CN101985036B - 淀粉样β(1-42)蛋白寡聚体、其衍生物及抗体、其制备方法和用途 - Google Patents
淀粉样β(1-42)蛋白寡聚体、其衍生物及抗体、其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及淀粉样β(1-42)蛋白神经调节的寡聚体、具体的生产方法(通过此方法能够以可重复方式高得率获得该寡聚体)、该寡聚体作为诊断性和治疗性试剂在寡聚体特异性抗体的产生中及发现及形成可与该寡聚体相互作用的物质中的用途。正如抗体本身和抗体或物质作为诊断或治疗剂的用途,也公开了产生抗体和发现物质的相应方法。本发明还涉及该寡聚体的衍生物以及基于淀粉样β(1-42)蛋白的简短形式的寡聚体、其产生和用途。
Description
本申请是发明人于2004年2月2日提交的题为“淀粉样β(1-42)蛋白寡聚体、其衍生物及抗体、其制备方法和用途”的中国专利申请200480008812.5的分案申请。
本发明涉及神经调节的淀粉样β(1-42)蛋白寡聚体、特定的生产方法(通过此方法能够以可重复方式以高得率获得该寡聚体)、该寡聚体作为诊断和治疗剂在产生寡聚体特异性抗体及寻找可与该寡聚体和其构成物相互作用的物质中的用途。
本发明同样也涉及所述寡聚体的衍生物(特别是交联的寡聚体和以淀粉样β(1-42)蛋白的截短形式为基础的寡聚体)、其制备及其用途。
正如抗体本身和所述抗体或物质作为诊断性或治疗性试剂的用途,也描述了产生抗体和发现物质的相应方法。
淀粉样β(1-42)蛋白,也简称为Aβ(1-42),是阿尔茨海默和唐氏(Down’s)综合征患者脑中的细胞外由蛋白质、脂类和碳水化合物和盐组成的不可溶性沉积物(老年或神经炎斑)的主要成分(C.L.Masters等,PNAS82,4245-4249,1985)。该蛋白质在水环境中趋于多聚化,可能以非常不同的分子形式存在。
不可溶性蛋白质的沉积与诸如阿尔茨海默氏病之类的痴呆病症的出现或进展之间的简单相关经证明是不令人信服的(R.D.Terry等,Neurobiol.Aging16,285-298(1995))。相反,突触和认知的偿失似乎与Aβ(1-42)的可溶形式更加相关(L.F.Lue等,Am.J.Pathol.155,853-862,(1999),C.A.McLean等,Ann.Neurol.46,860-866(1999))。
对于Aβ(1-42)的可溶性形式,主要有两种不同假说推测分子形式为阿尔茨海默氏病这类痴呆病症的原因。
首先,假设了Aβ(1-42)原纤维的细胞毒性作用。后者依然是可溶的、纤维状、相对高度聚集的Aβ(1-42)形式,具有150至250kDa范围分子量(Arispe等,PNAS90.567(1993),Lashuel等,Nature418,291(2002)),由于形成孔的特性,显然引起通过神经元细胞膜的不受控制的钙流入。
第二,已描述了具有15-30kDa范围内的寡聚Aβ(1-42)衍生物(M.P.Lambert等,PNAS95,6448-6453(1998))。在显示抑制海马部位神经元长时程增强速率的制品中,可以发现这些非纤维状寡聚体,也被称为淀粉样蛋白质衍生的、可扩散及导致痴呆(dementing)的配体(ADDL's for Amyloid Derived Dementing Ligands,见US-A6,218,506及WO01/10900,或for Amyloid Derived Diffusible Ligands,见Lambert等,前文)。
然而,对寡聚体的现有研究水平以对于实际相关种类的显著不确定性为特征。文献中的信息差异显著。因此,US-A6,218,506描述了具有3至12个亚基的ADDL,而在WO01/10900中描述的ADDL可能具有高达24个亚基。
更具体地,至少讨论了两种形式,这两种形式在非变性条件下的凝胶电泳分析中分别具有27至28kDa和23至24kDa(US-A6,218,506)或26kDa至28kDa(WO01/10900)和17kDa至27kDa(M.P.Lambert等,PNAS95,6448-6453(1998))范围内的分子量,以及在变性条件下的SDS凝胶电泳分析中分别为17kDa和22kDa(M.P.Lambert等,PNAS95,6448-6453(1998))和约22kDa至约24kDa和约18kDa至约19kDa(WO01/10900)的分子量。现也通过蛋白质印迹在阿尔茨海默患者的脑上检测到具有8kDa至12kDa范围分子量的SDS稳定的Aβ(1-42)寡聚体(C.A.McLean等,Ann.Neurol.46,860-866(1999))。
所述制备方法具有导致不均一寡聚体制品的缺点。
因此,现在还不可能以可重复的方式提供负责神经调节的Aβ(1-42)的分子形式,更不用说明确鉴定。
然而,可以从例如对阿尔茨海默患者自动免疫接种的最初临床研究过程衡量均一制品的重要性。由于形成的抗体也识别假定为细胞内衬(cell lining)所需的Aβ(1-42)形式,从而导致炎症反应,以Aβ(1-42)的聚集前形式接种,在一些患者当中引起相当的副作用(mengioencephalitis、出血)(D.Schenk.;Nat.Rev.Neurosci.3,824-828(2002))。
因此,特别以中和实际损害性蛋白质形式为目标的传统治疗,绝对需 要后者的鉴定和明确的制品。
另外,已报道了与阿尔茨海默氏病有关的Aβ(1-42)蛋白质的N末端截短形式的出现。除了Aβ(1-42)之外,早在1985年,在死亡的阿尔茨海默患者脑沉积物中也检测到N末端截短形式(C.Masters等,PNAS82,4245-4249(1985))。因此,也已知存在于脑中的特定蛋白酶(如中性溶酶(NEP24.11)或IDE(胰岛素降解酶的缩写))可以降解Aβ(1-42)(D.J.Selkoe,Neuron32,177-180,(2001))。
然而,N末端截短形式在阿尔茨海默氏病发病机理中的重要性尚不清楚(E.B.Lee等,JBS278,4458-4466(2003))。有趣的是,一些罹患散发性或家族性阿尔茨海默氏病或唐氏综合症的患者优先累积这些截短形式(J.等,PNAS91,8378-8382,(1994),C.Russo等,Nature405,531-532,(2000),T.C.Saido等,Neuron14,457-466,(1995))。相对新近的研究(N.Sergeant等,J.of Neurochemistry85,1581-1591(2003))显示,在死亡的阿尔茨海默患者脑中,60%的不可溶性Aβ肽基于N末端截短形式。
抗Aβ(1-42)蛋白质单体及其特定片段的抗体已有描述。
因此,WO03/016466和WO03/016467涉及单克隆抗体266及其缺乏CDR2中特定的糖基化位点的类似物。其人源化版本(Hu-266)也已知。这些文件也提及同抗体226一样识别Aβ(1-42)氨基酸13-28区域中表位的其它单克隆抗体。这些包括抗体4G8(也在Lue等,(1999)中提及,前文)和1C2。另外,McLean等,(1999)(前文)中提及抗体1E8,该抗体显然识别Aβ(1-42)氨基酸18-22区域中的表位。
此外,已知许多识别Aβ(1-42)N末端序列表位的其它抗体。这些包括可商业获得的单克隆抗体6E10(也在WO01/10900;等,(1994),前文;Sergeant等,(2003),前文中提及)和单克隆抗体3D6及10D5(见WO03/016467)和Ban50及NAB228(Lee等,(2003),如上)。
另外,必须提到单克隆抗体WO2、21F12和3D6以及多克隆血清ADA42(Sergeant等,(2003),前文)。
关于目前经历临床前试验的抗Aβ(1-42)抗体的概述可见于Schenk等,(2002),如上。
本发明基础提供引起Aβ(1-42)的神经调节作用,特别是神经元损伤性作用的分子形式为目的,该分子形式在诸如阿尔茨海默氏病和唐氏综合症这类痴呆病症中的有所增加存在;本发明使用特定方法,依靠可以从单体AB(1-42)蛋白质以均质制品形式获得的特定Aβ(1-42)寡聚体,以及依靠所述寡聚体的衍生物,特别是交联的寡聚体和基于截短Aβ(1-42)形式的寡聚体,来实现所述目的。制备方法使得在水性介质中难溶的肽合成的Aβ(1-42)蛋白质能够以高得率转变为明确可溶的寡聚体。
本发明因此涉及权利要求中详细定义的主题。
本发明因此涉及淀粉样β(1-42)蛋白的寡聚体,所述寡聚体在SDS凝胶电泳中具有约15kDa、20kDa、38kDa或48kDa的表观分子量,或涉及该寡聚体的衍生物,其分子量依据衍生而可能会或可能不会发生改变。
淀粉样β(1-42)蛋白是具有42个氨基酸的多肽,其通过蛋白质水解加工而来自淀粉样前体蛋白质(APP)。除了人类变体,这也包括出现在除人类之外的生物,特别是其它哺乳动物,尤其是大鼠中的淀粉样β(1-42)蛋白同种型。
根据具体的实施方案,本发明涉及人类淀粉样β(1-42)蛋白的寡聚体。人类淀粉样β(1-42)蛋白具体包括具有氨基酸序列SEQ ID NO:1的蛋白质,以及从所述序列衍生、特别是通过氨基酸交换而来的突变蛋白质及其等位基因变体。就此而言,必须非常具体地提到下列氨基酸置换:A21G、E22K、E22Q、E22G和D23N。因此,根据本发明,淀粉样β(1-42)蛋白的突变蛋白质或等位基因变体具体包括具有氨基酸序列SEQ ID NO:1的蛋白质,其中选自丙氨酸21、谷氨酸22和天门冬氨酸的一个或多个氨基酸被不同的氨基酸置换,这些氨基酸优选自甘氨酸、赖氨酸、谷氨酰胺和天门冬酰氨。根据本发明,特别重要的是在22位、特别是被谷氨酰胺或谷氨酸置换。
根据另一个具体的实施方案,本发明涉及大鼠淀粉样β(1-42)蛋白的寡 聚体。大鼠淀粉样β(1-42)蛋白具体包括具有氨基酸序列SEQ ID NO:2的蛋白质,以及从所述序列衍生、特别是通过氨基酸交换而来的突变蛋白质及其等位基因变体。就此而言,必须非常具体地提到的是那些对应于在人类序列中讨论的氨基酸置换。
可以通过已知的肽合成方法或重组制备淀粉样β(1-42)蛋白。另外,这些蛋白质许多可商业获得。以上同样应用于突变蛋白质和等位基因变体。
本发明的寡聚体可以通过淀粉样β(1-42)蛋白的寡聚化获得。寡聚化包括单体淀粉样蛋白质的非共价聚集,所以可以假定本发明的寡聚体由多个淀粉样β(1-42)蛋白单体组成。
依赖于寡聚化程度,本发明的寡聚体具有不同的分子量。因此可以通过变性凝胶电泳为该寡聚体指定表观分子量。在标准变性条件下(Tris-甘氨酸凝胶,4-20%,参照 UK,Nature227,680-685(1970)),使用下列标准蛋白质进行凝胶电泳时,所述表观分子量对于寡聚体A1约15kDa,对寡聚体A2约20kDa,对寡聚体B1约38kDa,对于寡聚体B2约48kDa,所述标准蛋白质在同一条件下具有下列表观分子量:肌球蛋白250为kDa,牛血清白蛋白为98kDa,谷氨酰胺氢化酶为64kDa,碳酸酐酶为36kDa,肌红蛋白为30kDa,溶茵酶为16kDa,抑肽酶为6kDa,胰岛素B链为4kDa(参照蓝色预染标准物)。根据另一方面,当在标准天然条件下(Tris-甘氨酸凝胶,4-20%),使用下列标准蛋白质进行凝胶电泳时,对于寡聚体B,分子量约为64至90kDa,标准蛋白质在同一条件下具有下列表观分子量:肌球蛋白250kDa,牛血清白蛋白98kDa,谷氨酰胺氢化酶64kDa,碳酸酐酶36kDa,肌红蛋白30kDa,溶茵酶16kDa,抑肽酶6kDa,胰岛素B链4kDa(参照蓝色预染标准物)。
根据另一方面,本发明的寡聚体以神经元细胞亲和性为特征。可以假定该寡聚体结合特定的细胞表面蛋白质,尤其是受体。
因此,本发明也涉及测定本发明的寡聚体与预先确定的细胞结构结合的方法,该方法包括:
i)允许本发明的寡聚体作用于所述细胞结构,以及
ii)测定本发明的寡聚体是否与该细胞结构结合。
根据另一方面,本发明的寡聚体以神经调节作用为特征。当允许至少一种本发明的寡聚体作用于神经元细胞(例如成神经细胞)瘤细胞的培养物时,这种神经调节作用特别能够以该细胞降低的存活率降低(神经毒性)而证明自身。这里,以本身已知的方式评价细胞存活率是可能的,例如,测定由本发明寡聚体的作用引起的凋亡程度。出于此目的,可以使用适宜的测定方法,例如基于3-[4,5-二甲基噻唑-2-基]-2,5-二苯基-四唑溴(MTT)的比色方法。这种神经调节作用能够以神经元发放速率的调节而具体地在体内表明其本身。
因此,本发明也涉及测定本发明的寡聚体的活性,尤其是神经毒性的方法,该方法包括:
i)允许本发明的寡聚体作用于细胞并且
ii)测定该细胞的状态是否被改变。
可以以上文说明的方式为所述方法提供本发明的寡聚体,例如,以任何上述组合物的形式提供。细胞及细胞结构能便利地以体外提供,特别是以细胞培养物或其他形式,就细胞结构而言,以匀浆物提供。这里,神经元细胞,特别是成神经细胞瘤细胞起测定神经毒性的作用。另外,体内(特别是作为生物体例如实验动物的部分)或离体提供细胞也是可能的。
通常在寡聚体作用之前和之后至少一次测定细胞状态。如果作用前状态和作用后状态之间的比较产生差异,则测试的寡聚体具有活性。
将要测定的状态类型取决于将要测定的活性类型。例如,可以通过测定存活率测定神经毒性。出于此目的,可以测定基于寡聚体作用前后的细胞总数的活细胞比例,并彼此比较。
基于上文测定结合或活性的方法,根据具体的实施方案,可以测试物质是否抑制本发明的寡聚体的结合和/或调节,即部分降低或基本完全抑制、或增强其活性。
出于此目的,原则上至少实施两次本方法,一次存在测试物质,还有一次没有测试物质。出于此目的,通常在提供寡聚体之后将所述测试物质 其加入寡聚体。
然而,如果旨在测定待测物质是否影响寡聚体的形成,宜于在寡聚体已经形成之前,即在提供寡聚体之前,将该物质加至用于寡聚体形成的反应物中。因此,可以在添加测试物质的情况下实施本发明制备方法,然后测定寡聚体是否形成及形成程度。出于此目的,可以测定这种方式获得的方法产物是否具有本发明寡聚体的特征,即,例如它们的分子量、结合能力和活性。
出于尽量突显神经调节作用的目的,必须优选本发明的寡聚体B。就此而言,也优选这些寡聚体的衍生物,尤其强调优选基于N末端截短的Aβ(1-42)蛋白质的寡聚体。
出于工艺流程的原因,可以以组合物的形式将寡聚体A和寡聚体B作为混合物生产,其中除了寡聚体,还含有小部分的其他多肽,特别是单体淀粉样β(1-42)蛋白,以及适当时聚集的淀粉样β(1-42)蛋白的更高分子量形式。这种组合物也是本发明的主题,特别以本发明的寡聚体的比例区别,基于从淀粉样β(1-42)蛋白衍生的蛋白质总量,至少为重量的70%、优选至少为90%重量、尤其是至少为95%。在寡聚体A的制备中,寡聚体A2(SDS凝胶中20kDa条带)的比例至少为重量的50%,优选为至少70%,尤其是至少为85%。在寡聚体B的制备中,寡聚体B1和B2(SDS凝胶中38kDa和48kDa条带)的比例至少为重量的60%,优选为至少75%,尤其是至少为90%。
可以衍生本发明的寡聚体。这类衍生的目的可以是,例如,调节所述寡聚体的物理化学性质(尤其是关于生物利用率)、提供具有检测标记的寡聚体或固定所述寡聚体,例如,可以将其偶联到支持物上。标记和固定对于诊断性应用尤其重要。
技术人员熟知蛋白质-生物化学领域常用的适当标记。这些包括荧光标记(例如特定的荧光蛋白和四甲基若丹明衍生物)、发光标记、显色标记、放射标记和磁性标记以及具有互补结合配偶体亲和力的标记,例如生物素和链霉亲和素衍生物。
关于表观分子量,必须考虑到,与未衍生、但是聚集数相同的寡聚体相比,寡聚体衍生物具有可能增加的表观分子量。因此,例如,基于在SDS凝胶中具有38kDa分子量的Aβ(1-42)寡聚体B1的生物素衍生物,具有42dDa的分子量。
根据本发明的特定实施方案,寡聚体是交联的。合适的交联剂为技术人员所公知,并且通常为双功能试剂,例如甲醛、戊二醛、辛二酸二琥珀酰亚胺酯、Dithiobis(玻珀酰亚胺丙酸酯)、二琥珀酰亚胺酒石酸酯、双磺基琥珀酰亚烷酒石酸酯、二甲基、二甲基pimelidate、二甲基suberimidate、二甲基-3,3’-dithiobispropionimidate、N-γ-maleinimidobutyloxy琥珀酰亚胺酯、琥珀酰亚胺-4(N-maleinimido甲基)-环己烷-1-羧酸酯、N-琥珀酰亚胺-(4-碘乙酰)aminobenzoat以及N-琥珀酰亚胺-3-(2-吡啶)丙酰酯。
这类交联的寡聚体具有的优点是稳定,而且其寡聚化通常不再可逆。它们因此特别适用于诊断性检验系统或作为生产寡聚体特异性抗体的免疫原。
本发明的寡聚体的衍生物也包括淀粉样β(1-42)蛋白片段的寡聚体。优选通过天然存在的蛋白酶作用获得的那些片段。与淀粉样β(1-42)蛋白相比,在生理缓冲液中非变性条件下通过蛋白质水解获得的片段特别地增加了蛋白质水解稳定性。优选通过内肽酶作用获得的片段。根据本发明的特定实施方案,所述片段可以通过胰蛋白酶、糜蛋白酶、嗜热菌蛋白酶、弹性蛋白酶、木瓜蛋白酶或蛋白内切蛋白酶GluC的作用获得。根据另一方面,优选其本发明寡聚体通过神经调节作用区分的片段。对于此方面的进一步说明,参考本发明淀粉样β(1-42)蛋白寡聚体的神经调节作用的相关注释。
