
A.用于Fab PK分析的方法学
对1A1 Fab而言,使用夹层ELISA测定血浆1A1 Fab的浓度。用山羊抗人IgGκ包被平板,向平板中加入标准物、对照样品和研究样品,然后在室温下温育1小时。山羊抗人IgG用于检测,随后使用OPD用于比色反应。平板在A493的吸光度处读数,参考A700。
从标准曲线测定来自血浆样品的浓度,使用4/5-参数算法从小鼠血浆中1A1 Fab的已知量制备所述标准曲线;用于Fab和Fab-5K PEG测定的范围为0.003到0.3μg/mL;用于Fab-10K PEG测定的范围为0.006到0.2及0.04到0.4μg/mL;用于Fab 20K PEG测定的范围为0.02-0.4和0.04-0.4μg/mL。结果清晰地证实,添加PEG分子并增加PEG分子的大小提高了聚乙二醇化Fab相比于非聚乙二醇化1A1 Fab(24小时后检测不到)在血浆中的停滞(对20K聚乙二醇化1A1 Fab而言,96小时后为77ng/ml)。
B.1A1AβELISA测定
ELISA与如上对m266所述的基本上相同。通过稀释血浆将样品制备成样品稀释液,以产生以下:20%血浆、0.5M胍、5mM Tris pH 8.0、0.5X蛋白酶抑制剂混合物、20μg/ml 1A1和PBS。在这些测定法中测定的Aβ肽为全长Aβ1-40或Aβ1-42。在15、30和60分钟,650nm处监测比色反应的进行。结果呈现于下文表2中。
表2.药物动力学结果:用于Aβ40(pg/ml)的平均血浆浓度
时间(h) | 1A1Fab | 1A1Fab+5KD PEG | 1A1Fab+10KD PEG | 1A1Fab+20KD PEG |
1481218244896168240 | 136.8153.696.65131.9105.9114.7106.8 | 117.1208.2198.4133.695.1288.4893.96 | 116.3281.6406.3585.1170.7113.7110.8 | 111.2529.21243642177.9125.4200 |
以实施例1中与m266 Fab相似的方式,来自表2的数据证实,共价结合PEG分子的人源化Fab也提供允许灵活的给药方案同时防止抗体-抗原复合体在血浆循环中延时积累的理想PK/PD谱。
实施例3
鼠类266和人源化1A1Fab类似物的纯化
使用两步层析法策略纯化来自转染有小鼠266Fab或人源化1A1Fab及类似物的细胞的培养上清,所述两步层析法策略由阳离子交换层析后接使用Superdex 75树脂(GE Healthcare)的大小排阻层析组成。收集后,使用TFF浓缩培养上清并在4℃对20倍过量体积的10mM醋酸钠(pH5)进行透析过夜。通过离心去除沉淀物并将上清装载到充满10mM醋酸钠(pH5)的SP琼脂糖(GE Healthcare)填充床上。继续用含有更大量NaCl的10mM醋酸钠(pH5)洗涤柱子,直至在大约90到110mM NaCl处洗脱Fab片段。鉴定并收集含有活性Fab的柱级分。使用离心浓缩装置(Millipore)减少体积并交换缓冲液(PBS)。将终体积调整到13ml并装载到Superdex 75分筛柱(sizing column)上。鉴定并收集含在大约50kD洗脱馏分的Fab用于进一步的表征和聚乙二醇化。
实施例4
体外聚乙二醇化和表征
封闭从细胞培养物纯化的1A1-Fab上的N56C半胱氨酸用于聚乙二醇化。使用Pierce’s Reduce-ImmTM固定还原性小珠来选择性还原N56C半胱氨酸。从生产商提供的柱子中提取还原性小珠并以分批方式使用。首先用Reduce-IMM平衡缓冲液#1(磷酸钠+EDTA,pH 8.0)中的8ml 10mM DTT激活~4ml小珠30分钟。然后用PBS洗涤所述小珠3次。向小珠中加入PBS pH 7.4中1.7mg/ml的18ml 1A1 N56C Fab并向混合物中加入10mMEDTA。在室温下旋转并温育所述混合物4-5小时。使用HandeeTM树脂分离剂从小珠中分离Fab并用PBS洗涤小珠。将Fab与洗涤物组合,并与5倍摩尔过量的PEG-马来酰亚胺(来自NOF的20kPEG;来自Sunbio的10kPEG;来自Nektar的5kPEG)反应1小时。反应混合物对4L 10mM醋酸钠缓冲液pH 5.0进行透析,使得可在用10mM醋酸钠缓冲液(pH5.0)平衡的SP琼脂糖柱上捕获Fab和Fab-PEG。用盐梯度洗脱未反应的Fab和Fab-PEG。它们在50mM到70mM NaCl之间被洗脱。利用PBS作为流动相通过大小排阻层析(Superdex75柱,GE Healthcare)进一步纯化蛋白质。还原反应可扩大和缩小。类似方法可用于制备聚乙二醇化的鼠类266 Fab N56C。
利用大小排阻层析分析样品,以确定向Fab中添加PEG。用TSKG3000PW XL(Tosoh Bioscience)柱进行大小排阻层析法。该柱使用在214nm操作的Agilent HP1100系列分析HPLC,利用PBS加0.35M NaCl(pH7.4)以0.5ml/分钟运行。此外,用SDS-PAGE分析样品。10μg纯化的材料装载到4-12%NuPageBis-Tris凝胶上并用SimplyBlueTM SafeStain染色。
实施例5
用Biacore测定动力学常数
