CN1158134A - 激活促红细胞生成素受体的抗体 - Google Patents
激活促红细胞生成素受体的抗体 Download PDFInfo
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- CN1158134A CN1158134A CN95195117A CN95195117A CN1158134A CN 1158134 A CN1158134 A CN 1158134A CN 95195117 A CN95195117 A CN 95195117A CN 95195117 A CN95195117 A CN 95195117A CN 1158134 A CN1158134 A CN 1158134A
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Abstract
描述了激活促红细胞生成素受体并刺激红细胞生成的抗体及其片段。还描述了产生抗体的杂交瘤细胞系以及治疗贫血的方法和组合物。
Description
发明领域
本发明涉及识别促红细胞生成素受体的抗体。更具体地说,本发明涉及激活促红细胞生成素受体并刺激红细胞生成的抗体。
发明背景
促红细胞生成素(EPO)是一种涉及红细胞样祖细胞生长和成熟为红细胞的糖蛋白激素。EPO在胎儿期由肝脏产生,在成人时由肾脏产生并刺激红血细胞样前体产生红细胞。EPO产生减少,一般在成人中作为肾衰竭的结果而出现,并引起贫血。已用遗传工程技术生产EPO,包括从用偏码促红细胞生成素的基因转化的宿主细胞表达和分泌该蛋白质。重组EPO的施用在治疗贫血中是很有效的。例如,Eschbach等。(新英格兰医学杂志.316.73(1987))描述了EPO在校正由慢性肾衰竭引起的贫血中的应用。
Miyake等描述了人尿EPO的纯化(生物化学杂志.252 5558(1977))。在美国专利4,703,008号中介绍了编码促红细胞生成素基因的鉴定,克隆,和表达。在Lai等人的美国专利4,667,016中描述了一种从细胞培养基中纯化重组EPO的方法。
对EPO刺激红细胞生成的机制还知之甚少。但已清楚EPO通过与特定的细胞表面受体的结合激活细胞的生长和/或分化,但尚未完全了解激活的具体机制以及受体和相关蛋白的结构。没想促细胞生成素受体(EPO-R)以多聚体复合物存在。沉降研究表明其分子量是330±48KDa(Mayeux等,欧洲生物化学杂志194,271(1990))。交联研究提示受体复合物由至少两种不同的多肽组成,一种66-72KDa,和一种85KDa以及100KDa的种类(Mayeux等,生物化学杂志,266,23380(1991));McCaffery等,生物化学杂志,264,10507(1991))。EPO受体的免疫沉淀还检测到了一种不同的95KDa的蛋白(Miura & Ihle血液81、1739(1993))。另外的交联研究发现了三种含110,130和145KDa复合物的EPO。110和145KDa复合物含EPO受体是因为它们可被抗该受体的抗体免疫沉淀(Miura & Ihle,上文)。羧基末端截短形式的EPO受体的表达导致110KDa复合物但不是145KDa复合物的检测。这表明较大分子量的复合物包含110KDa复合物中的多肽和另外的35KDa的蛋白。
通过对小鼠和人EPO受体的克隆和表达,对EPO受体结构和功能有了进一步的了解。(D’Andrea等。细胞57,277(1989));Jones等,血液76,31(1990);Winkelmann等。血液76,24(1990);PCT申请WO 90/08822号;D’Andrea等的美国专利5,278,065号)。全长的人EPO受体是一个483个氨基酸的跨膜蛋白,带有一个约224氨基酸的细胞外结构域和一个25个氨基酸的信号肽。人受体表现出与小鼠受体大约82%的氨基酸序列同源性。证明在哺乳动物细胞中表达的克隆的全长EPO受体(66-72KDa)具有与红细胞样祖细胞中的天然受体相似的结合EPO的亲和性(100-300nM)。因此这种形式被认为包含主要的EPO结合决定簇并被称为EPO受体。作为交联复合物的一部分它被观察到的85和100KDa蛋白与EPO受体不同,但其与EPO亲缘相近因为EPO可与其交联。85和100KDa的蛋白彼此相关并且85KDa的蛋白可能是100KDa蛋白的蛋白降解产物(Sawyer生物化学杂志264,13343(1989))。