从结构上,优选片段特别在于它们是通过去除淀粉样β(1-42)蛋白N末端序列而衍生的特征。根据一方面,该N末端序列可以是具有淀粉样β(1-42)蛋白N末端序列的上至23个、优选具有上至21个、特别是具有上至19个氨基酸的片段。因此,根据本发明,优选序列包含相邻氨基酸24至42、优选22至42、特别是20至42个连续氨基酸的Aβ(1-42)蛋白质片 段。根据另一方面,将要去除的N末端序列可以是具有至少7个、优选具有至少9个、特别是具有至少11个Aβ(1-42)蛋白质N末端序列氨基酸的序列。因此,根据本发明,特别优选N末端截短了7至23个、优选9至21个、特别是11至19个氨基酸的淀粉样β(1-42)蛋白的片段。这些片段对应于式Aβ(x-42),其中x为8至24,优选10至22,特别是12至20。根据本发明,Aβ(x-42)片段因此优选自下列片段:Aβ(8-42)、Aβ(9-42)、Aβ(10-42)、Aβ(11-42)、Aβ(12-42)、Aβ(13-42)、Aβ(14-42)、Aβ(15-42)、Aβ(16-42)、Aβ(17-42)、Aβ(18-42)、Aβ(19-42)、Aβ(20-42)、Aβ(21-42)、Aβ(22-42)、Aβ(23-42)和Aβ(24-42)。特殊的片段是通过嗜热菌蛋白酶作用获得的淀粉样蛋白质β(20-42)片段和通过GluC内切蛋白酶作用获得的淀粉样蛋白质β(12-42)片段。
本发明的衍生物还有来自具有1个或2个额外C末端氨基酸的淀粉样蛋白质β(12-42)的那些形式。其实施例因此包括Aβ(1-43)蛋白质的寡聚体、或其对应于上述内容的衍生物。
制备寡聚体的发明方法可以基本包括三个步骤。其第一步是任选但是有利的,第二步是寡聚体A和B的制备绝对需要的,第三步起到制备本发明的寡聚体B的作用。
步骤1涉及蛋白质的解折叠。出于此目的,可以允许例如六氟异丙醇(HFIP)这类氢键断裂试剂作用于蛋白质。当作用温度为约20至50℃、特别是约35至40℃时,数分钟的作用时间(例如约10至60分钟)足以。随后,于易于与水性缓冲液混合的适宜有机溶剂(例如二甲亚砜(DMSO))中溶解蒸发干燥的残余物(优选以浓缩形式),产生至少部分解折叠的蛋白质悬浮液。如果需要,储备的悬浮液可以于低温(例如在约-20℃)临时贮存。
上述步骤1的备选方案是,可以在弱酸性、优选水溶液中溶解蛋白质,例如约10mM的HCl水溶液。通常孵育数分钟后,经离心去除不溶性组分。于10000g数分钟为宜。优选于室温(即在20至30℃范围内的温度)实施这些方法步骤。离心后获得的上清液含有淀粉样β(1-42)蛋白,并可以 于低温(例如在约-20℃)临时贮存。
步骤2涉及产生寡聚体A的蛋白质寡聚化。出于此目的,任选地允许去垢剂作用于至少部分解折叠的蛋白质,直至产生足够的寡聚体A。
优选使用离子去垢剂,特别是阴离子去垢剂。
根据具体的实施方案,使用式(I):
R-X
的去垢剂,其中R基为具有6至20个、优选10至14个碳原子的未分支或分支烷基,或具有6至20个、优选10至14个碳原子的未分支或分支链烯基,
X基为酸性基团或其盐,X优选自-COO-M+、-SO3-M+,特别是-OSO3 -M+,M+是氢阳离子、或者无机或有机阳离子,优选自碱性金属和碱土金属阳离子以及铵阳离子。
具有式(I)的去垢剂是有利的,其中R是未分支烷基,必须特别提及的是1-烷基。优选十二烷基硫酸钠(SDS)。也可以有利地使用月桂酸和油酸。去垢剂十二烷酰肌氨酸的钠盐(也称为肌氨酰NL-30或)也格外有利。
去垢剂作用时间尤其依赖于进行寡聚化的蛋白质是否解折叠,如果是,还有解折叠的程度。根据步骤1,如果预先以氢键断裂试剂(即,特别是以六氟异丙醇)处理蛋白质,当作用温度约为20至50℃、特别是约为35至40℃时,数小时范围内的作用时间是足够的,有利地为约1至20、特别是约2至10小时的作用时间。如果以较少解折叠或基本没有解折叠的蛋白质开始,以相应较长的作用时间为宜。如果淀粉样β(1-42)蛋白经过预处理,例如,根据上文指出的步骤1的备选方案,或者将所述蛋白质直接引入步骤2,当作用温度约为20至50℃、特别是约为35至40℃时,约5至30小时、特别是约10至20小时范围内的作用时间足以。孵育后,宜通过离心去除不溶性组分。于10000g数分钟为宜。
所选择的去垢剂浓度取决于所使用的去垢剂。如果使用SDS,浓度0.01至1%、优选0.05至0.5%比重的范围,例如约质量0.2%的比重,经证明 是有利的。如果使用月桂酸或油酸,略高的浓度是有利的,例如,在0.05至2%、优选0.1至0.5%比重的范围,例如约0.5%的比重。
去垢剂作用应在接近于生理范围的盐浓度下发生。因此,尤其以50至500mM、优选100至200mM范围内、特别是140mM的NaCl浓度为宜。
产生的含有寡聚体A的溶液,即特别是含有寡聚体A1和/或A2的溶液,可以于低温(例如在约-20℃)临时贮存。该溶液可以用于例如本发明的用途或进一步的反应以产生寡聚体B、或在第一步之后的进一步处理或纯化步骤,所述步骤具体实现了至少一种本发明的寡聚体A的进一步浓缩。具体地,通过色谱方法,可以从存在于溶液中的其他蛋白质组分(尤其是来自淀粉样β(1-42)蛋白的那些组合)取出寡聚体。特别适用于此目的的是亲和色谱方法,该方法可以使用例如特异性抗体,即也特别为本发明的寡聚体特异性抗体。
步骤3涉及产生寡聚体B的寡聚化。优选地,选择含有寡聚体A的组合物作为这个步骤的反应物。在此方面,根据本发明,寡聚体A是制备寡聚体B的重要中间物。如果该组合物来自步骤2,其通常含有去垢剂以及生理范围的盐浓度。宜降低去垢剂的作用和盐浓度。这可以通过降低去垢剂和盐的浓度而解决,例如通过稀释,宜使用水或低盐浓度缓冲液,例如Tris-HCl,pH7.3。在约2至10范围内、有利地在3至8范围内、特别是约为4的稀释因子经证明为适宜的。也可以添加中和所述去垢剂作用的物质实现去垢剂作用的降低。这些物质的实例包括能够络合去垢剂的物质,如能够在纯化和提取措施过程中稳定细胞的物质,例如特定的EO/PO阻断共聚物,特别是以商品名F68的阻断共聚物。烷氧基化、特别是乙氧基化烷基酚,例如X系列的乙氧基化t-辛基酚(特别是 X100)、3-(3-(胆酰胺基丙基二甲氨基)-1-丙磺酸盐或烷氧基化、特别是乙氧基化山梨聚糖脂肪酯,例如系列的那些,特别是20,在特定的临界微囊浓度附近或之上的浓度范围内。
随后,孵育该溶液直至产生足够的寡聚体B。当作用温度约为20至 50℃、特别是约为35至40℃时,数小时范围内、优选约10至30小时范围内、特别是约15至25小时范围内的作用时间足以。然后可以浓缩溶液,并且可能的残余物可以通过离心去除。这里也证明以10000g数分钟为宜。离心后获得的上清液含有寡聚体B。
产生的含有寡聚体B(即,特别是寡聚体B1和B2)的溶液,可于低温(例如在约-80℃)临时贮存。该溶液可以用于例如本发明的用途、或进一步处理或纯化步骤可以跟随第一步。对此,参考与寡聚体A有关的相应测量的注释。
出于基本完全去除用于制备寡聚体的去垢剂的目的,可以实施进一步处理或纯化步骤。例如,可以首先从含有去垢剂的溶液沉淀寡聚体B、分离并重新溶解于不合去垢剂的介质中。沉淀蛋白质的措施为技术人员所熟知。根据本发明,添加水-甲醇中的乙酸经证明是有利的。偿未结合具体的机制,去垢剂或去垢剂的变种以及盐浓度看似将蛋白质置于确定的溶解形式,正如可以通过例如变性或天然凝胶电泳或凝胶渗透层析所检测的,该形式与溶解于水性生理缓冲液中的蛋白质起始形式明显不同。这是令人惊讶的,由于去垢剂通常能够去聚集,即,使亚基、蛋白质聚集体去组装,根据本发明,从倾向于聚集的单体开始获得了确定的寡聚体。
通过实施本发明的上述方法,在已经经过适当衍化的淀粉样β(1-42)蛋白上有利地制备了本发明的寡聚体衍生物。另外,衍生寡聚体也是可能的,但是这不应改变所述寡聚体的结构。合适的蛋白质化学手段为技术人员所公知。
可以用本身已知的方式实施本发明的寡聚体或其衍生物的交联。例如,如果使用戊二醛作为交联剂,可以使用戊二醛溶液处理从本发明方法步骤2或3产生的溶液。于室温数小时后,寡聚体已与戊二醛反应。该反应然后可以用本身已知的方式,通过将过量的戊二醛与试剂反应而被终止,所述试剂普遍已知用于此目的,例如氨基乙醇。取决于本发明的寡聚体A或B是否交联,获得分别称为A-CL和B-CL的本发明交联寡聚体溶液。
特别是对于任选交联的寡聚体B或其衍生物,在其合成之后,再次增 加盐浓度而不损害所述寡聚体的稳定性是可能的。这对于该寡聚体的应用是重要的,例如,在生理条件对应用有利的情况下(细胞应用、体内应用)。
原则上,通过寡聚化从相应的单体开始,或者通过蛋白质水解从所述淀粉样β(1-42)蛋白的寡聚体开始,都可以制备淀粉样β(1-42)蛋白片段的寡聚体。因此,根据第二个方法的变体,可以使用蛋白酶处理由本发明的方法步骤2或3产生的溶液。当达到需要的蛋白质水解程度时,以公知的方式失活蛋白酶。然后可以按照此处已经说明的程序分离产生的寡聚体,如果需要,通过另外的处理和纯化步骤进一步处理。
通过其均一性和稳定性区分本发明的寡聚体以及包含它们的组合物。它们在生理介质(例如生理盐溶液)中是可溶的,并通过其稍微的球状外观而不同于纤维状形式。它们具有的优点是具有的空间结构显然不同于其他Aβ(1-42)形式,特别是单体、非毒性寡聚体、包含Aβ(1-42)的前体分子APP、原纤维和纤维。它们因此特别适于在体内和体外产生特异性抗体,使例如特异性的自动免疫成为可能。就此而言,关键是仅仅将那些导致疾病的寡聚体用于免疫。其他形式的Aβ(1-42)例如单体分子或较小的寡聚体,在生物体中对于重要的信号功能可能是必需的。同样,去除推定为对细胞衬里重要的纤维状堆积物,可能损伤生物体。
以同样的方法,使用本发明的同源性寡聚体或含有该寡聚体的组合物,可以实施可能的治疗,例如被动免疫、寡聚体形式的去稳定剂的使用以及受体(部分)激动剂和拮抗剂的使用。因此,同源性寡聚体制备也使用于被动免疫的多克隆或单克隆抗体的特异性生产成为可能。使用本发明的寡聚体及含有这些寡聚体的组合物,也可能发现与疾病有关并受该寡聚形式影响的受体分子或信号分子。
由于其涉及淀粉样β蛋白质相关的生理过程,本发明的寡聚体具有诊断性和治疗性价值。因此,本发明涉及本发明的寡聚体及其衍生物(任选以相应组合物的形式)在诊断性体外和体内检测方法中的用途。
淀粉样β蛋白质相关的生理过程包括与淀粉样蛋白质沉积(淀粉样蛋白病)有关的那些。这些包括那些导致神经组织结构改变,并且对例如阿 尔茨海默病和唐氏综合征而言重要的那些过程,以及影响其他组织、诸如淀粉样微血管病变(例如嗜刚果红淀粉样蛋白血管病(CAA))的那些过程。
本发明还涉及本发明的寡聚体及其衍生物(任选以相应组合物的形式)在产生寡聚体特异性抗体中的用途。
这里,以Aβ(1-42)蛋白质的截短形式为基础的发明性寡聚体具有特别的重要性:由于缺失N末端,它们可以产生显然比Aβ(1-42)蛋白质更具选择性的免疫反应。尽管经典Aβ(1-42)蛋白质中的高免疫原性N末端显著地产生对该分子此区域的特异性抗体,例如根据制造商信息针对N末端(6E10:Aβ(1-17)以及BAM-10Aβ(1-12))的抗体6E10(F.Signet)和BAM-10(Sigma,St.Louis),可以使用本发明以截短的Aβ(1-42)形式为基础的寡聚体获得的抗体识别寡聚体特异性区域,从而达到与其他Aβ(1-42)形式相当的选择性。由于接种后产生的更强选择性的免疫应答(与APP、单体形式、原纤维、纤维、斑点(plaque)相比)也必然伴有更少的副反应(例如脑出血、原位单体形式的生理神经营养活性的损伤),这个优点尤其可以利用到主动接种当中。特别地,用于被动免疫(即基因治疗)的单克隆抗体的更高的生产和选择性也是可能的。
此用途的一个方面是产生治疗框架内寡聚体特异性抗体。
本发明因此还涉及本发明的寡聚体及其衍生物在治疗领域中、特别是作为疫苗的用途。
这样的疫苗通常为药物组合物,包括至少一种本发明的寡聚体和/或至少一种本发明的寡聚体的衍生物。为此目的,特别可以使用本发明的任何组合物,其包括两个或多个寡聚体,或使用不同组合物的组合。因此,特别可以使用寡聚体B,即B1和B2,或其衍生物进行接种。该组合物可以进一步包括生理上适宜的载体,以及任选地还含有赋形剂,例如免疫刺激物。
尽管原则上可以选择任何适宜的载体,载体的类型通常取决于给药途径。因此本发明的疫苗可以特别地以适用于肠胃外(例如静脉内、肌肉内和皮下)给药途径的形式进行配制。在这些情况下,载体优选包括水、盐、 醇、脂肪、腊和/或缓冲液。
本发明的疫苗可以使用多种免疫刺激物中的任一种。例如可以包含佐剂。大多数佐剂含有保护抗原免于迅速代谢的物质,例如氢氧化铝或矿物油、以及来源于脂质A、百日咳球杆菌(Bordadella pertussis)或结核分支杆菌(Mycobacterium tuberculosis)的蛋白质。适宜的佐剂通常可以商业获得,例如完全或不完全弗氏佐剂、AS-2、铝盐,例如氢氧化铝(适当时以凝胶形式)或磷酸铝、钙盐、铁盐或锌盐、酰化酪氨酸的不溶性悬浮液、酰化糖、阳离子或阴离子衍生的多聚糖、聚磷腈、可生物降解的微球体、单磷酰脂质A。细胞因子例如GM-CSF或白介素2、7或12同样可以用作佐剂。
本发明进一步涉及生产抗体的方法,该方法包括:
i)以至少一种本发明的寡聚体、其衍生物或组合物免疫宿主;并
ii)获得应答该免疫产生的含有抗体的宿主血清。
根据生产抗体方法的特定实施方案,给以免疫混合物进行免疫,免疫混合物包括本发明的多种寡聚体或寡聚体的衍生物的混合物。具体地,在该方法过程中,给以寡聚体组分不同的多个免疫混合物尤其有利。
如果将要使用的寡聚体或寡聚体衍生物没有或仅有弱的免疫原性,可以将其偶联至载体而增加其免疫原性,优选偶联至载体蛋白质,例如钥孔槭血蓝蛋白(KLH)、鲎蛛血蓝蛋白(LPH)、牛血清白蛋白(BSA)或卵清蛋白(OVA)。许多公知用于此目的的可能的偶联可供技术人员利用。可能有利的实例是与戊二醛反应,例如,将寡聚体或寡聚体衍生物与适当的肽或肽混合物在水或水溶液中孵育。该反应可以在环境温度(即通常在室温)下方便地进行。然而,降低或略升高温度也可能有利。该反应通常在几小时内产生所需的结果,例如,2小时的反应时间在常用范围内。戊二醛浓度常在ppm至%范围内,宜从10ppm高至1%、优选从10ppm至0.5%。反应参数的优化在技术人员技能内,并应考虑到寡聚体A和B或寡聚体衍生物在所选的反应条件下稳定。
优选组合将要使用的成分而制备免疫混合物。孵育最初产生的组分混和物是有利的。这通常在环境温度(即室温)下方便地进行。然而,冷却 或略加热该混和物可能有利。孵育期常从几分钟至数小时,1小时的孵育时间经证明是有利的。
除了抗原,免疫混合物常含有额外的赋形剂,特别是常用于免疫的佐剂,例如弗氏佐剂。更特别地,将完全弗氏佐剂用于初次免疫,而以不完全弗氏佐剂进行任何额外的免疫。将抗原(免疫原)(优选以上述组分混和物的形式)添加至赋形剂中制备免疫混合物,抗原通常被乳化。
适宜的宿主特别为啮齿动物或兔。以免疫混合物注射这些或其他适宜的宿主,优选皮下注射。可以使用免疫测定测量抗体效价,例如竞争性使用羊抗宿主IgG绵羊抗血清和经标记的寡聚体。因此可以在免疫末期确定特定的宿主是否适于产生抗体。例如,如果进行4次免疫,可以在第三次免疫后测定抗体效价,然后从具有足够抗体效价的动物获得抗体。
优选在数周或数月的时期后从宿主采集血清而获得产生的抗体。最后,将动物放血。可以从用本身已知的方式获得的血液中获得含有所需抗体的血清。如果需要,这样获得的全部血清可以由技术人员进一步纯化,将存在于其中的抗体成分、特别是识别寡聚体的抗体浓缩。
根据本发明方法的特定实施方案,选择至少一种血清抗体,该抗体特异性识别用作免疫原的寡聚体或其衍生物,或者在用作免疫原的组合物中存在的至少一种寡聚体或其衍生物。在本文中,特异性指抗体对免疫原比对其他物质(特别是与相关蛋白质、尤其是与单体淀粉样β(1-42)蛋白、以及比本发明的寡聚体具有更高分子量的寡聚或多体淀粉样β(1-42)蛋白聚集物相比)具有更高的结合亲和力。也可以用此方式获得寡聚体特异的单克隆抗体。然而,出于此目的,优选从宿主脾组织摘除、并从由此获得的脾淋巴细胞开始,以常用方式建立产生单克隆抗体的杂交瘤。
抗体生产详述
B淋巴细胞共含有由几十亿种不同抗体特异性组成的抗体所有组成成分,是哺乳动物免疫系统的一部分。对特定抗原的正常免疫应答指从所述所有组成成分选择一种或多种的该抗原特异性结合抗体。免疫应答的成功至少部分建立在抗体特异性识别(以及最终消除)刺激性抗原并忽视所述 抗体的环境中其他分子的能力。
特异性识别一个特定靶抗原的抗体的有效性导致单克隆抗体技术的发展。目前标准化杂交瘤技术允许对目的抗原具有单独特异性的抗体的产生。更近来,重组抗体技术,例如抗体文库的体外筛选得到发展,这些技术也允许具有对目的抗原的单独特异性的抗体的产生。
在本发明的方法中,允许目的抗原体内或体外作用于抗体所有组成成分。
根据一个实施方案,以抗原体内免疫动物而使抗原作用于所有组成成分。体内途径可以进一步包括从动物淋巴细胞建立大量杂交瘤,并选择分泌与该抗体特异性结合的抗体的杂交瘤。将要免疫的动物可以是,例如小鼠、大鼠、兔、鸡、骆驼或绵羊,或任何上述动物的转基因版本;例如,具有人类免疫球蛋白基因的转基因鼠,其在抗原刺激后产生人类抗体。其他可以被免疫的动物类型包括重度联合免疫缺损(SCID)小鼠,其已用人类外周单核血细胞(嵌合hu-PBMC SCID小鼠)或用淋巴结细胞或其前体细胞重构,以及以致死全身辐射处理,然后使用重度联合免疫缺损(SCID)小鼠的骨髓瘤细胞抵抗辐射,并随后移植人类功能淋巴细胞的小鼠(“Tremera”系统)。可免疫的另一种动物类型,是基因组中编码目的抗原的内源性基因经例如同源重组而关闭(敲除)的动物(例如小鼠),以致以抗原免疫后,所述动物将此抗原识别为外来物。对技术人员而言,此方法产生的多克隆或单克隆抗体,显然以使用已知的筛选方法为特征并进行选择,筛选方法包括(但是不限于)ELISA技术。