Biacore2000仪器也用于测定结合动力学。Biacore利用表面等离子共振的光学特性来检测葡聚糖生物传感器基质内相互作用分子的蛋白质浓度的变化。除了如指出的,所有试剂和材料均购买于BiacoreAB(Upsala,Sweden)。所有测量均在25℃进行。样品溶解在HBS-EP缓冲液(150mM氯化钠、3mM EDTA、0.005%(w/v)表面活性剂P-20和10mM HEPES,pH7.4)中。使用胺偶联试剂盒,以8000反应单位(Ru)的水平将山羊抗人κ抗体固定在CM5传感器芯片的流动细胞1到4上。
使用多轮分析循环来评估结合。每一循环以50μL/分钟的流速进行,并由以下步骤组成:注射~20μL,10μg/mL旨在捕获400-500Ru的抗体结合组合物,注射250μL人Abeta(1-40)(对每一循环而言,在200nM开始并使用两倍连续稀释),接着是20分钟的分离,和使用~30μL 10mM盐酸甘氨酸pH1.5再生。使用BIAevaluation软件中的“1∶1(Langmuir)结合”模型评估每一循环的结合与分离。结果显示N56C位点处的聚乙二醇化对结合人abeta的Fab的亲和力几乎没有影响。
实施例6
用KinExA测定平衡常数
由于抗原Fab复合体的低解离速率,KinExA分析用作正交方法以通过平衡结合分析测定结合亲和力。KinExA 3000仪器(Sapidyne Inst.Inc.)用作测定结合动力学。简言之,所述抗原共价偶联琼脂糖小珠并在该仪器上检测游离Fab/Fab-PEG与小珠的结合。为了测定Kd,含有Fab/Fab-PEG(对1A1-Fab-20kPEG而言为20pM或500pM,对1A1Fab而言为5pM或50pM)与减少的连续稀释抗原人类可溶性Abeta(1-40)(0-10nM)的各管在含有1mg/ml BSA的PBS中,于37℃温育30-50小时,以保证平衡的完成。温育后,根据生产商的说明书在KinExA 3000上测定各平衡样品中的游离Fab/Fab-PEG。使用KinExA 3000软件通过n曲线分析测定Kd值。结果证实,1A1 Fab与鼠类266 Fab(240pM)相比,以大于~10倍的亲和力紧密结合人abeta(19pM)。此外,N56C位点处20K PEG的共价结合对1A1-Fab(12pM)的亲和力没有影响。
实施例7
使用基于细胞的ELISA的淀粉样蛋白前体蛋白质(APP)的结合分析
为了评估266 Fab/mAb与Abeta前体蛋白质APP的交叉反应性,使用了稳定表达APP(氨基酸1-751)的HEK 293细胞。通过将APP(1-751)基因克隆进含有新霉素抗性标记的质粒中来产生这些细胞。将重组质粒转染进HEK 293中,并以200μg/ml G418选择细胞,以产生过表达稳定细胞系。对于结合测定,75,000 APP 751细胞置于PDL包被的96孔板的每一个孔中。在生长培养基(DMEM F12、5%FBS、10mM Hepes pH7.5、200μg/ml G418)中温育2天后,除去液体并在含10mg/ml BSA的PBS(含Ca/Mg)中加入20μg/ml的Fab或mAb。结合在4℃进行2小时,并用10mg/ml BSA洗涤细胞3次。在PBS/BSA(Southern Biotech)中加入对人或小鼠轻链特异的第二抗体(辣根过氧化物酶(hrp)缀合的抗1κ轻链)。PBS/BSA中1∶5000的稀释用于抗人轻链,1∶2000用于抗小鼠轻链。在4℃温育1小时后,细胞用PBS/BSA洗涤5次。作为Fab/mAb结合APP功能的Hrp活性通过加入底物TMD 10分钟进行测定。将反应转移至澄清的96孔板并测量650nm处的吸光度。数据表明,聚乙二醇化(5kD、10kD和20kD)1A1-Fab和m266-Fab赋予Abeta肽优于APP的选择性。
序列表
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Claims (11)
1.包含抗体片段的分子,所述抗体片段在第13-28位氨基酸之间特异结合人Aβ肽,其中所述抗体片段包含轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:2,并且其中聚乙二醇分子共价结合所述抗体片段重链可变区的CDR,其中所述聚乙二醇分子具有0.5kD至30kD的分子量。
2.权利要求1的分子,其中所述抗体片段为Fab片段。
3.权利要求1或2中任一项的分子,其中所述聚乙二醇分子共价结合所述重链可变区SEQ ID NO:2第56位氨基酸处的半胱氨酸。
4.权利要求1的分子,其中所述聚乙二醇分子具有约20kD的分子量。
5.包含具有轻链可变区SEQ ID NO:1和重链可变区SEQ ID NO:2的抗体Fab片段的分子,其中所述抗体Fab片段在SEQ ID NO:2的第56位处共价结合20kD的聚乙二醇分子。
6.组合物,其包含权利要求1-5中任一项的分子。
7.权利要求6的组合物,其还包含可药用载体。
8.根据权利要求1至5中任一项的分子在制备用于治疗或预防与Aβ肽活性相关的病症的药物中的用途。
9.根据权利要求1至5中任一项的分子在制备用于治疗或预防选自阿尔茨海默氏病、唐氏综合症、脑淀粉样血管病CAA、认知缺陷、中风、脑溢血和一般性精神衰弱的病症的药物中的用途。
10.根据权利要求9的分子的用途,其中阿尔茨海默氏病为临床前阿尔茨海默氏病。
11.根据权利要求9的分子的用途,其中阿尔茨海默氏病为临床阿尔茨海默氏病。
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