根据另一实施方案,以抗原筛选重组抗体文库,使抗原体外作用于抗体所有组成成分。可以在例如噬菌体表面或在酵母细胞表面或在细菌表面表达重组抗体文库。在不同的实施方案中,重组抗体文库是例如scFv文库或Fab文库。根据另一实施方案,以RNA-蛋白质融合物表达抗体文库。
生产本发明抗体的另一途径,包括组合体内和体外途径。例如,以抗原体内免疫动物使所述抗原作用于抗体所有组成成分,然后以该抗原体外筛选从该动物淋巴细胞制备的重组抗体文库或单结构域抗原文库(例如含 有重和/或轻链)。根据另一条途径,用抗原体内免疫动物,使所述抗原作用于抗体所有组成成分,然后把该动物淋巴细胞产生的重组抗体文库或单结构域文库进行亲和力成熟。根据另一条途径,用抗原体内免疫动物,使所述抗原作用于抗体所有组成成分,然后选择分泌目的抗体的单个抗体产生细胞,并从所选细胞cDNA获得重链和轻链可变区(例如通过PCR),在哺乳动物宿主细胞内体外表达重链和轻链可变区(称此为选择淋巴细胞抗体方法或SLAM),从而可进一步选择并操纵所选的抗体基因序列。另外,可以通过在宿主细胞内表达重链和轻链的抗体基因、并选择那些分泌具有所需结合亲和力的抗体的哺乳动物细胞,通过表达克隆选择单克隆抗体。
因此,本发明一方面为筛选和反筛选提供明确的抗原。因此,根据本发明,可以选择那些多克隆和单克隆抗体,所述抗体结合本发明的寡聚体或其衍生物,而非其他形式的Aβ(1-42)蛋白质、APP、淀粉样纤维或淀粉样斑点以及大量其他无关抗原和组织。
技术人员充分了解,抗体选择以充分确定的抗原为基础。反之,不充分确定的抗原在使用时并不具有足够的选择性。简言之,体外作用和选择与亲和层析相似,使用的所需抗原的“配体”已除去以不充分的亲和力结合抗原的那些。因此,在具有其他抗体的巨大集合体中,所需抗体的富集程度与抗原质量直接相关。令人惊讶的是,本发明的寡聚体及其衍生物是可用于浓缩合适的相关选择性抗体,并将其从识别Aβ(1-42)蛋白质相关的其他形式及其他无关抗原的抗体有效取出的抗原。
本发明的生产抗体的方法可用于生产多种类型的抗体。这些基本包括人类抗体、嵌合抗体、人源化抗体和CDR移植抗体及其抗原结合部分。
本发明生产抗体的方法说明如下。在此指出体内途径、体外途径或二者的组合之间的区别。
体内途径
从体内产生的抗体生产细胞开始,可以通过标准技术生产单克隆抗体,例如最初由Kohler和Milstein(1975,Nature256:495-497)(也见Brown 等(1981)J.Immunol127:539-46;Brown等(1980)J Biol Chem255:4980-83;Yeh等(1976)PNAS76:2927-31;以及Yeh等(1982)Int.J.Cancer29:269-75)描述的杂交瘤技术。生产单克隆抗体杂交瘤的技术广为人知(一般见R.H.Kenneth,《Monoclonal Antibodies:ANew Dimension In Biological Analyses》,Plenum Publishing Corp.,New York,New York(1980);E.A.Lerner(1981)Yale J.Biol.Med.,54:387-402;M.L.Gefter等,(1977)Somatic Cell Genet.,3:231-36)。简而言之,将不死细胞系(一般为骨髓瘤)与哺乳动物淋巴细胞(一般为脾细胞或淋巴结细胞或外周血淋巴细胞)融合,其中哺乳动物经本发明的寡聚体或其衍生物免疫;筛选得到的杂交瘤细胞培养上清液,以鉴定产生对本发明的寡聚体或其衍生物具有特异性的单克隆抗体的杂交瘤。许多广为人知的融合淋巴细胞和不死细胞系的方法中的任一种都可应用于此目的(也见G.Galfre等,(1977)Nature266:550-52;Gefter等,Somatic Cell Genet.,引用如上;Lerner,Yale J.Biol.Med.,引用如上;Kenneth,《Monoclonal Antibodies》,引用如上)。此外,技术人员将理解,这类方法有许多改变,这些改变同样有用。一般,不死细胞系(例如骨髓瘤细胞系)来自与淋巴细胞相同的哺乳动物物种。例如,可以将以本发明的免疫原制品免疫的小鼠淋巴细胞与小鼠不死细胞系融合,建立小鼠杂交瘤。优选的不死细胞系是小鼠骨髓瘤细胞系,该细胞系对含有次黄嘌来呤、氨基喋呤和胸苷的培养基(HAT培养基)敏感。大量骨髓瘤细胞系中的任何一种可以标准(standardwise)地用作融合配偶体,例如P3-NS1/1-Ag4-1、P3-x63-Ag8.653或Sp2/O-Agl4骨髓瘤系。这些骨髓瘤细胞系可以从美国典型培养物保藏中心(ATCC),Rockville,MD获得。通常,使用聚乙二醇(PEG)将HAT敏感的小鼠骨髓瘤细胞系与小鼠脾细胞融合,然后使用HAT培养基选择融合产生的杂交瘤细胞,从而杀死非融合的、以及非生产性融合的骨髓瘤细胞(非融合的脾细胞因其未经转化而在数天后死亡)。通过筛选杂交瘤培养物上清液中特异性识别本发明的寡聚体及其衍生物的单克隆抗体,鉴定产生这类抗体的杂交瘤细胞,例如,使用标准ELISA测定法选择那些可以特异性结合本发明的寡聚体或 其衍生物的抗体。
根据需要的抗体类型,多种宿主动物可以用于体内免疫,可以使用自身表达目的抗原的内源性版本的宿主。另外,可以使用造成目的抗原的内源性版本缺陷的宿主。例如,通过相应内源性基因的同源重组在特定内源性蛋白质造成缺陷的小鼠(即敲除小鼠)已证实产生针对用来免疫的蛋白质的体液应答,从而能够用于对该蛋白质的高亲和力单克隆抗体的生产(见,例如Roes,J.等,(1995)J.Immunol.Methods183:231-237;Lunn,M.P.等,(2000)J.Neurochem.75:404-412)。
对于生产抗本发明的寡聚体或其衍生物的非人类抗体,许多非人类哺乳动物细胞是抗体生产的适宜宿主。尽管对于杂交瘤的生产优选小鼠,适且的宿主包括小鼠、大鼠、小鸡、骆驼、兔和山羊(及其敲除版本)。另外,表达人类抗体所有组成成分的非人类宿主动物可用于基本上生产对人类抗原具有双特异性的人类抗体。这种非人类动物包括含有人类免疫球蛋白转基因的转基因动物(例如小鼠)(嵌合hu-PBMC SCID小鼠)以及在下文中更详细说明的人/小鼠辐射嵌合体。
根据一个实施方案,经本发明的寡聚体及其衍生物免疫的动物是非人的哺乳动物(优选小鼠),所述动物为人类免疫球蛋白基因转基因动物,以致该非人类哺乳动物在抗原刺激后产生人类抗体。一般,将具有人类生殖细胞系构型的重链和轻链免疫球蛋白转基因引入内源性重链和轻链基因座被失活的动物。如果以抗原(例如以人类抗原)刺激这类动物,产生来自人类免疫球蛋白序列的抗体。可以通过标准杂交瘤技术从这类动物的淋巴细胞制造人类单克隆抗体。关于具有人类免疫球蛋白的转基因小鼠及其在人类抗体生产中的用途的进一步描述,见例如US5,939,598、WO96/33735、WO96/34096、WO98/24893和WO99/53049(Abgenix Inc.),以及US5,545,806、US5,569,825、US5,625,126、US5,633,425、US5,661,016、US5,770,429、US5,814,318、US5,877,397以及WO99/45962(Genpharm Inc.);也见MacQuitty,J.J.和Kay,R.M.(1992)Science257:1188;Taylor,L.D.等,(1992)Nucleic Acids Res.20:6287-6295;Lonberg,N.等,(1994) Nature368:856-859;Lonberg;N.和Huszar,D.(1995)Int Rev.Immunol.13:65-93;Harding,F.A.和Lonberg,N.(1995)Ann.N.Y.Acad.Sci.764:536-546;Fishwild,D.M.等,(1996)Nature Biotechnology14:845-851;Mendez,M.J.等,(1997)Nature Genetics15:146-156;Green,L.L.和Jakobovits,A.(1998)J.Exp.Med.188:483-495;Green,L.L.(1999)J.Immunol.Methods231:11-23;Yang,X.D.等,(1999)J.Leukoc.Biol.66:401-410;Gallo,M.L.等,(2000)Eur.J.Immunol.30:534-540。
根据另一实施方案,经本发明的寡聚体及其衍生物免疫的动物可以是重度联合免疫缺损小鼠,该小鼠以人类外周单个核血细胞或淋巴结细胞或其前体重建。已证实,这类被称为嵌合hu-PBMC SCID小鼠的小鼠在抗原刺激下产生人类免疫球蛋白应答。关于这些小鼠及其在生产抗体中的用途的进一步描述,见例如Leader,K.A.等,(1992)Immunology76:229-234;Bombil,F.等,(1996)Immunobiol.195:360-375;Murphy,W.J.等,(1996)Semin.Immunol.8:233-241;Herz,U.等,(1997)Int.Arch.Allergy Immunol. 113:150-152;Albert,S.E.等,(1997)J.Immunol.159:1393-1403;Nguyen,H.等,(1997)Microbiol.Immunol.41:901-907;Arai,K.等,(1998)J.Immunol.Methods217:79-85;Yoshinari,K.和Arai,K.(1998)Hybridoma 17:41-45;Hutchins,W.A.等,(1999)Hybridoma18:121-129;Murphy,W.J.等,(1999)Clin.Immunol.90:22-27;Smithson,S.L.等,(1999)Mol.Immunol.36:113-124;Chamat,S.等,(1999)J.Infect.Diseases180:268-277;以及Heard,C.等,(1999)Molec.Med.5:35-45。
根据另一实施方案,以本发明的寡聚体及其衍生物免疫的动物是以致死全身辐射处理,然后以重度联合免疫缺损小鼠(SCID)小鼠骨髓细胞抵抗辐射,继之移植人类功能性淋巴细胞的小鼠。经目的抗原免疫所述小鼠,使用这类被称为Trimera系统的嵌合体通过标准杂交瘤技术生产人类单克隆抗体。关于这些小鼠及其在生产抗体中的用途的进一步描述,见例如Eren,R.等,(1998)Immunology93:154-161;Reisner,Y和Dagan,S.(1998)Trends Biotechnol.16:242-246;Ilan,E.等,(1999)Hepatology29:553-562; 以及Bocher,W.O.等,(1999)Immunology96:634-641。
体外途径
作为经免疫和选择而产生本发明抗体的备选方案,可以用本发明的寡聚体及其衍生物筛选重组免疫球蛋白文库,从而分离与寡聚体及其衍生物特异性结合的免疫球蛋白文库成员由此鉴定和分离本发明抗体。生产和筛选展示文库的试剂盒可商业获得(例如Pharmacia Recombinant Phage Antibody System,目录号24-9400-01;以及StratagenePhage Display Kit,目录号240612)。在许多实施方案中,展示文库是scFv文库或Fab文库。筛选重组抗体文库的噬菌体展示技术已被充分描述。用于产生和筛选抗体展示文库的方法和化合物的特别有利的实例,可见于例如McCafferty等,WO92/01047、US5,和EP589877(具体描述了scFv展示);Ladner等,US5,223,409、US5,403,484、US5,571,698、US5,837,500和EP436597(例如,描述pIII融合);Dower等,WO91/17271、US5,427,908、US5,580,717和EP589877(具体描述Fab展示);Winter等,International Publication WO92/20791和EP368,684(具体描述免疫球蛋白可变结构域序列的克隆-);Griffiths等,US5,885,793和EP589887(具体描述使用重组文库分离针对人类抗原的人抗体);Garrard等,WO92/09690(具体描述噬茵体表达技术);Knappik等,WO97/08320(描述人类重组抗体文库HuCal);Salfeld等,WO97/29131(描述针对人类抗原(人肿瘤坏死因子)的重组人抗体的产生,以及重组抗体的体外亲和力成熟)以及Salfeld等,U.S.Provisional Application No.60/126,603和以此为基础的专利申请(也描述了针对人类抗原(人白介素12)重组人类抗体的产生,以及重组抗体的体外亲和力成熟)。
关于重组抗体文库筛选的进一步描述,可见于科学出版物中,例如Fuchs等,(1991)Bio/Technology9:1370-1372;Hay等,(1992)Hum Antibod Hybridomas3:81-85;Huse等,(1989)Science246:1275-1281;Griffiths等,(1993)EMBO J12:725-734;Hawkins等,(1992)J Mol Biol226:889-896;Clarkson等,(1991)Nature352:624-628;Gram等,(1992)PNAS 89:3576-3580;Garrad等,(1991)Bio/Technology9:1373-1377;Hoogenboom等,(1991)Nuc4cid Res19:4133-4137;Barbas等,(1991)PNAS88:7978-7982;McCafferty等,Nature(1990)348:552-554;以及Knappik等,(2000)J.Mol.Biol.296:57-86。
作为使用噬菌体展示文库系统的替代方案,可以在酵母细胞或细菌细胞表面表达重组抗体文库。WO99/36569描述了在酵母细胞表面表达的文库的制备和筛选方法。WO98/49286更详细地描述了在细菌表面表达的文库的制备和筛选方法。
一旦组合文库的目标抗体得到鉴定,就使用标准分子生物学技术分离编码该抗体轻链和重链的DNA,例如通过对展示包装(例如噬茵体)的DNA PCR扩增,所述展示包装已在文库的筛选中被分离。可用于制备PCR引物的抗体轻链和重链基因的核苷酸序列为技术人员所公知。在例如Kabat,E.A.,等,(1991)《Sequences of Proteins of Immunological Interest》,第五版,U.S.Department of Health and Human Services,NIH Publication No.91-3242以及人类生殖细胞系VBASE序列数据库中描述了许多这样的序列。
可以通过在宿主细胞内重组表达免疫球蛋白轻链和重链基因,产生本发明的抗体或抗体部分。为了重组表达抗体,以一个或多个重组携带编码该抗体免疫球蛋白轻链和重链的DNA片段的表达载体转染宿主细胞,从而在宿主细胞内表达轻链和重链,并优选将其分泌至培养宿主细胞的培养基中。可从此培养基中分离抗体。使用标准重组DNA方法以获得重链和轻链抗体基因。在例如Sambrook、Fritsch及Maniatis(编),《Molecular Cloning;ALaboratory Manual》,第二版,ColdSpring Harbor,N.Y.,(1989);Ausubel,F.M.等(编)《Current Protocols in Molecular Biology》,Greene Publishing Associates,(1989)以及Boss等的US4,816,397中描述了这类方法。
一旦获得编码目的抗体VH和VL片段的DNA片段,就可以使用标准重组DNA技术对该DNA片段进行进一步操作,例如为了将可变区基因 转变为抗体链全长基因、Fab片段基因或scFv基因。这些操作包括将编码vL或VH的DNA片段与编码另一蛋白质(例如抗体怛定区域柔性接头)的另一DNA片段有效连接。术语“有效连接的”在此理解为指两个DNA片段以某种方式彼此连接,在这种方式中,于框内保持了两个DNA片段编码的氨基酸序列。
将编码VH区的DNA与编码重链恒定区(CH1、CH2及CH3)的另一个DNA分子有效连接,可以将分离的编码VH区的DNA转变为重链全长基因。人类重链恒定区基因序列广为人知(见例如Kabat,E.A.等,(1991)《Sequencesof Proteins of Immunological Interest》,第五版,U.S.Department of Health and Human Services,NIH Publication No.91-3242),可以通过标准PCR扩增方法获得跨越该区的DNA片段。重链恒定区可以是来自IgGl、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD的恒定区,优选IgG1或IgG4恒定区。为了获得重链Fab片段的基因,可以将编码VH的DNA与仅编码重链恒定区CH1的另一个DNA分子有效连接。
将编码vL的DNA与编码轻链恒定区CL的另一个DNA分子有效连接,可以将分离的编码VL区的DNA转变为轻链全长基因(以及Fab轻链基因)。人类轻链恒定区基因序列广为人知(见例如Kabat,E.A.等,(1991)《Sequences of Proteins of Immunological lnterest》,第五版,U.S.Department of Health and Human Services,NIH Publication No.91-3242),可以通过标准PCR扩增方法获得跨越该区的DNA片段。轻链恒定区可以是恒定κ或λ区,优选恒定κ区。
为了产生scFv基因,可以将编码VH和VL的DNA片段与编码柔性接头(例如氨基酸序列(Gly4-Ser)3)的另一个片段有效连接,使VH和VL序列以连续的单链蛋白质表达(见Bird等,(1988)Science242:423-426;Huston等,(1988)Proc.Nail.Acad.Sci.USA85:5879-5883;McCafferty等,Nature(1990)348:552-554)。
可以使用上述方法从单结构域文库分离对本发明的寡聚体或其衍生物 具有特异性的单结构域VH和VL。具有所需特异性的两个VH单结构域链(具有或不具有CH1)或两个VL链、或一个VH和一个VL链的一对可用于从身体中去除本发明的寡聚体或其衍生物。
为了表达本发明的重组抗体或抗体部分,可以将编码部分或全长轻链和重链的DNA插入表达载体,以便将基因与转录和翻译控制序列有效连接。本文中,术语“有效连接的”理解为指抗体基因以某种方式连接于载体内,在这种方式中,载体内的转录和翻译控制序列完成其调节该抗体基因转录和翻译的预期功能。
选择表达载体和表达控制序列,以便与使用的宿主细胞兼容。可以将抗体轻链基因与抗体重链基因插入到单独的载体中,或将两个基因插入到相同的表达载体中,这是通常的情况。使用标准方法将抗体基因插入表达载体中(例如抗体基因和载体上的互补限制性切割位点的连接,或者,若不存在限制性切割位点时的平末端连接)。轻链和重链序列插入前,表达载体可已经携带抗体恒定区序列。例如,一条途径是将VH和VL序列转化为全长抗体基因,通过将其插入到已经分别编码重链和轻链恒定区的表达载体中,从而将VH片段和CH片段在载体内有效连接,也将VL片段和CL片段在载体内有效连接。另外,或作为备选,重组表达载体可以编码便于抗体链从宿主细胞分泌的信号肽。可将该抗体链的基因克隆到载体中,从而将信号肽在框内与抗体链基因N末端有效连接。信号肽可以是免疫球蛋白信号肽或异源信号肽(即非免疫球蛋白蛋白质的信号肽)。除了抗体链基因,本发明的表达载体可以具有控制抗体链基因在宿主细胞内表达的调节序列。术语“调节序列”旨在包括控制抗体链基因转录和翻译的启动子、增强子和其它表达控制元件(例如聚腺苷酸化信号)。在例如Goeddel;《Gene Expression Technology:Methods InEnzymology185》,Academic Press,San Diego,CA(1990)中描述了这类调节序列。技术人员将理解,包括调节序列选择在内的表达载体的设计,依赖于诸如将转化的宿主细胞的选择、所需的蛋白质表达强度等因素。哺乳动物宿主细胞中表达的优选调节序列包括在哺乳动物细胞中导致强蛋白质表达的病毒元件,例如来自 自巨细胞病毒(CMV)(例如CMV启动子/增强子)、猿猴病毒40(SV40)(例如SV40启动子/增强子)、腺病毒(例如腺病毒主要晚期启动子(AdMLP))和多瘤病毒的启动子和/或增强子。关于病毒调节元件及其序列的进一步描述,见例如Stinski,US5,168,062、Bell等,US4,510,245及Schaffner等,US4,968,615。
除了抗体链基因和调节序列,本发明的重组表达载体可以具有额外序列,例如那些调节载体在宿主细胞中复制的序列(如复制起点)和选择性标记基因。选择性标记基因便于选择被引入载体的宿主细胞(见例如Axel等的US专利No4,399,216、4,634,665和5,179,017)。例如,常见的是选择性标记基因赋予引入载体的宿主细胞药物抗性,例如G418、潮霉素或氨甲蝶呤抗性。优选的选择性标记基因包括二氢叶酸还原酶(DHFR)基因(用于以甲氨蝶呤选择/扩增的dhfr-宿主细胞中)和neo基因(用于G418选择)。
为了表达轻链和重链,以标准技术将编码重链和轻链的表达载体转染宿主细胞中。术语“转染”的不同形式旨在包括常用于将外源DNA引入原核或真核宿主细胞的多种技术,例如电穿孔、磷酸钙沉淀、DEAE-葡聚糖转染等。尽管理论上可以在原核或真核细胞内表达本发明的抗体,优选在真核细胞中、特别是哺乳动物细胞中表达抗体,因为在这类真核细胞、特别是哺乳动物细胞中,正确折叠以及组装和分泌免疫活性抗体的概率高于原核细胞。对于活性抗体的高得率生产,抗体基因的原核表达经报导是低效的(Boss,M.A.和Wood,C.R.(1985)Immunology Today6:12-13)。
优选以哺乳动物细胞表达本发明的重组抗体,这些细胞包括CHO细胞(包括在Urlaub和Chasin,(1980)Proc.Natl.Acad.Sci.USA77:4216-4220中描述的dhfr-CHO细胞,如R.J.Kaufman和P.A.Sharp(1982)Mol.Biol.159:601-621中所描述,其与DHFR选择标记一起使用)、NS0骨髓瘤细胞、COS细胞和SP2细胞。把编码抗体基因的重组表达载体引入哺乳动物细胞时,培养宿主细胞直至抗体在该宿主细胞中表达,或优选将抗体分泌至宿主细胞生长的培养基中从而生产抗体。可以使用标准蛋白质 纯化方法将抗体从培养基中分离。
也可以使用宿主细胞生产完整抗体的部分,例如Fab片段或scFv分子。上述方法的改变当然也包括在本发明内。例如,可能需要以编码本发明的抗体轻或重链的DNA(但是并非两者)转染宿主细胞。如果存在非结合目的抗原所需的轻或重链,则可通过重组DNA技术将本发明这类轻链或这类重链的DNA或两者部分或全部去除。由这类截短的DNA分子表达的分子也包括在本发明的抗体内。另外,也可以通过标准化学方法将本发明的抗体与第二个抗体交联,生产双功能抗体,该抗体的一条重链和一条轻链为本发明的抗体,另一条重链和一条轻链对不同于目的抗原的抗原具有特异性。
在优选的重组表达本发明抗体或其抗原结合部分的系统中,通过磷酸钙介导的转染方法将编码抗体重链和抗体轻链的重组表达载体引入dhfr-CHO细胞中。在重组表达载体内,抗体重链和轻链基因在每种情况下与CMV增强子AdMLP-启动子调节元件有效连接,以影响该基因的强转录。重组表达载体也携带DHFR基因,通过使用甲氨蝶呤选择/扩增,该基因可用于选择被载体转染的宿主细胞。培养选择的转化宿主细胞,以表达重链和轻链,并从培养基中分离完整抗体。使用标准分子生物学技术制备重组表达载体,转染宿主细胞,选择转化体,培养所述宿主细胞,并从培养基中获得抗体。因此本发明涉及合成本发明的重组抗体的方法,该方法通过在合适的培养基中培养本发明的宿主细胞,直至合成本发明的重组抗体。该方法可进一步包括从所述培养基分离重组抗体。
作为通过噬菌体展示筛选重组抗体文库的备选方案,技术人员公知的其它方法可用于筛选大的组合文库,以鉴定本发明的抗体。在一种类型的备选表达系统中,以RNA-蛋白质融合物的形式表达重组抗体文库,如Szostak和Roberts,o98/31700、以及Roberts,R.W.和Szostak,J.W.(1997)Proc.Natl.Acad.Sci.USA94:12297-12302中所描述。在这个系统中,体外翻译合成的在3′末端携带嘌呤霉素(肽受体抗生素)的RNA产生mRNA与由其编码的肽或蛋白质的共价融合物——合成mRNA。因此,以编码的 肽或蛋白质(例如抗体或其部分)的特性为基础,例如,所述抗体或其部分对本发明的寡聚体或其衍生物的结合,可以浓缩mRNA的复杂混合物(例如重组文库)中的特定mRNA。可以以上述方式通过重组表达(例如在哺乳动物宿主细胞中)通过对这些库筛选而获得的编码抗体或其部分的核酸序列,另外,还可以通过更多轮筛选mRNA-肽融合物,将突变引入初始的选择序列,或以上述方法使用重组抗体的体外亲和力成熟方法,进行亲和力成熟。
体内和体外途径的组合
也可以使用体内和体外途径的组合产生本发明的抗体,例如在一个方法中,首先让本发明的寡聚体或其衍生物在宿主动物内体内作用于抗体所有组成成分,以刺激产生结合寡聚体或衍生物的抗体,然后在一种或多种体外技术的帮助下实现进一步的抗体选择和/或抗体成熟(即优化)。根据一个实施方案,这种组合方法可以包括:首先以本发明的寡聚体或其衍生物免疫非人类动物(例如小鼠、大鼠、兔、小鸡、骆驼、山羊或其转基因版本或嵌合小鼠),以刺激对所述抗原的抗体应答,然后使用淋巴细胞的免疫球蛋白序列制备和筛选噬茵体展示文库,其中淋巴细胞已经寡聚体或衍生物的作用体内刺激。可以用上述与体内途径有关的方式实施此组合方法的第一步,而用上述与体外途径有关的方式实施此组合方法的第二步。以随后体外筛选噬茵体展示文库的超免疫非人类动物的优选方法包括由BioSite Inc.描述的那些,见例如WO98/47343、WO91/17271、US5,427,908和US5,580,717,其中噬菌体展示文库从所述经过刺激的淋巴细胞制备。
根据另一实施方案,这种组合方法可以包括:首先以本发明的寡聚体或其衍生物免疫非人类动物(例如小鼠、大鼠、兔、小鸡、骆驼、山羊或其转基因本或嵌合小鼠),刺激对所述寡聚体或其衍生物的抗体应答,然后通过筛选杂交瘤(从例如免疫动物制备)选择产生具有所需特异性抗体的淋巴细胞。从选择的克隆分离抗体或单结构域抗体基因(通过标准克隆方法,例如反转录聚合酶链反应),并将其进行体外亲和力成熟,从而改进所选抗体(可能不只一种)的结合特性。可以用上述与体内途径有关的方式 实施此组合方法的第一步,而用上述与体外途径有关的方式实施此组合方法的第二步,特别是使用体外亲和力成熟方法,例如在WO97/29131和WO00/56772中描述的那些。
在另一组合方法中,使用技术人员公知为选择淋巴细胞抗体方法(SLAM)的程序从单个分离的淋巴细胞产生重组抗体,在US5,627,052、WO92/02551和Babcock,J.S.等,(1996)Proc.Natl.Acad.Sci.USA93:7843-7848中描述了该方法。在此方法中,首先以本发明的寡聚体或其衍生物体内免疫非人类动物(例如小鼠、大鼠、兔、小鸡、骆驼、山羊或其转基因本、或嵌合小鼠),以刺激对该寡聚体或衍生物的免疫反应,然后使用抗原特异性溶血噬斑试验选择分泌目的抗体的单个细胞。为此目的,可以使用诸如生物素的接头将寡聚体或衍生物或结构相关的目的分子与绵羊红细胞偶联,从而可以使用溶血噬斑试验鉴定分泌具有合适的特异性抗体的细胞。在鉴定分泌目的抗体的细胞之后,通过反转录酶PCR从细胞获得轻链和重链可变区的cDNA,然后可以在哺乳动物细胞(例如COS或CHO细胞)中将该可变区与合适的免疫球蛋白恒定区(例如,人恒定区)联合表达。通过涂布转染细胞,则可以使宿主细胞经历进一步体外分析和体外选择,例如,为了分离表达具有所需特异性的抗体的细胞,其中宿主细胞由来自体内选择的淋巴细胞的扩增的免疫球蛋白序列转染。扩增的免疫球蛋白序列可被进一步体外操作。
与上述程序类似,也可以制备对本发明的寡聚体或其衍生物及结构相关的合成分子具有特异性的抗体。这包括:
i)提供抗原,该抗原具有与本发明的寡聚体或其衍生物及结构相关的合成分子所共有的结构特点;
ii)将抗体所有组成成分暴露于该抗原;
iii)从该抗体所有组成成分选择与两个结构相关分子结合的抗体,从而获得具有所需特异性的抗体。
本发明涉及可以通过上面方法获得的寡聚体特异性抗体及其在制备治疗淀粉样β相关精神障碍的药物或在制备诊断淀粉样β相关精神障碍的组 合物中的用途。
根据本发明获得的抗体特别包括可以通过上面的方法获得的抗血清。抗血清可以是全血清,即去除细胞和凝血成分后从宿主获得的血液,或该血清的级份,在此级份中,特别含有浓缩的免疫球蛋白级份,优选浓缩的、识别寡聚体的免疫球蛋白级份。可以使用上述方法结合抗体纯化获得这种级份。
本发明的抗血清是多克隆的,即它们含有具有不同特异性、通常不同类和亚类的抗体,通常具有所有L链同种型,识别多个蛋白质表位。
使用本发明的多种寡聚体作为免疫原时,本发明的抗血清通常交叉反应。
根据另一方面,根据本发明获得的抗体也包括单克隆抗体,特别是嵌合和人源化抗体,及其寡聚体结合片段。
本发明涉及与本发明的寡聚体或其衍生物结合的蛋白质和特别是抗体,即对本发明的寡聚体或其衍生物具有特异性的抗体。本发明也涉及所述蛋白质或抗体的部分,特别是其抗原结合部分,即与本发明的寡聚体或其衍生物结合的蛋白质或抗体部分。
优选以致对本发明的寡聚体或其衍生物的特异性结合具有特定的结合动力学(即高亲和力、低解离、低解离速率、强中和活性)的本发明抗体。
对于对本发明的寡聚体或其衍生物具有的亲和力在KD=10-6-10-12M范围内的蛋白质,特别是抗体。特别优选高亲和力蛋白质、特别是高于Kd=10-8M的亲和力、高于Kd=10-9M的亲和力、高于Kd=10-10M的亲和力或高于Kd=10-11M的亲和力结合的抗体。
根据另一方面,优选以相对较低的亲和力、尤其是以低于Kd=10-8M的亲和力结合其它Aβ(1-42)蛋白质形式(特别是单体Aβ(1-42)蛋白质和/或Aβ(1-40)蛋白质)的那些蛋白质,特别是抗体。
因此,根据本发明,优选以高于单体Aβ(1-42)蛋白质和/或Aβ(1-40)蛋白质的亲和力结合寡聚体或其衍生物的那些蛋白质,特别是抗体。特别优选10、100、或1000的亲和力比值。
根据另一方面,可以选择本发明的抗体,以致以0.1s-1或更低的koff速率常数与寡聚体或其衍生物结合。对于1×10-2s-1或更低、1×10-3s-1或更低、1×10-4s-1或更低、1×10-5s-1或更低的速率常数koff,按照指出的顺序优选性逐渐增加。
另外,可以选择本发明的抗体,以致以1×10-6M或更低的IC50抑制本发明的寡聚体或其衍生物的活性,特别是神经毒性活性。对于1×10-7M或更低、1×10-8M或更低、1×10-9M或更低、1×10-10M或更低、或1×10-11M或更低的抑制常数IC50,按照指出的顺序优选性逐渐增加。
抗体优选为分离的抗体。根据另一方面,抗体为中和抗体。本发明的抗体包括单克隆和重组抗体。根据许多实施方案,抗体可以包括完全来自单一物种的氨基酸序列,例如人类抗体或小鼠抗体。根据其它实施方案,抗体可以是嵌合抗体或CDR移植物或人源化抗体的其他形式。
术语“抗体”意指由4个多肽链,即两个重(H)链和两个轻(L)链组成的免疫球蛋白分子。链通常通过二硫键与另一个连接。每条重链由该重链的可变区(此处缩写为HCVR或VH)和该重链的恒定区组成,重链恒定区由CH1、CH2和CH3三个结构域组成。每条轻链由该轻链的可变区(此处缩写为LCVR或VL)和该轻链的恒定区组成,轻链恒定区由CL结构域组成。VH和VL去可以进一步划分为被称为互补性决定区域(CDR)的高变区和散布的、被称为构架区(FR)的保守区。每个VH和VL区由三个CDR和四个FR组成,它们按照下列顺序从N端向C端排列:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
术语抗体的“抗原结合部分”(或简称为“抗体部分”)指对本发明的寡聚体或其衍生物具有特异性的一个或多个抗体片段,所述片段仍然能够特异地结合该寡聚体或其衍生物。完全抗体的片段经显示能够执行抗体的抗原结合功能。依照术语抗体的“抗原结合部分”,结合片段的实例包括(i)Fab片段,即由VL、VH、CL和CH1结构域组成的单价片段;(ii)F(ab')2片段,即包括两个Fab片段的二价片段,其中Fab片段通过二硫桥在铰链区中彼此连接;(iii)由VH和CH1结构域组成的Fd片段;(iv)由抗体单 臂的FL和VH结构域组成的Fv片段;(v)由VH结构域或VH、CH1、CH2、DH3、或VH、CH2、CH3组成的dAb片段(Ward等,(1989)Nature 341:544-546);以及(vi)分离的互补性决定区域(CDR)。尽管Fv片段的两个结构域(即VL和VH)由分离的基因编码,可以使用合成接头和重组方法进一步将其彼此连接,使它们可能以单个蛋白质链制备,在该蛋白质链中,VL和VH区组合以形成单价分子(公知为单链Fv(ScFv);见例如Bird等,(1988)Science242:423-426;以及Huston等,(1988)Proc.Natl.Acad.Sci.USA85:5879-5883)。术语抗体的“抗原结合部分”也旨在包括这类单链抗体。单链抗体的其它形式例如“双抗体”也包括于此。双抗体是二价双特异性抗体,在双抗体中,VH和VL结构域表达在单个多肽链上,但是使用对于能够组合在相同链上的两个结构域而言太短的接头,从而迫使该结构域与不同链的互补结构域配对,并形成两个抗原结合位点(见例如Holliger,P.等,(1993)Proc.Natl.Acad.Sci.USA90:6444-6448;Poijak,R.J.等,(1994)Structure2:1121-1123)。
另外,抗体或抗原结合部分可以是较大的免疫黏附素球蛋白分子的部分,其中免疫黏附素分子由该抗体或抗体部分与一个或多个其它蛋白质或肽的共价或非共价联合而形成。与这类免疫黏附素分子相关的是链霉亲和素核心区的使用,以制备四聚体scFv分子(Kipriyanov,S.M等,(1995)Human Antibodies und Hybridomas6:93-101),以及半胱氨酸、标记肽和C末端多组氨酸标签的使用,以产生二价和生物素化的scFv分子(Kipriyanov,S.M.等,(1994)Mol.Immunol.31:1047-1058)。使用常规技术例如以木瓜蛋白酶或胃蛋白酶消化,可以从完整抗体产生诸如Fab和F(ab′)2片段的抗体部分。另外,使用标准重组DNA技术可以获得抗体、抗体部分和免疫黏附素分子。“对本发明的寡聚体或其衍生物具有特异性的分离的抗体”指对本发明的寡聚体或其衍生物具有特异性的抗体,其基本不合具有不同抗原特异性的其它抗体,即,尤其不含有与上述Aβ(1-42)的其它形式特异性结合的抗体的抗体。
术语“中和抗体”指与特定抗原的结合引起该抗原生物活性的抑制的 抗体。使用适宜的体外或体内测定法,测量抗原的一个或多个生物活性的指标,可以评价该抗原生物活性的抑制。
术语“单克隆抗体”指来自杂交瘤的抗体(例如由杂交瘤分泌的抗体,其中杂交瘤通过杂交瘤技术(例如根据Kohler和Milstein的标准杂交瘤方法)制备)。因此将来自杂交瘤产生并对本发明的寡聚体或其衍生物具有特异性的抗体称为单克隆抗体。
术语“重组抗体”指通过重组方法生产、表达、产生或分离的抗体,例如使用转染入宿主细胞的重组表达载体表达的抗体;从重组组合抗体文库分离的抗体;从人类免疫球蛋白基因转基因动物(例如小鼠)分离的抗体(见例如Taylor,L.D.等,(1992)Nucl.Acids Res.20:6287-6295);或者以其它方法生产、表达、产生或分离的抗体,在这些方法中将特定的免疫球蛋白基因序列(例如人类免疫球蛋白基因序列)与其它DNA序列组装。重组抗体包括,例如嵌合的、CDR移植物和人源化抗体。
术语“人类抗体”指其可变和恒定区对应于或来自人类生殖细胞系免疫球蛋白序列的抗体,例如Kabat等所描述(见Kabat等,(1991)《Sequences of Proteins of Immunological Interest》,第五版,U.S.Department of Health und Human Services,NIH Publication No.91-3242)。然而,本发明的人类抗体可以在例如CDR、特别是CDR3中含有不是由人类生殖细胞系免疫球蛋白序列编码的氨基酸残基(例如通过体外随机或定点诱变、或通过体内体细胞突变引入的突变)。本发明的重组人类抗体具有可变区,也可以含有来自人类生殖细胞系免疫球蛋白序列的恒定区(见Kabat,E.A.等,(1991)《Sequences of Proteins of Immunological Interest》,第五版,U.S.Department of Health und Human Services,NIH Publication No9l-3242)。然而,根据具体的实施方案,将这类重组人类抗体进行体外诱变(或如果使用人类Ig序列转基因动物,则为体内体细胞诱变),以致重组抗体VH和VL区的氨基酸序列成为非天然体内存在于人类生殖细胞系抗体所有组成成分的序列,尽管其涉及或来自人类生殖细胞系VH和VL序列。根据具体的实施方案,这种重组抗体是选择诱变或回复突变或两者的结果。
术语“回复突变”指包括用对应于同源生殖细胞系抗体序列的生殖细胞系残基替换人类抗体的部分或全部体细胞突变氨基酸的方法。将本发明的人类抗体重链和轻链序列分别在VBASE数据库中与生殖细胞系序列比较,以鉴定具有最高同源性的序列。突变编码这些确定的异化氨基酸的核苷酸位点,将本发明的人类抗体的印记(imprint)回复为生殖细胞系序列。应当研究以此方式鉴定为候选的回复突变的各个氨基酸对抗原结合的直接或间接重要性,不应将突变后削弱该人类抗体所需性质的氨基酸整合到最终的人类抗体中。为了保持回复突变的氨基酸数目尽可能少,尽管有异于最接近的生殖细胞系序列,与第二个生殖细胞系序列的相应氨基酸序列相同的那些氨基酸位点应当保持不变,条件是所述第二个生殖细胞系序列在相关氨基酸两侧以至少10个、优选12个氨基酸与本发明的人类抗体序列相同并线性对应。可以在抗体优化的任何阶段实施回复突变。
术语“嵌合抗体”指含有一个物种重链和轻链可变区序列,但是其中VH和/或VL的一个或多个CDR区序列被另一个物种的CDR序列置换的抗体,例如具有小鼠重链和轻链可变区、其中一个或多个小鼠CDR(例如CDR3)被人类CDR序列置换的抗体。
术语“人源化抗体”指含有非人类物种(例如小鼠、大鼠、兔、小鸡、骆驼、山羊)重链和轻链可变区序列、但是其中至少部分VH和/或VL序列经过改变从而更加“与人类似”,即与人类生殖细胞系可变区序列更加相似。一种类型的人源化抗体是CDR移植物抗体,其中将人类CDR序列插入非人类VH和VL序列中,以置换相应的非人类CDR序列。
测量抗体结合动力学的一种方式是使用表面等离振子共振方法。术语“表面等离振子共振”指一种光学现象,通过这种现象,使用例如BIAcore系统(Pharmacia Biosensor AB,Uppsala,Sweden and Piscataway,NJ),以生物传感器矩阵检测蛋白质浓度的变化,可以分析生物特异性相互作用。进一步描述,见U.等,(1993)Ann.Biol.Clin.51:19-26;U.等,(1991)Biotechniques11:620-627;Johnsson,B.等,(1995)J.Mol.Recognit.8:125-131;以及Johnnson,B.等,(1991)Anal.Biochem.198: 268-277。
术语“Koff”指抗体从抗体/抗原复合物解离的解离速率常数。
术语“Kd”指特定的抗体-抗原相互作用解离常数。
本发明的抗体的结合亲和力可以使用标准体外免疫测定例如ELISA或BIAcore分析进行评价。
除了抗体,蛋白质可以是T细胞受体衍生的分子或T细胞受体衍生的受体结构域或该受体结构域与免疫球蛋白Fc部分的融合蛋白质。
本发明也涉及药物制剂(组合物),该制剂含有本发明的蛋白质,特别是本发明的抗体,以及任选的可药用载体。本发明的药物组合物可以进一步含有至少一种额外的治疗剂,例如一种或多种治疗可被本发明抗体减轻的疾病的治疗剂。例如,如果本发明的抗体结合本发明的寡聚体,药物组合物可以进一步含有一种或多种额外的治疗紊乱的治疗剂,其中所述寡聚体的活性对紊乱是重要的。
药物适宜载体包括任何溶剂、分散介质、包衣、抗茵和抗真菌剂、等渗和延迟吸收剂等,只要它们是生理学相容的。药物接受载体包括例如水、盐溶液、磷酸缓冲盐溶液、右旋糖、甘油、乙醇等,及其组合。在许多情况下,优选使用等渗剂,例如糖、多元醇(如甘露醇或山梨醇),或还有氯化钠。药物适宜载体可以进一步含有相对少量的辅助物质,例如润湿剂或乳化、防腐剂或缓冲液,它们将增加抗体的半衰期或功效。
药物组合物可适于例如胃肠外施用。在此,优选将抗体制备为具0.1-250mg/ml的抗体含量的可注射溶液。可以以液体或冻干形式制备可注射溶液,药剂形式为铅玻璃(flint glass)或小瓶、安瓿或填充注射器。缓冲液可以含有L-组氨酸(1-50mM,优选5-10mM),具有5.0-7.0、优选6.0的pH。其它适宜的缓冲液包括(但不限于)琥珀酸钠、柠檬酸钠、磷酸钠或磷酸钾缓冲液。可以使用氯化钠以调节溶液张力至0-300mM(对于液体剂量形式优选150mM)浓度。对于冻干剂量形式也可以包括抗冻剂,例如蔗糖(例如0-10%,优选0.5-1.0%)。其它合适的防冻剂是海藻糖和乳糖。对于冻干剂量形式也可以包括填充物例如甘露糖(例如1-10%,优 选2-4%)。稳定剂例如L-蛋氨酸(例如51-50mM,优选5-10mM)可以用于液体和冻干剂量形式。其它合适的填充物为甘氨酸和精氨酸。也可以使用表面活性剂例如聚山梨醇酯80(例如0-0.05%,优选0.005-0.01%)。其它表面活性剂为聚山梨醇酯20和BRIJ表面活性剂。
本发明的组合物可以有多种形式。这包括液体、半固体和固体剂量形式,例如液体溶液(如可注射及可灌输溶液)、分散剂或悬浮液、片剂、丸剂、粉末、脂质体和栓剂。优选的形式取决于欲施用的类型和治疗应用。一般,优选对可注射或可灌输溶液形式的组合物,例如与人类被动免疫的其它抗体相似的组合物。优选的给药途径是胃肠外(例如静脉内、皮下腹膜内、肌肉内)。根据优选的实施方案,通过静脉灌输或注射给予抗体。根据另一个优选的实施方案,通过肌肉内或皮下注射给予抗体。
治疗组合物一般必须是无菌并在制备和储存条件下稳定的。可以将组合物配制为溶液、微乳液、分散剂、脂质体或其它适于高活性物质浓度的有序结构。可以以要求的量将活性化合物(例如抗体)引入合适的溶剂(根据需要,适当时为上述成分的一种或组合),然后无菌过滤该溶液,从而制备无菌的可注射溶液。分散剂通常是通过将活性物质引入含有基本分散介质(恰当时还有其它所需成分)的无菌赋形剂中而制备。在制备无菌可注射溶液的无菌冻干粉末的情况下,真空干燥和喷雾干燥是优选的制备方法,该方法从前面无菌过滤溶液生产活性成分、合适时还有其它所需成分的粉末。通过使用例如卵磷脂这类包衣、在分散剂情况下保持要求的颗粒尺寸或使用表面活性剂,可以保持溶液合适的流动性。通过将延缓吸收的制剂(例如单硬脂酸盐和明胶)额外引入组合物,可以实现可注射组合物的延长吸收。
尽管对于许多治疗应用,优选的给药类型是皮下注射、静脉注射或静脉灌注,本发明的抗体可以通过许多技术人员公知的方法给药。技术人员将理解,给药途径和/或类型取决于所需的结果。根据具体的实施方案,可以用保护化合物免于迅速释放的载体制备活性化合物,例如具有受控释放的制剂,包括植入物、经皮膏药以及微胶囊化的释放系统。可以使用可生 物降解的、生物相容的多聚体,例如乙烯-醋酸乙烯酯共聚物、聚酸酐、聚乙二醇酸、胶原质、聚原酸酯、聚乳酸。制备这类制剂的方法为技术人员所熟知,见例如《Sustained and Controlled Release Drug Delivery Systems》,J.R.Robinson,编,Marcel Dekker,Inc.,New York,1978。
根据具体的实施方案,本发明的抗体可以在例如惰性稀释剂或可同化的食用载体中经口给药。也可以将抗体(如果需要,还有其它成分)封闭在硬或软明胶胶囊中、压缩成片剂或直接加入食物中。对于经口治疗给药,可以将抗体与赋形剂混和,以可吞吃片剂、含片剂、胶囊、酏剂、悬浮剂、糖浆剂等形式使用。如果旨在将本发明的抗体通过非胃肠外途径给药,有必要从防止其失活的材料中选择包衣。
本发明的抗体优选能够体外或体内中和其结合的本发明的寡聚体或其衍生物的活性。因此,在例如含有所述寡聚体或其衍生物的细胞培养物中,或在存在该寡聚体或其培养物的人类个体或哺乳动物中,该抗体可以用于抑制本发明的寡聚体或其衍生物的活性。根据一个实施方案,本发明涉及抑制本发明的抗体或其衍生物的活性的方法,该方法包括:允许本发明的抗体作用于寡聚体或其衍生物,以致抑制该寡聚体或其衍生物的活性。例如,可以在体外抑制该活性。例如,可以将本发明的抗体加入细胞培养物中,其中的细胞培养物含有或可疑含有本发明的寡聚体或其衍生物,从而抑制培养物中寡聚体或其衍生物的活性。另外,可以在个体体内抑制寡聚体或其衍生物的活性。
本发明进一步涉及在个体内抑制本发明的寡聚体或其衍生物的活性的方法,其中个体遭受紊乱,该紊乱涉及淀粉样β蛋白质,本发明的寡聚体或其衍生物的活性尤其重要。该方法包括将至少一种本发明的抗体施用于个体,以抑制抗体结合的寡聚体或其衍生物活性。所述个体优选为人类。本发明的抗体可以出于治疗性目的施用于人类个体。另外,本发明的抗体可以出于兽医目的或在具体疾病的动物模型框架内施用于非人类哺乳动物。这类动物模型对于评价本发明抗体的治疗性功效(例如测试给药的剂量和时程)可能有用。
本发明的寡聚体及其衍生物参与其中的紊乱,特别包括在其发展和/或过程中涉及本发明的寡聚体或其衍生物的紊乱。这尤其指那些紊乱:本发明的寡聚体或其衍生物是该病症明显或推定的病理生理学原因,或是参与该紊乱的发展或过程的因素。因此,这里包括那些病症,在这些病症中,本发明的寡聚体或其衍生物的活性的抑制可以缓解病症的症状和/或进展。通过例如在遭受具体病症个体的生物学液体中本发明的寡聚体或其衍生物浓度的升高(例如在血清、血浆、CSF、尿等中浓度升高)可以证实这些紊乱。这可以使用例如本发明的抗体检测。本发明的寡聚体及其衍生物在与许多病症相关的病理学中起到重要作用,在这些病症中,涉及神经变性因素、认知缺陷、神经毒性因素和炎性因素。
本发明的抗体可以与一种或多种对于上述病症的治疗备用的额外治疗剂一起施用。
本发明的药物组合物一般含有至少一种本发明的治疗有效量或预防有效量的抗体。根据所需治疗,例如是否需要治疗性或预防性处理,可以选择并调整剂量方案。例如,根据治疗情况的需要,可以给以单次给药、随时间多次单独给药,或增加或降低的剂量。为了方便给药并保证剂量统一,以单次剂量形式以配制胃肠外组合物尤其有利。
本发明的抗体的治疗有效量或预防有效量可以在例如0.1-20mg/kg并优选1-10mg/kg范围内,但是不限于此。根据欲缓解病症的类型和严重性,这些量当然可以变化。
在抗体的诊断性使用框架内,特异性寡聚体的定性或定量测定特别用于诊断疾病相关的淀粉样β(1-42)形式。在本文中,特异性指能够以足够的灵敏度检测特定寡聚体或寡聚体混合物的可能性。本发明的抗体有利地具有低于10ng/ml样品、优选低于1ng/ml样品、并特别优选低于100pg/ml样品的灵敏度,意味着可以通过本发明的抗体检测到至少在各例中表明的每毫升样品中的寡聚体浓度,以及有利的更低浓度。
根据免疫学实施测定。在原则上可以使用任何使用到抗体的分析性或诊断性测定,包括凝集和沉淀技术、免疫测定、免疫组织化学方法和免疫 印迹方法,例如蛋白质印迹或斑点印迹方法。在此也包括体内方法,例如成像方法(imaging method)。
在免疫测定中的用途是有利的。竞争性免疫测定和夹层免疫测定都是合适的,竞争性免疫测定即抗原和标记的抗原(示踪物)竞争抗体结合,夹层免疫测定即通常使用标记的第二抗体检测特异性抗体对抗原的结合。这些试验可以是同相的,即没有分离成固相和液相,或是多相的,即通过例如固相结合的抗体将结合的标记与未结合的标记分开。根据标记和测量方法,不同的多相和同向免疫测定形式可以分成具体的类别中,例如RIA(放射免疫测定)、ELISA(酶联免疫吸附测定)、FIA(荧光免疫测定)、LIA(发光免疫测定)、TRFIA(时间分辨的FIA)、IMAC(免疫活化)、EMIT(酶放大的免疫测试)、TIA(turbodimetric免疫测定)、I-PCR(免疫-PCR)。
关于本发明的寡聚体测定,优选竞争性免疫测定法,其中标记的寡聚体(示踪物)与样品将要定量的寡聚体样品竞争结合使用的抗体。借助于标准曲线,可以从置换的示踪物的量测定样品中抗原的量,即寡聚体的量。
可用于这些目的的标记中,经证实酶是有利的。例如,可以使用基于过氧化物酶(特别是辣根过氧化物酶)、碱性磷酸酶和β-D-半乳糖酶的系统。可获得这些酶的特异性底物,特异性底物的转化可以通过例如光度法监测。合适的底物系统以下列为基础:对于碱性磷酸酶,对硝基苯磷酸盐(p-NPP)、5-溴-4-氯-3-吲哚磷酸/氮蓝四唑(BCIP/NBT)、Fast-Red/萘酚-AS-TS磷酸;对于过氧化物酶,2,2-联氮-双(3-乙基苯并噻唑啉-6-磷酸)(2,2-Azino-bis-(3-ethylbenzthiazolin-6-sulfonsiure))(ABTS)、邻苯二胺(OPT)、3,3′,5,5’-四甲基对二氨基联苯(3,3’,5,5’-Tetramethylbenzidine)(TMB)、邻联茴香胺、5-氨基水杨酸、3-二甲基氨基苯酸(DMAB)和3-甲基-2-苯并噻唑啉酮腙(3-methyl-2-benzothiazolinhydrazone)(MBTH);对于β-D-半乳糖酶,邻硝基苯-β-D-半乳糖苷(o-NPG)、对硝基苯-β-D-半乳糖苷和4-Methylumbelliphenyl-β-D-半乳糖苷(MUG)。在许多情况下,这些底物系统可商业获得即用形式,例如以片剂形式,其可 以含有其它试剂,例如适宜的缓冲液等。
使用的示踪物是标记的寡聚体。在这一方面,可以通过标记待测寡聚体并将其用作示踪物而测定特定的寡聚体。
可以用本身已知的方式将标记与寡聚体偶联,以制备示踪物。通过类推提及上文关于衍生本发明寡聚体的注释。另外,可以获得许多为与蛋白质缀合而经过适当修饰的标记,例如生物素、抗生物素蛋白、extravidin或链霉亲和素缀合的酶、马来酰亚胺活化的酶等。这些标记可以与寡聚体或根据需要与经适当衍生的寡聚体直接反应而生成示踪物。例如,如果使用链霉亲和素过氧化物酶缀合物,则首先需要求生物素化寡聚体。这相应地应用于相反的顺序。关于这个目的,合适的方法也为技术人员所公知。
如果选择多相免疫测定形式,可以通过将抗原-抗体复合物结合到支持物上而将其分离,例如,通过与支持物偶联的抗独特型抗体(如抗兔IgG抗体)来分离。支持物、特别是以适宜的抗体包被的微滴定板是已知的,并且部分可商业获得。
本发明进一步涉及免疫测定套装,该套装具有至少一种上述抗体以及其它组分。该套装通常为包装单位形式,是实施本发明的寡聚体测定的组合物。为了操作尽可能简单的目的,优选以基本即用的形式提供上述手段。有利的安排是以试剂盒形式提供免疫测定。试剂盒通常包括用于分开排列组分的多个容器。可以以即用稀释液、以用于稀释的浓宿物,或者以用于溶解或悬浮的干物质或冻干物提供所有组分;个别或所有成分可以冷冻或在室温保存直至使用。血清优选在例如-20℃迅速冷冻,以致在这种情况下,免疫测定在使用前不得不优选保持在结冰温度之下。
与免疫测定提供的其它组分取决于该免疫测定的类型。通常,与抗血清一起提供标准蛋白、示踪物(可能需要,也可能不需要)以及对照血清。另外,也包括微滴定板(优选抗体包被的)、用于例如测试、洗涤或底物转变的缓冲液和酶底物本身。
免疫测定、作为实验室和医院中的辅助的抗体的生产和使用的通用原则可见于例如《Antibodies,A Laboratory Manual》(Harlow,E.和Lane,D. 编,Cold Spring Harbor Laboratory,Cold Spring Harbor,NY,1988)。
其它目标还有抑制本发明的寡聚体聚集或加速其解聚的物质。这类物质特别代表了治疗上述淀粉样β相关紊乱(例如阿尔茨海默病)的可能的治疗剂。
本发明因此也涉及表征物质或物质混合物的方法,该方法包括
i)以适当的方式提供该物质或物质混合物;
ii)允许该物质或物质混合物作用于至少一种本发明的寡聚体、其衍生物或组合物;以及
iii)测定该物质或该物质混合物的特定部分是否与所述寡聚体、其衍生物或存在于所述组合物中的至少一种寡聚体或其衍生物结合。
也可以在生物来源的混合物中实施该方法,例如细胞制品和细胞提取物,从而鉴定对本发明的寡聚体具有亲和力的天然结合配偶体,例如细胞表面抗原,特别是受体和可溶性配体,例如特定的蛋白质和中介体。
不仅是物质的结合,其与本发明的寡聚体的相互作用及对寡聚体的影响也可以是该方法的主题。因此特别可以测定是否:
-该物质能够调节、特别是抑制淀粉样β蛋白质向本发明的寡聚体的聚集;
-该物质能够调节、特别是增强本发明的寡聚体的解聚;
-本发明的寡聚体引起结合配偶体的功能改变,例如对受体具有激动、部分激动、拮抗或逆激动作用。
所述方法通常为体外筛选方法,可以用于从多种不同物质中选择对进一步应用最有前景的那些物质。例如,可以通过组合化学方法建立包括大量潜在活性物质的广泛的物质文库。可以自动化筛选具有所需活性的物质的组合文库。筛选机器人用于有效地分析单个的测定,这些测定优选排列在微滴定板上。本发明因此也涉及筛选方法,即一级和二级筛选方法,优选其中至少应用一种下述方法。如果应用若干方法,它们可以随时间变化或同时应用于同一样品,或应用于待测物质的不同样品。
实施这类方法的特别有效的技术是闪烁亲近测定法,简称为SPA,该 技术为药物筛选领域所公知。实施该测定的试剂盒和组分可以商业获得,例如从Amersham Pharmacia Biotech。原则上,将溶解的或膜固定的受体固定在含有闪烁物质的小的荧光微球体上。当例如放射性配体与结合受体结合时,由于闪烁物质和放射性配体的空间亲近,闪烁物质得到刺激并发光。
实施这类方法的另一个特别有效的技术是药物筛选领域公知的FlashPlateR技术。实施该测定的试剂盒和组分可以商业获得,例如从NENR Life Science Products。该原则也基于闪烁物质包被的微滴定板(96或384孔)的。
本发明涉及物质或物质混合物的一部分,及其在制备药剂或制备组合物中的用途;作为与寡聚体、其衍生物或与存在于相应组合物中的至少一种寡聚体或其衍生物结合的配体,其中物质或物质混合物的部分可以根据本方法确定,其中药剂用于淀粉样β相关的紊乱(特别是痴呆)的治疗,其中组合物组合物用于诊断淀粉样β相关的紊乱(特别是痴呆)。
下面的实施例旨在说明本发明,而非限制其范围。
附图说明:
图1显示Aβ(1-42)寡聚体A制品(泳道A)、Aβ(1-42)寡聚体B制品(泳道B)、标准蛋白质(分子量标志蛋白质,泳道C)的SDS PAGE;
图2显示Aβ(1-42)寡聚体A制品(泳道A);Aβ(1-42)寡聚体A-CL制品(泳道A’);Aβ(1-42)寡聚体B制品(泳道B);Aβ(1-42)寡聚体B-CL制品(泳道B’);标准蛋白质(分子量标志蛋白质,泳道C)的SDS PAGE;
图3显示生物素Aβ(1-42)寡聚体B制品(泳道A);标准蛋白质(分子量标志蛋白质,泳道B)的SDS PAGE;
图4显示荧光素Aβ(1-42)寡聚体B制品(泳道A);标准蛋白质(分子量标志蛋白质,泳道B)的SDS PAGE;
图5显示与含有Aβ(1-42)寡聚体B的制品比较,含有Aβ(1-42)冻干物的溶液的凝胶渗透层析;
图6显示Aβ(1-42)寡聚体B制品(泳道A);标准蛋白质(分子量标 志蛋白质,泳道B)的NATIVE PAGE;
图7显示(a)单体Aβ(1-42)蛋白质、(b)Aβ(1-42)寡聚体A以及(c)Aβ(1-42)寡聚体B对人类成神经细胞瘤细胞系IMR-32表面的结合;
图8以%显示以Aβ(1-42)寡聚体B处理鼠皮质神经元后的神经毒性作用,误差条对应于95%置信区间;
图9显示以胰蛋白酶(泳道2)、糜蛋白酶(泳道3)、嗜热菌蛋白酶(泳道4)、弹性蛋白酶(泳道5)、木瓜蛋白酶(泳道6)处理的或未经处理的(泳道7)Aβ(1-42)制品;以及标准蛋白质(分子量标志蛋白质,泳道1)的SDS PAGE;
图10显示下列反应性的斑点印迹100pmol(A行)、10pmol(B行)、l pmol(C行)、0.1pmol(D行)、0.01pmol(E行)的实施例6b的Aβ(1-42)寡聚体B制品(列1)、实施例1a的HFIP处理的Aβ(1-42)单体(列2)、实施例15a的嗜热菌蛋白酶剪切的Aβ(1-42)寡聚体B制品(列3)、实施例14a的戊二醛交联的Aβ(1-42)寡聚体B制品(列4)、根据M.P.Lambert等,J.Neurochem.79,595-605(2001)在4℃或室温或37℃(分别为列5、列6和列7)制备的ADDL、溶解于0.1%NH4OH中的Aβ(1-42)(列8)、实施例27的Aβ(1-42)原纤维制品(列9)、以及Sigma的PBS稀释的APP(列10),其中具有a)单克隆抗体6E10;b)实施例25d的多克隆抗血清(d1);c)实施例25c的多克隆抗血清(c1);d)实施例25a的多克隆抗血清(a1);以及e)实施例25a的多克隆抗血清(a2);
图11显示实施例15a的Aβ(1-42)寡聚体B制品(泳道B);实施例15b的Aβ(1-42)寡聚体C制品(泳道C);实施例6b的Aβ(1-42)寡聚体B制品(泳道A);以及标准蛋白质(分子量标志蛋白质,泳道D)的SDS PAGE;
图12显示与实施例14a的Aβ(1-42)寡聚体B-CL制品比较,实施例6b的Aβ(1-42)寡聚体B制品的凝胶渗透层析;
图13显示单体Aβ(1-42)蛋白质(左);Aβ(1-42)寡聚体(12体,A);以及Aβ(1-42)寡聚体(12体,C)和Aβ(20-42)寡聚体(12体,B)的图示表示,后二者可以通过蛋白酶水解剪切获得;
图14以Aβ(1-42)寡聚体B对海马区作用的时间函数显示兴奋性突触后电位(fEPSP);
图15显示描述与非特异性结合荧光相比(b),Aβ(1-42)寡聚体B与大鼠海马神经元结合的免疫荧光图像(a)。
除非另外指出,本发明寡聚体的浓度以单体Aβ(1-42)多肽的摩尔浓度表示。术语β淀粉样(1-42)蛋白质相当于术语淀粉样β(1-42)蛋白。除非另外指出,使用的蛋白质(多肽)为人类来源。
实施例
实施例1
a)人类Aβ(1-42)贮存悬浮液的制备
将2mg人类β淀粉样(1-42)蛋白质(简称:Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国)溶解于800μl1,1,1,3,3,3-六氟-2-丙醇中,在Eppendorf管中于37℃孵育30分钟。继之在真空浓缩器(Speed Vac)中蒸发至干燥。以88μl DMSO吸收残余物,得到5mM Aβ(1-42)贮存悬浮液,可保存于-20℃。
b)大鼠Aβ(1-42)贮存悬浮液的制备
将2mg大鼠β淀粉样(1-42)蛋白质(简称:大鼠Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国)溶解于800μl1,1,1,3,3,3-六氟-2-丙醇中,在Eppendorf管中于37℃孵育30分钟。继之在真空浓缩器(Speed Vac)中蒸发至干燥。以88μl DMSO吸收残余物,得到5mM大鼠Aβ(1-42)贮存悬浮液,可保存于-20℃。
实施例2
a)具有l5kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的制备;使用SDS
将690μl PBS缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)添加到60μl实施例1a的贮存溶液中,以75μl2%强度的十二烷基硫酸钠(SDS) 溶液调节该混合物至SDS含量为0.2%。继之为在37℃、5小时的孵育,以及于10000g、10分钟的离心。该Aβ(1-42)寡聚体A制品(约400μM Aβ(1-42))可以保存于-20℃。
b)具有15kDa和20kDa分子量的大鼠Aβ(1-42)寡聚体[大鼠Aβ(1-42)寡聚体A]的制备;使用SDS
将690μl PBS缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)添加到60μl实施例1b的贮存溶液中,以75μl2%强度的十二烷基硫酸钠(SDS)溶液调节该混合物至SDS含量为0.2%。继之为在37℃、5小时的孵育,以及于10000g、10分钟的离心。该大鼠Aβ(1-42)寡聚体A制品(约400μM大鼠Aβ(1-42))可以保存于-20℃。
c)具有15kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的制备;使用月桂酸
将690μl PBS缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)添加到60μl实施例1a的贮存溶液中,以75μl5%强度的月桂酸溶液调节该混合物至月桂酸含量为0.5%。继之为在37℃、5小时的孵育,以及于10000g、10分钟的离心。该Aβ(1-42)寡聚体A制品(约400μM Aβ(1-42))可以保存于-20℃。
d)具有15kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的制备;使用油酸
将690μl PBS缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)添加到60μl实施例1a的贮存溶液中,以75μl5%强度的油酸溶液调节该混合物至油酸含量为0.5%。继之为在37℃、5小时的孵育,以及于10000g、10分钟的离心。该Aβ(1-42)寡聚体A制品(约400μM Aβ(1-42))可以保存于-20℃。
e)具有15kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的制备;使用十二烷酰肌氨酸
将690μl PBS缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)添加到60μl实施例1a的贮存溶液中,以75μl5%强度的十二烷酰肌氨酸溶液 调节该混合物至油酸含量为0.5%。继之为在37℃、5小时的孵育,以及于10000g、10分钟的离心。该Aβ(1-42)寡聚体A制品(约400μM Aβ(1-42))可以保存于-20℃。
实施例3
a)制备具有15kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的备选方法
在220μ110mM水HCl溶液中吸收1mg人类β淀粉样(1-42)蛋白质(简称:Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国),并于室温孵育10分钟。通过于10000g离心5分钟去除不溶性成分。上清液(1mMAβ(1-42))含有Aβ(1-42)蛋白质,并如下进行进一步加工:
将9μl PBS缓冲液和1μl2%强度的SDS溶液添加至1μl上清液中,混合物于37℃孵育16h。hAβ(1-42)寡聚体A制品(100μM)可以保存于-20℃。
b)制备具有15kDa和20kDa分子量的大鼠Aβ(1-42)寡聚体[大鼠Aβ(1-42)寡聚体A]的备选方法
在10mM水HCl溶液220μl中吸收1mg大鼠β淀粉样(1-42)蛋白质(简称:大鼠Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国),并于室温孵育10分钟。通过于10000g离心5分钟去除不溶性成分。上清液(1mM大鼠Aβ(1-42))含有大鼠Aβ(1-42)蛋白质,并如下进行进一步加工:
将9μl PBS缓冲液和1μl2%强度的SDS溶液添加至1μl上清液中,混合物于37℃孵育16h。大鼠Aβ(1-42)寡聚体A制品(100μM)可以保存于-20℃。
实施例4
a)制备具有15kDa和20kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体A]的备选方法
将1mg人类β淀粉样(1-42)蛋白质(简称:Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国)溶解于44μl1%SDS/H2O中(5mM Aβ(1-42))。将5μl该溶液与40μl PBS及5μl2%SDS混和,于37℃孵育16h。通过于10000g离心5分钟去除不溶性成分。由此获得的Aβ(1-42)寡聚体A制品(500μM Aβ(1-42))可以保存于-20℃。
b)制备具有15kDa和20kDa分子量的大鼠Aβ(1-42)寡聚体[大鼠Aβ(1-42)寡聚体A]的备选方法
将1mg大鼠β淀粉样(1-42)蛋白质(简称:大鼠Aβ(1-42);肽合成材料、冻干物,来自Bachem,德国)溶解于44μl1%SDS/H2O中(5mM大鼠Aβ(1-42))。将5μl该溶液与40μlPBS及5μl2%SDS混和,于37℃孵育16h。通过于10000g离心5分钟去除不溶性成分。由此获得的大鼠Aβ(1-42)寡聚体A制品(500μM大鼠Aβ(1-42))可以保存于-20℃。
实施例5
a)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的制备
将根据实施例2a获得的Aβ(1-42)寡聚体A溶液以2.475ml水稀释(0.05%SDS含量,0.1mM Aβ(1-42)),于37℃孵育20小时。该Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
b)具有38kDa和48kDa分子量的大鼠Aβ(1-42)寡聚体[大鼠Aβ(1-42)寡聚体B]的制备
将根据实施例2a获得的大鼠Aβ(1-42)寡聚体A溶液以2.475ml水稀释(0.05%SDS含量,0.1mM大鼠Aβ(1-42)),于37℃孵育20小时。该大鼠Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
c)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的备选制备
将根据实施例2c获得的Aβ(1-42)寡聚体A溶液以2.475ml水稀释 (0.125%月桂酸含量,0.1mM Aβ(1-42)),于37℃孵育20小时。该Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
d)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的备选制备
将根据实施例2d获得的Aβ(1-42)寡聚体A溶液以2.475ml水稀释(0.125%油酸含量,0.1mM Aβ(1-42)),于37℃孵育20小时。该Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
e)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的备选制备
将根据实施例2e获得的Aβ(1-42)寡聚体A溶液以2.475ml水稀释(0.125%十二烷酰肌氨酸含量,0.1mM Aβ(1-42)),于37℃孵育20小时。该Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
f)具有38kDa和48kDa分子量的无SDS Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的制备
将根据实施例6b获得的Aβ(1-42)寡聚体B制品10μl与4%/33%/63%比例的乙酸/甲醇/水混合物250μl混和,在冰上于0℃孵育30分钟。离心(10000g,10分钟)后,去除上清液,将沉淀的蛋白质以200μl缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)吸收。以此方法获得的制品含有无SDS形式的溶解的Aβ(1-42)寡聚体B,可保存于-20℃。
实施例6
a)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]的透析和浓缩
将根据实施例5a制备的Aβ(1-42)寡聚体B与30ml含有F68(BASF)的PBS混和,并于Amicon Centriprep YM,30KD中浓缩至3ml。通过离心(10000g,5分钟)去除可能存在的残余物。取出上清 液。该Aβ(1-42)寡聚体B制品的等分试样可以于-80℃冷冻并保存用于进一步研究。
b)具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]浓缩物的制备
将根据实施例5a制备的Aβ(1-42)寡聚体B72.6ml经30KDCentriprep YM Tube(Amicon)浓缩为2ml。于10000g离心10分钟去除浓缩物。取出上清液,并于6℃在透析管中对1l缓冲液(5mM磷酸钠,35mM氯化钠,pH7.4)透析16h。于10000g离心10分钟去除透析物。取出上清液并于-80℃保存用于进一步研究。
实施例7
生物素-Aβ(1-42)贮存悬浮液的制备
将0.5mg生物素-β-淀粉样(1-42)蛋白质(简称:生物素-Aβ(1-42);肽合成材料、冻干物,AnaSpec)溶解于200μl1,1,1,3,3,3-六氟-2-丙醇中,在Eppendorf管中于37℃孵育30分钟。继之在真空浓缩器(Speed Vac)中蒸发至干燥。以20.5μl DMSO吸收残余物,得到5mM生物素Aβ(1-42)贮存悬浮液,可保存于-20℃。
实施例8
具有17kDa和22kDa分子量的生物素-Aβ(1-42)寡聚体[生物素-Aβ(1-42)寡聚体A]的制备
将2μl实施例7的贮存悬浮液与23μl PBS缓冲液(20mM磷酸钠,140mM NaCI,pH7.4)混和,以2.4μl2%强度的十二烷基硫酸钠(SDS)溶液调节至SDS含量为0.2%。继之为于37℃孵育6小时。通过于10000g、5分钟的离心去除不可溶成分。以此方法获得的生物素-Aβ(1-42)寡聚体A制品可以保存于-20℃。
实施例9
具有42kDa和52kDa分子量的生物素-Aβ(1-42)寡聚体[生物素-Aβ(1-42)寡聚体B]的制备
以82μl水稀释(含0.05%SDS0.1mM Aβ)根据实施例8获得的生物素-Aβ(1-42)寡聚体A,于37℃孵育16小时。通过于10000g、5分钟的离心去除不可溶成分。生物素-Aβ(1-42)寡聚体B制品可以于-20℃冷冻并保存用于进一步研究(图3)。
实施例10
荧光素-Aβ(1-42)贮存悬浮液的制备
将0.5mg荧光素-β-淀粉样(1-42)蛋白质(简称:荧光素-Aβ(1-42);肽合成材料、冻干物,AnaSpec)溶解于200μl1,1,1,3,3,3-六氟-2-丙醇中,在Eppendorf管中于37℃孵育30分钟。继之在真空浓缩器(Speed Vac)中蒸发至干燥。以20.5μl DMSO吸收残余物,产生5mM荧光素Aβ(1-42)贮存悬浮液,可保存于-20℃。
实施例11
具有17kDa和22kDa分子量的荧光素-Aβ(1-42)寡聚体[荧光素-Aβ(1-42)寡聚体A]的制备
将2μl实施例10的贮存悬浮液与23μl PBS缓冲液(20mM磷酸钠,140mM NaCI,pH7.4)混和,以2.4μl2%强度的十二烷基硫酸钠(SDS)溶液调节至SDS含量为0.2%。继之为于37℃孵育6小时。通过于10000g、5分钟的离心去除不可溶成分。以此方法获得的荧光素-Aβ(1-42)寡聚体A制品可以保存于-20℃。
实施例12
具有42kDa和52kDa分子量的荧光素-Aβ(1-42)寡聚体[荧光素-Aβ(1-42)寡聚体B]的制备
以82μl水稀释(0.05%SDS含量,0.1mM Aβ)根据实施例11获得 的荧光素-Aβ(1-42)寡聚体A,于37℃孵育16小时。荧光素-Aβ(1-42)寡聚体B制品可以于-80℃冷冻并保存用于进一步研究(图4)。
实施例13
实施例2a的Aβ(1-42)寡聚体A的交联[Aβ(1-42)寡聚体A-CL]
以7.5μ1PBS、0.2%SDS将10μl根据实施例2a制备的Aβ(1-42)寡聚体A稀释为100μM Aβ(1-42)含量。将1μl新制的10mM戊二醛水溶液添加至该溶液中,继之于室温搅拌3h。向样品中添加1μl100mM的氨基乙醇水溶液(pH7.4)并搅拌1h,以饱和过量的戊二醛。以此方法获得的制品含有交联Aβ(1-42)寡聚体A,将其称为Aβ(1-42)寡聚体A-CL制品。
实施例14
a)实施例5a的Aβ(1-42)寡聚体B的交联[Aβ(1-42)寡聚体B-CL]
将10μl根据实施例5a制备的Aβ(1-42)寡聚体B与1μl新制的10mM戊二醛水溶液混和,并于室温搅拌3h。向样品中添加1μl100mM的氨基乙醇水溶液(pH7.4)并搅拌1h,以饱和过量的戊二醛。以此方法获得的制品含有交联Aβ(1-42)寡聚体B,将其称为Aβ(1-42)寡聚体B-CL制品。
b)交联Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B-CL]的备选方案
将72.6ml根据实施例5a制备的Aβ(1-42)寡聚体B与7.26ml新制的10mM戊二醛水溶液混和,并于室温搅拌2h。向样品中添加726μl缓冲液(20mM磷酸钠,140mM NaCl,500mM氨基乙醇,pH74溶液(pH7.4)并搅拌30分钟,以饱和过量的戊二醛。通过15ml30kDa Centriprep管将反应混合物浓缩至3ml。经10000g、10分钟的离心去除浓缩物。去除上清液,并在透析管中与6℃对1L5mM磷酸钠、35mM NaCl,pH7.4透析16h。随后经10000、10分钟的离心去除透析物,取出上清液并可于-80℃保存以进一步研究。以此方法获得的制品含有交联的Aβ(1-42)寡聚体B,将其称为Aβ(1-42)寡聚体B-CL制品。
实施例15
a)以嗜热菌蛋白酶切割,从Aβ(1-42)寡聚体B制备截短的Aβ(20-42)寡聚体:
将1.59ml根据实施例6b制备的Aβ(1-42)寡聚体B制品与38ml缓冲液(50mM MES/NaOH,pH7.4)及1mg/ml的嗜热茵蛋白酶水溶液(Roche)200μl混和。将反应混合物于室温搅拌20h。然后加入80μl100mM EDTA水溶液(pH7.4),另外以400μl1%强度的SDS溶液将混合物调节至SDS含量为0.01%。以15ml30kDa Centriprep管将反应混合物浓缩至约1ml。将浓缩物与9ml缓冲液(50mM MES/NaOH,0.02%SDS,pH7.4)混和,再次浓缩至1ml。将浓缩物在透析管中于6℃对1L缓冲液(5mM磷酸钠,35mM NaCl)透析16h。以2%强度的SDS水溶液将透析物调节至SDS含量为0.1%。通过于10000g、10分钟的离心取出样品,并去除上清液。
进一步分析由此得到的材料(SDS聚丙烯酰胺凝胶电泳;见图11);对产生的截短的寡聚体的质谱分析显示,该寡聚体由截短的Aβ(20-42)组成。
b)以蛋白质内切酶GluC切割,从Aβ(1-42)寡聚体B制备截短的Aβ(12-42)寡聚体:
将2ml根据实施例6b制备的Aβ(1-42)寡聚体B制品与38ml缓冲液(5mM磷酸钠,35mM氯化钠,pH7.4)及1mg/ml的GluC蛋白质内切酶水溶液(Roche)150μl混和。将反应混合物于室温搅拌6h,随后加入另外150μl1mg/ml的GluC蛋白质内切酶水溶液(Roche)。将反应混合物于室温搅拌另外16h,继之以添加8μl5M DIFP溶液。以15ml30kDaCentriprep管将反应混合物浓缩至约1ml。将浓缩物与9ml缓冲液(5mM磷酸钠,0.02%SDS,pH7.4)混和,再次浓缩至1ml。将浓缩物在透析管中于6℃对1L缓冲液(5mM磷酸钠,35mM NaCl)透析16h。以1%强度的SDS水溶液将透析物调节至SDS含量为0.1%。通过于10000g、10分钟的离心取出样品,并去除上清液。
进一步分析由此得到的材料(SDS聚丙烯酰胺凝胶电泳;见图11);对产生的截短的寡聚体的质谱分析显示,该寡聚体由截短的Aβ(12-42)组成。
Aβ(1-42)寡聚体的特征
实施例16
SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)
在变性条件下,根据标准条件在4-20%强度的Tris-甘氨酸SDS PAGE中分析实施例2a、5a、9、12、13和14a的制品,所有这些制品含有寡聚体A和B,从而表征变性条件下的分子量。
上述SDS PAGE的评价(图1)揭示,起始蛋白质Aβ(1-42)依然以约4kDa的条带存在于寡聚体A制品中,而实施例2a的寡聚体A1和A2(在图1中分别表示为1和2)在约15kDa(较弱条带)和在约20kDa(主要条带)处可见。
在寡聚体B制品中,可以检测到相对较少的起始蛋白质Aβ(1-42)(在约4kDa处的相对弱条带)。相反,实施例5a的寡聚体B1和B2(在图1中分别表示为3和4)出现在约38kDa和约48kDa(见箭头)。对于实施例9和12的生物素和荧光素衍生的寡聚体B,出现约42kDa和约52kDa的相应较高的分子量(图3、4)。
分别含有交联产物A-CL和B-CL的制品(实施例13和14a;图2)的分析显示,两个样品基本保持其寡聚化程度(见条带A与条带A’,以及条带B与B′)。与寡聚体A和B相比略微不同的迁移行为可以由轻微改变的SDS结合能力和N末端氨基基团和赖氨酸残基各自的修饰解释。
截短的Aβ(20-42)寡聚体(实施例15a)的分析显示,以嗜热茵蛋白酶蛋白酶水解去除N末端肽,使38/48kDa双条带(图11,泳道A)转变为28/38kDa双条带(图11,泳道B)。相似地,截短的Aβ(12-42)寡聚体(实施例15a)的分析显示,以Glu-C蛋白质内切酶蛋白酶水解去除N末端肽,使38/48kDa双条带(图11,泳道A)转变为33/40kDa双条带(图11, 泳道C)。
实施例17
凝胶渗透层析
为了更详细地研究非变性条件下的分子量行为,使用Superose12HR10/30柱,以FPLC流程方法实施凝胶渗透层析(GPC)。在4℃进行GPC。
将柱子以5个体积的PBS缓冲液(流速0.5ml/min,UV检测,214nm)平衡,并首先使用蛋白质标准标定。随后,分析实施例5a的Aβ(1-42)寡聚体B制品(图5,底部)以及用于比较的相同浓度的Aβ(1-42)冻干物,其中Aβ(1-42)冻干物新鲜称量,溶于PBS,于10000g、5分钟的离心去除不溶性成分后进行分析(图5,上部)。
评价显示,Aβ(1-42)寡聚体B制品具有蛋白质级份,该蛋白质级份以在约50kDa上下的分子量范围内的主峰为特征。而在变性条件下的SDSPAGE中,该蛋白质级份略有别于单体Aβ(1-42)蛋白质,其中单体Aβ(1-42)蛋白质以约16kDa上下的分子量范围内的主峰为特征。
为了研究实施例6b的Aβ(1-42)寡聚体B和实施例14a的Aβ(1-42)寡聚体B-CL在非变性条件下的分子量行为,使用Superose12HR10/30柱,以FPLC流程方法于室温实施凝胶渗透层析(GPC)。将柱子以5个体积的PBS缓冲液(流速0.5ml/min,UV检测,214nm)平衡,并首先使用蛋白质标准标定。随后,以PBS缓冲液将实施例6b的Aβ(1-42)寡聚体B稀释为1mg/ml,以PBS缓冲液将实施例14a的Aβ(1-42)寡聚体B-CL稀释为1mg/ml,分析两个混合物(图12)。
评价显示,SDS含量降低的Aβ(1-42)寡聚体B制品具有蛋白质级份,该蛋白质级份以在约100kDa上下的分子量范围内的主峰为特征。与此相比,具有降低的SDS含量的Aβ(1-42)寡聚体B-CL制品具有蛋白质级份,该蛋白质级份以约60kDa上下的分子量范围内的主峰为特征。
实施例18
实施例5a的Aβ(1-42)寡聚体B的天然聚丙烯酰胺凝胶电泳(NATIVE PAGE)
在非变性条件下,在4-20%强度的Tris-甘氨酸中分析实施例5a含有寡聚体B的制品,从而表征天然条件下的分子量。
以非离子去垢剂Triton X-100中和制品中存在的去垢剂。为此目的,将1μl(4%Triton X-100)移取到10μl实施例5a的制品中,并于室温孵育5分钟。随后,取10μl与相同体积的天然样品缓冲液(4ml1M Tris、pH6.8、8ml甘油、1ml溴酚蓝于50ml H2O中)混和并电泳(运行缓冲液:7.5g Tris、36g甘氨酸于2.5L H2O中)。
天然PAGE的评价显示,起始蛋白质Aβ(1-42)依然以约28kDa的条带(泳道A,表示为1)存在于寡聚体B制品中,而实施例5a制品的主要条带在表观分子量64-90kDa处可见(泳道A,表示为2)(图6)。
重要的是,还可以天然分子量分析方法,例如凝胶渗透层析(见实施例17)或天然凝胶电泳(见实施例18),为本发明的寡聚体(特别是寡聚体B)指定分子量。
混和SDS之后,可以在SDS PAGE中指定约38和48kDa分子量的寡聚体B,在天然凝胶中,基于选择的标准蛋白质,其被检测为约64-90kDa分子量范围内的条带。由于寡聚体的迁移行为除了其大小之外,基本尤其电荷本质决定,不能期望该方法提供分子量的精确读数。然而,该结果导致确定的寡聚体种类存在的结论。
实施例19
a)在生理缓冲液中,Aβ(1-42)寡聚体B于多种蛋白质浓度的稳定性
在下列条件下,于PBS缓冲液中测试根据实施例6b获得的Aβ(1-42)寡聚体B的稳定性。
用PBS以2×稀释步骤将5mg/ml稀释至0.08mg/ml。于室温将所有获得的溶液孵育24小时。继之与冷冻的对照制品比较,分析SDS PAGE 中的条带模式。在所有样品中,条带模式一致。
b)在生理缓冲液中,Aβ(1-42)寡聚体B于多种温度并在不同时期后的稳定性
以PBS缓冲液将根据实施例6b获得的Aβ(1-42)寡聚体B稀释至0.5mg/ml,并于室温或于37℃孵育24h或96h。
随后,在SDS PAGE中分析条带模式。在所有样品中,条带模式一致。
实施例20
用多种蛋白酶(胰蛋白酶、糜蛋白酶、嗜热菌蛋白酶、弹性蛋白酶、木瓜蛋白酶)酶解Aβ(1-42)寡聚体B
以缓冲液(20mM磷酸钠,140mM NaCl,pH7.4)将根据实施例6b获得的Aβ(1-42)寡聚体B制品的等分试样稀释至0.5mg/ml,并在下列条件下于37℃和pH7.4与占图9所示各种蛋白酶溶液重量1/50的蛋白酶溶液孵育20h,蛋白酶溶液如图9所示。任何在SDS-PAGE中分析1μl反应混合物。
SDS PAGE显示,在选择的限制性蛋白质水解条件下,从Aβ(1-42)寡聚体B制品开始,所有蛋白酶将约为38/48kDa的双带恢复为约32/28kDa的双带。
实施例21
Aβ(1-42)寡聚体B在大鼠血浆中的稳定性
将4μl根据实施例6b制备的Aβ(1-42)寡聚体B制品76μl大鼠血浆于室温孵育0h、1h、2h、4h和8h。在干冰中冷冻终止孵育。
随后,添加SDS样品缓冲液后,将所有样品在SDS PAGE中分析。继之通过在蛋白质印迹中对Aβ(1-42)寡聚体B染色进行稳定性评价。抗Aβ(1-42)抗体6E10(Signet)用于检测。通过与碱性磷酸酶偶联的抗小鼠IgG抗体并添加底物NBT/BCIP使条带显影。所观察条带的分子量和强度经2h、4h和8h时期几乎保持不变。
结果提示,Aβ(1-42)寡聚体B具有高的血浆稳定性。据此,血浆中的生物半衰期在约8小时和更长的范围内。
实施例22
分别具有分子量17kDa和22kDa[生物素-Aβ(1-42)寡聚体A]以及42kDa和52kDa[生物素-Aβ(1-42)寡聚体B]的生物素-Aβ(1-42)寡聚体对人类神经细胞表面的结合
通过FACScan(Beckton Dickinson)研究人类β-淀粉样(1-42)蛋白质和两种Aβ(1-42)寡聚体A和Aβ(1-42)寡聚体B对人类成神经细胞瘤细胞系IMR-32(ATCC编号:CCL-127)的结合。将IMR-32细胞悬浮液(1.5×106细胞/0.1ml PBS)与生物素标记的人类β淀粉样(1-42)蛋白质(肽合成材料,冻干物,AnaSpec)以及含有生物素标记的寡聚体A和B的制品(分别为实施例8和9)与37℃孵育20分钟。随后以缓冲液(PBS加1%BSA)清洗细胞,并与偶联到链霉亲和素异硫氰酸酯上的荧光素(Sigma)于室温孵育20分钟。在使用缓冲液的清洗步骤之后,通过FACScan分析对IMR-32细胞表面的结合。虚线表示不存在生物素标记的组分时的背景荧光。个别制品的添加导致细胞相关荧光的强烈增加并以实线表示。寡聚体A(图7B)和B(图7C)对细胞表面的结合与单体β-淀粉样(1-42)蛋白质的结合(图7A)截然不同。数据表示所述寡聚体对人类细胞表面的特定结合位点。
实施例23
脑室内(icv)给药后,在大鼠脑匀浆物中检测Aβ(1-42)寡聚体B
以脑室内快速给药施用10nmol根据实施例6b获得的Aβ(1-42)寡聚体B10。分别在15和120分钟后制备脑。同样制备未处理的对照动物的脑。为了这个目的,各例中,将1g大鼠脑与9ml破碎缓冲液A(200ml5mM磷酸钠,35mM NaCl,300mM蔗糖,调节至pH7.4,并与4片来自Roche的蛋白酶抑制品混合物混和)在50ml Falcon管中混和,并在 冰上以超声破碎2分钟。静置溶液20分钟,然后简短震荡,并分装为8×1ml的等分试样(=匀浆物)。
为了实施定量检测,首先在PBS中制备一系列标准Aβ(1-42)寡聚体B制品,制品浓度在1.58ng/μl-0.005ng/μl范围内。
另外,作为阳性对照,也处理未处理大鼠对照脑的匀浆物,并以相同的方法在此脑匀浆物中制备一系列标准Aβ(1-42)寡聚体B。然后将匀浆物进行100000g、1h的超离心,上清液用于随后的分析。
根据对两个系列的标准物(PBS与对照脑匀浆物上清液)测量的值的比较,可以如下定量确定Aβ(1-42)寡聚体B的含量,首先在阳性对照中比较,然后也与经处理的大鼠的脑样品中比较。
1)斑点印迹检测
将1μl标准样品系列和经处理动物的样品提取物液滴加于硝化纤维纸上,使用抗体6E10(抗Aβ(1-42);Signet)检测Aβ(1-42)。使用偶联到抗小鼠IgG上的碱性磷酸酶并添加染色试剂NBT/BCIP进行染色。
尽管在未处理大鼠的脑提取物中没有可检测的Aβ(1-42)(0.0lnmol/g),通过与相应浓度的阳性对照比较染色强度,在处理后15分钟处死的大鼠中可以检测到约0.4nmol/g Aβ(1-42),以相同的方法,在处理后120分钟处死的大鼠中仍然可以检测到约0.2nmol/g。由此得到外源性给药的Aβ(1-42)寡聚体B约105分钟的平均生物半衰期。
2)蛋白质印迹检测
所有斑点印迹分析的样品液同样以蛋白质印迹分析。蛋白质印迹也以mMAb6E10(抗Aβ(1-42);Signet)、并另外以偶联到抗小鼠IgG上的碱性磷酸酶和添加染色试剂NBT/BCIP显色。
抗Aβ(1-42)反应性条带仅仅出现在38/48kDa区域,相当于Aβ(1-42)寡聚体B的表观分子量,即,即使给药2小时后,寡聚体结构仍然在体内保持。
另外,通过与阳性对照中相应于Aβ(1-42)寡聚体B的强染色条带的比较染色强度,在15分钟大鼠(0.4nmol/g)和120分钟大鼠(0.2nmol/g)的脑 中,可以评估出与点印迹方法中相同的浓度。
实施例24
具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]对鼠皮质神经元的神经毒性作用
按照文献制备鼠皮质神经元并与神经胶质细胞以混和培养物培养(Choi等,(1987)J.Neurosci.7,357-368)。从脑膜和下脑区(lower brain region)用器械取下发育第14-15天胚胎的皮质。在0.05%强度的胰蛋白酶溶液中于37℃孵育5-7分钟,将细胞彼此分离,随后用开口减小的巴斯德吸管将上述溶液吹打数次。确定细胞数量后,于0.5ml维持培养基(含有0.8mM谷氨酰胺、18mM葡萄糖、23mM NaHCO3和10%马血清的基本必需培养基)中以每2cm2430000个细胞将细胞接种于细胞培养材料上,细胞培养材料以多聚L鸟氨酸和层黏连蛋白包被。在潮湿的细胞培养孵育器中于37℃、5%CO2中实施培养和后续的孵育。培养3-5天后,使用(+)-5-氟-2′-脱氧尿苷/尿苷混合物(各10μM)培养1天,以中断神经胶质细胞的繁殖。培养14天后,研究Aβ(1-42)寡聚体B的毒性作用。为此目的,将细胞在脑细胞缓冲液(120mM NaCl,5.4mM KCl,1.8mM CaCl2,15mM葡萄糖,25mM HEPES,pH7.2)中孵育15分钟。将对照细胞1与300μM L-谷氨酰胺在脑细胞缓冲液中孵育相同时间。将Aβ寡聚体储备液以无血清培养基(含有0.8mM谷氨酰胺、20mM葡萄糖、26mM NaHCO3)稀释至多个终浓度,并孵育24小时。在无血清培养基中平行孵育以L谷氨酰胺处理的对照细胞1。另一组细胞(对照细胞2)仅用脑细胞缓冲盐和无血清培养基处理。24h后去除细胞培养物上清液,残余细胞在蒸馏水中孵育20分钟而被破坏,酶学测定两种溶液中的乳酸脱氢酶(LDH)活性。为了评价,测定细胞培养物上清液的LDH活性与上清液和残余细胞的LDH活性总和的比,形成四重测定的平均值。实施3次实验,每个实验条件存在一式四份(n=12)。将谷氨酰胺处理的细胞(对照细胞1)平均值设定为100%神经元死亡,将既没有以L谷氨酰胺也没有以Aβ寡聚体处 理的细胞(对照细胞2)的平均值设定为0%神经元死亡,将以Aβ寡聚体处理的细胞的值加以相应转换。在各例所用的浓度下测定的所有神经毒性平均值显示,具有38kDa和48kDa分子量的Aβ(1-42)寡聚体[Aβ(1-42)寡聚体B]截然不同的毒性作用,见图8。
实施例25
抗体的产生
用于免疫的混合物含有所有佐剂(Biogenes),基本成分如下:
95%石蜡油
2.4%吐温40
0.1%胆固醇
0.1%脂多糖
将佐剂与抗原溶液以2:1比例混和直至获得稳定乳剂。乳剂经注射并形成稳定释放抗原的储库。
a)以实施例15a的截短的Aβ(20-42)寡聚体B免疫兔产生多克隆抗血清
根据标准程序,以实施例15a的未缀合的Aβ(20-42)寡聚体B制品免疫两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心,从中获得抗血清。将以此方式获得的血清称为血清(a1)。
在标准条件下,使用戊二醛将2mg实施例15a的Aβ(20-42)寡聚体B制品与LPH偶联,根据标准程序,以该缀合的Aβ(1-42)寡聚体B免疫另两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(a2)。
b)以实施例15b的截短的Aβ(12-42)寡聚体B免疫兔产生多克隆抗血清
根据标准程序,以实施例15b的未缀合的Aβ(12-42)寡聚体B制品免疫两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(b1)。
在标准条件下,使用戊二醛将2mg实施例15b的Aβ(12-42)寡聚体B制品与LPH偶联,根据标准程序,以该缀合Aβ(12-42)寡聚体B免疫另两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(b2)。
c)以实施例14a的Aβ(20-42)寡聚体制品B-CL免疫兔产生多克隆抗血 清
根据标准程序,以实施例14a的未缀合的Aβ(1-42)寡聚体B-CL制品免疫两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(c1)。
d)以实施例6b的Aβ(1-42)寡聚体制品B免疫兔产生多克隆抗血清
根据标准程序,以实施例6b的未缀合的Aβ(1-42)寡聚体B制品免疫两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(d1)。
在标准条件下,使用戊二醛将2mg实施例6b的Aβ(1-42)寡聚体B制品与LPH偶联,根据标准程序,以该缀合Aβ(1-42)寡聚体B制品免疫另两只兔:
第1天初次免疫,并取免疫前血清
第2天第一次加强
第14天第二次加强
第28天第三次加强并采血
第35天取血
将兔血于室温放置,随后于室温离心从中获得抗血清。将以此方式获得的血清称为血清(d2)。
实施例26
实施例25的免疫多克隆血清关于与多种Aβ(1-42)形式交叉反应的特征描述
为了描述多克隆抗血清的特征,在100pmol/μl-0.01pmol/μl范围内,于PBS中制备多种Aβ(1-42)形式的连续稀释物。在各例中,将1μl样品加至硝化纤维膜:使用实施例25的适当的兔血清实施检测。使用mMAb6E10(Signet)实施检测以对比。偶联到抗兔IgG的碱性磷酸酶(用于比较:偶联到抗小鼠IgG的碱性磷酸酶)加染色试剂NBT/BCIP的添加一起用于染色。图10显示以此方式获得的斑点印迹,可以用数字评价如下:
抗原 | 6E10 | 血清(a1) | 血清(a2) | 血清(d1) | 血清(c1) |
寡聚体 | 0.1 | 0.5 | 10 | 0.1 | 0.5 |
单体 | 0.1 | 10 | 100 | 0.1 | 1 |
Fibril | 1 | 100 | >100 | 0.5 | 1 |
APP | 0.01 | >1 | 1 | 0.1 | 0.1 |
Aβ(1-40)* | 0.1 | 100 | n.d. | 0.5 | 10 |
*未在图10中显示
表中数字显示可见抗原的最小量[pmol;除了APP,各例以单体为基础](检出限)
与以实施例15a的未缀合Aβ(20-42)寡聚体免疫获得的兔血清(a1),的免疫学反应比较明确显示,抗这些寡聚体的抗体仅与其它形式例如原纤维、APP和单体微弱地交叉反应。相反,观察到与Aβ(1-42)寡聚体B(见行1)的显著的强烈交叉反应以及此外与稳定CL抗原的交叉反应。该数据显示在此提出的寡聚体形式与APP、单体和原纤维结构相比截然不同的结构。抗体与寡聚体特异的结构结合。
与以实施例15a的缀合Aβ(20-42)寡聚体免疫获得的兔血清(a2)的免 疫学反应也显示,抗这些寡聚体的抗体仅与其它形式例如原纤维和单体微弱地交叉反应。相反,观察到与Aβ(1-42)寡聚体B(见行1)的弱交叉反应,以及此外与稳定CL抗原和APP的交叉反应。该数据同样显示在此提出的寡聚体形式与单体和原纤维结构相比截然不同的结构。
与以实施例6b的Aβ(1-42)寡聚体免疫获得的兔血清(d1)、以及以实施例14a的Aβ(1-42)寡聚体B-CL免疫获得的兔血清(c1)的免疫学反应比较显示,抗这些寡聚体的抗体与其它形式(例如原纤维、APP和单体)的交叉反应与同Aβ(1-42)寡聚体B的反应一样强烈(见行1)。这些抗体展示与mMAb6E10(Signet)相当的免疫反应,并非结合寡聚体特异的结构。
相应地,以实施例6b的Aβ(1-42)寡聚体免疫小鼠,以本身已知的方式建立单克隆抗体。在此,10个杂交瘤中的2个分泌单克隆抗体,其结合峰型图(特别是就上面测试的反应性而言)与抗血清(a1)相似。
实施例27
Aβ(1-42)原纤维的制备
以300μl缓冲液(20mM磷酸钠、140mM NaCl,pH7.4)将100μl溶于0.1%NH4OH中的2mg/ml的Aβ(1-42)溶液稀释至0.5mg/ml,并以0.1M HCl调节至pH7.4(100μM Aβ(1-42))。将样品于37℃孵育24h,然后通过于10000g、10分钟的离心取出。将得到的蛋白质残留物以400μl缓冲液(20mM磷酸钠、140mM NaCl,pH7.4)重悬浮。以此方法获得的Aβ(1-42)原纤维制品可以于-20℃保存并用于进一步研究。
实施例28
Aβ(1-42)寡聚体B-CL(实施例14b)对海马切片的作用
将400μM厚度的海马横切片于34℃在染毒(gassed)Ringer's溶液(NaCl124mM,KCl4.9mM,MgSO41.3mM,CaCl2.5mM,KH2PO41.2mM,NaHCO325.6mM,葡萄糖10mM)灌注下置于浸渍切片槽中。随后,在单极刺激电极的辅助下刺激谢弗侧枝(Schaffer collateral),记录 辐射层中的兴奋性突触后电位(fEPSP)。以产生最大fEPSP的刺激水平的30%给以测试脉冲。每10分钟施以三次100个脉冲,诱导长时程增强,单个脉冲具有200μs的宽度(强烈的强直收缩)。产生的fEPSP增强至少记录240分钟。实施例14b的寡聚体B-CL(500nM)的加入(rinsing-in)在第一次痉挛前20分钟开始,于最后痉挛10分钟后停止。测定fEPSP的升高(斜率),并作为对时间的函数作图。
图14显示,Aβ(1-42)寡聚体的洗入(washing-in)抑制海马长时程增强,特别是在维持期。因此,Aβ(1-42)寡聚体BCL影响神经细胞的信息储存(细胞己忆)。
实施例29
Aβ(1-42)寡聚体B(实施例5b)与大鼠原代海马神经元的结合
研究实施例5b的Aβ(1-42)寡聚体B与大鼠原代海马神经元的结合。
将海马神经元在含有B27添加物的Neurobasal培养基中于多聚L赖氨酸包被的盖玻片上培养,并在培养的第14天使用。向新鲜培养基添加200nM(总单体Aβ浓度)寡聚体并于37℃孵育15分钟,使寡聚体与神经元细胞膜结合。去除含有Aβ的培养基后,实施使用培养基的两步清洗步骤,然后将细胞于3.7%的甲醛中固定。在细胞的进一步清洗之后,在PBS缓冲液中,于室温以10%正常驴血清封闭非特异性结合位点90分钟。于室温将6E10(来自小鼠)以1:2000稀释液作为第一抗体应用2h。再次清洗细胞,并与抗小鼠并偶联荧光色素Cy3的第二抗体(来自驴)于室温孵育2h。细胞经过再次清洗之后,以包埋介质将含有神经元的盖玻片固定于载玻片上。在荧光显微镜下描绘具有结合的Aβ寡聚体的海马神经元。使用的对照为混合物,该混合物中免除了第一抗体6E10。该对照因此展示了不基于Aβ的非特异性荧光。
如图15a所示,寡聚体以斑点方式与神经元细胞表面结合。相反,由于第二抗体与神经元的结合,没有第一抗体的对照仅展示低的非特异性荧光(图15b)。
Claims (27)
1.淀粉样β(1-42)寡聚体,其在SDS凝胶电泳中具有约38KDa和/或48KDa的表观分子量,其中所述寡聚体是通过下述方法获得的,所述方法包括步骤:
(a)将单体Aβ(1-42)蛋白与去垢剂接触,以形成Aβ(1-42)寡聚体A,其在SDS凝胶电泳中具有约15KDa和/或20KDa的表观分子量;
(b)降低去垢剂作用并继续孵育所述Aβ(1-42)寡聚体A,以形成Aβ(1-42)寡聚体B,所述寡聚体A在SDS凝胶电泳中具有约15KDa和/或20KDa的表观分子量,所述寡聚体B在SDS凝胶电泳中具有约38KDa和/或48KDa的表观分子量;
其中去垢剂为式R-X的化合物,其中R基为具有6至20个碳原子的未分支或分支烷基,或具有6至20个碳原子的未分支或分支链烯基,X基为酸性基团或其盐。
2.淀粉样β(1-42)寡聚体,其在SDS凝胶电泳中具有约15KDa和/或20KDa的表观分子量,,其中所述寡聚体是通过下述方法获得的,所述方法包括步骤:
将单体Aβ(1-42)蛋白与去垢剂接触,以形成Aβ(1-42)寡聚体A,其在SDS凝胶电泳中具有约15KDa和/或20KDa的表观分子量
其中去垢剂为式R-X的化合物,其中R基为具有6至20个碳原子的未分支或分支烷基,或具有6至20个碳原子的未分支或分支链烯基,X基为酸性基团或其盐。
3.根据权利要求1或2的寡聚体,其中X选自-COO-M+、-SO3 -M+,和-OSO3 -M+,且M+是氢阳离子、或者选自碱性金属和碱土金属阳离子以及铵阳离子的无机或有机阳离子。
4.权利要求3的寡聚体,其中所述去垢剂为十二烷基硫酸钠。
5.制备淀粉样β(1-42)寡聚体的方法,所述方法包括将单体淀粉样β(1-42)蛋白与去垢剂接触,降低去垢剂作用并继续孵育,且回收寡聚体, 其中所述去垢剂为式R-X的化合物,其中R基为具有6至20个碳原子的未分支或分支烷基,或具有6至20个碳原子的未分支或分支链烯基,且X基为酸性基团或其盐。
6.制备淀粉样β(1-42)寡聚体的方法,所述方法包括将单体淀粉样β(1-42)蛋白与去垢剂接触,且回收寡聚体,其中所述去垢剂为式R-X的化合物,其中R基为具有6至20个碳原子的未分支或分支烷基,或具有6至20个碳原子的未分支或分支链烯基,且X基为酸性基团或其盐。
7.根据权利要求5或6的方法,其中X选自-COO-M+、-SO3 -M+,和-OSO3 -M+,且M+是氢阳离子、或者选自碱性金属和碱土金属阳离子以及铵阳离子的无机或有机阳离子。
8.权利要求7的方法,其中所述去垢剂为十二烷基硫酸钠。
9.权利要求1-4中任一项的寡聚体,其中所述寡聚体是标记的、固定的或交联的。
10.权利要求9的寡聚体,其中所述寡聚体用荧光标记、发光标记、显色标记、放射标记或磁性标记来标记。
11.制品,其包含权利要求1的淀粉样β(1-42)寡聚体或根据权利要求5的方法产生的淀粉样β(1-42)寡聚体,所述制品中淀粉样β(1-42)寡聚体的比例为总淀粉样β(1-42)蛋白的至少60%重量。
12.权利要求11的制品,其中所述制品中淀粉样β(1-42)寡聚体的比例为总淀粉样β(1-42)蛋白的至少75%重量或至少90%重量。
13.制品,其包含权利要求2的淀粉样β(1-42)寡聚体或根据权利要求6的方法产生的淀粉样β(1-42)寡聚体,所述制品中淀粉样β(1-42)寡聚体的比例为总淀粉样β(1-42)蛋白的至少50%重量。
14.权利要求13的制品,其中所述制品中淀粉样β(1-42)寡聚体的比例为总淀粉样β(1-42)蛋白的至少70%重量或至少85%重量。
15.药物组合物,其包含权利要求1-4、9或10的任一项的寡聚体、根据权利要求5或6的方法产生的淀粉样β(1-42)寡聚体,或权利要求11至14中任一项的制品,以及任选地生理上适宜的载体。
16.权利要求1-4、9或10的任一项的寡聚体、根据权利要求5或6的方法产生的淀粉样β(1-42)寡聚体,或权利要求11至14中任一项的制品在制备用于诊断性体内或体外检测方法的组合物中的用途。
17.权利要求1-4、9或10的任一项的寡聚体、根据权利要求5或6的方法产生的淀粉样β(1-42)寡聚体,或权利要求11至14中任一项的制品在制备用于治疗淀粉样蛋白病的药物中的用途。
18.与权利要求1的淀粉样β(1-42)寡聚体结合的抗体,或其抗原结合部分,其中所述抗体能够通过用寡聚体免疫宿主获得。
19.抗体或其抗原结合部分,所述抗体与权利要求1的淀粉样β(1-42)寡聚体以比单体淀粉样β(1-42)蛋白更高的亲和力结合。
20.与权利要求2的淀粉样β(1-42)寡聚体结合的抗体,或其抗原结合部分,其中所述抗体能够通过用寡聚体免疫宿主获得。
21.抗体或其抗原结合部分,所述抗体与权利要求2的淀粉样β(1-42)寡聚体以比单体淀粉样β(1-42)蛋白更高的亲和力结合。
22.权利要求19的抗体或其抗原结合部分,其以高于KD=10-8M的亲和力、以高于KD=10-9M的亲和力、以高于KD=10-10M的亲和力或以高于KD=10-11M的亲和力与所述寡聚体蛋白结合。
23.权利要求19的抗体或其抗原结合部分,其以低于KD=10-8M的亲和力与单体淀粉样β(1-42)蛋白和/或单体淀粉样β(1-40)蛋白结合。
24.权利要求19的抗体或其抗原结合部分,其中所述抗体对所述寡聚体蛋白的亲和力比对单体淀粉样β(1-42)蛋白和/或单体淀粉样β(1-40)蛋白的亲和力高至少10倍、比对单体淀粉样β(1-42)蛋白和/或单体淀粉样β(1-40)蛋白的亲和力高至少100倍,或比对单体淀粉样β(1-42)蛋白和/或单体淀粉样β(1-40)蛋白的亲和力高至少1000倍。
25.权利要求18-24中任一项的抗体或其抗原结合部分,其中所述抗体是多克隆抗体或单克隆抗体,人类抗体;嵌合抗体;CDR移植物抗体,或人源化抗体。
26.药物组合物,其包含权利要求18-25中任一项的抗体或其抗原结 合部分,以及任选的可药用载体。
27.权利要求18-25中任一项的抗体或其抗原结合部分在制备用于治疗淀粉样蛋白病的药物中的用途。
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