已经产生了只含细胞外结构域的可溶(截短)形式的EPO受体并且发现其结合EPO的亲和性约为1nM,或者是说比全长受体低3-10倍(Harris等,生物化学杂志267,15205(1992);Yang & Jones血液82,1713(1993))。尚不知道与全长蛋白相比其亲和活性降低的原因。有一种可能是其它蛋白种类也可能是EPOR复合物的部分并参与EPO的结合而因此增加了亲和性。支持这种可能性的是Dong &Goldwasser的观察结果(实验血液学21,483(1993))即低亲和性EPO受体的细胞系与不结合EPO的CHO细胞系的融合导致了表达高EPO结合亲和性的EPO受体的杂种细胞系的产生。另外,通过Scatchard分析测定用全长EPOR转染的CHO细胞系,其导致了具有高和低两种亲和性受体的细胞系的产生。EPOR拷贝数的扩增增加了低亲和性但不是高亲和性的结合。这些结果与CHO细胞中有限量的蛋白可从低亲和性EPOR转换为高亲和性EPOR是一致的。
EPO受体的激活引起几种生物效应。其中三种活性是刺激增殖,刺激分化和抑制凋亡(Liboi等,美国国家科学院院刊90,11351(1993);Koury科学248,378(1990))。引起增殖活化和分化活化的信号转导通路看来是可分离的(Noguchi等,分子细胞生物学,8,2604(1988);Patel等,生物化学杂志,267,21300(1992);Liboi等ibid)。一些结果提示一种辅助蛋白对介导分化信号是必须的(Chiba等,自然362,646(1993);Chiba等,美国国家科学院院刊90,11593(1993))。但是对辅助蛋白在分化中的作用有不同的认识,因为组成性激活形式的受体既能刺激增生又能刺激分化(Pharr等,美国国家科学院院刊90,938(1993))。
EPO受体的活化可能是因为其二聚体化。即,EPO可以以两个EPO受体分子的交联形式起作用。有证据支持这种建议。小鼠EPO受体129位精氨酸突变为半胱氨酸导致受体的组成性活化,大概是由于在两个受体亚基之间形成了二硫键(Yoshimuar等.自然348,647(1990))。另外在细胞中发现了EPOR的多聚体复合物(Miura & Ihle生物化学生物物理学报306,200(1993))。但是,对纯化的EPO可溶性受体的稳定的多聚体形式的分离尚未有报道。另外,EPOR的二聚体化可能是必须的,但其自己却不足以完全活化细胞。例如,二聚体化可引起增殖信号但不是分化信号。即,需要辅助蛋白传递分化信号。
EPO受体二聚体化和活化之间可能的关系可被用于鉴定与EPO不同但却活化受体的化合物。例如,抗体具有两个相同的抗原结合位点。一个抗EPOR抗体可结合两个EPOR分子并可使之彼此邻近以发生二聚体化。为了在体内起作用,这些抗体必须识别细胞表面的EPOR并以一种可引起信号转导路径活化的方式结合。另外,希望活化同时导致红系祖细胞的增殖和分化。已报道可以以相似的方式理解人生长激素受体(Fuh等.科学256,1677(1992))和上皮生长因子(Schreiber等.美国国家科学院院刊78,7535(1981))。
希望能鉴定具有活化EPO受体并刺激红细胞生成的特征的分子。为了这样做,对EPO受体活化和信号转导机制的理解是重要的。一种证明该机制的方式可能是通过对能识别EPO受体以活化受体并刺激红细胞生成的抗体进行鉴定而实现的。这类抗体可做治疗或诊断用并且还可用于探测EPO受体。
下面的参考文献描述了与小鼠或人EPO受体结合的抗体:
D’Andrea等.在造血生成的生物学Wiley-Liss,Inc.(1990)153-159页中,产生了抗小鼠EPO受体氨基末端和羧基末端肽的多克隆抗肽抗体。Western印迹证明该抗体可与小鼠受体反应。
Bailey等,实验血液学,21,1535-1543(1993)制得了抗合成肽的多克隆抗肽抗体,该合成肽是与小鼠EPO受体胞外和胞质结构域同源的。通过测定苯肼处理的小鼠中脾细胞对3H胸腺嘧啶核苷的掺入,未检测到这些抗体对受体的活化。
Bayne等.血液82,2088-2095(1993)制得了抗人EPO受体的一个氨基末端肽的多克隆抗体。已证明该抗体可与人血清中可溶形式的受体反应。
D’Andrea等,血液82,46-52(1993)制得了抗人EPO受体的单克隆抗体。该抗体可与用人EPO cDNA克隆转染的Ba/F3细胞结合并且一些抗体抑制EPO的结合并中和EPO依赖的生长。
Fisher等.血液82,197A(1993)应用与上文D’Andrea等所述的相同的单克隆抗体将其区分为其生长和成熟对EPO依赖和不依赖的红系祖细胞。
未有报道前文参考文献所述的抗体可活化EPO受体或刺激红系祖细胞的生长和/或成熟。
因此,本发明的目的即是产生可识别EPO受体并与之结合以使受体活化的抗体。本发明另外的目的是产生可与EPO受体结合并刺激红细胞产生的抗体,它是通过刺激红系祖细胞的增殖和/或分化产生红细胞而刺激红细胞生成的。该类抗体可用于治疗贫血或可用于以EPO受体功能紊乱为特征的疾病。另外,该类抗体可导致用于治疗贫血的治疗剂的鉴定。发明概述
本发明涉及激活促红细胞生成素的抗体或其片段。通过对识别人EPO受体的抗体的筛选,发现了两个称为Mab71和Mab73的刺激UT7-EPO细胞增殖的抗体,该EPO信赖的细胞系在缺乏添加的EPO时不增殖。另外Mab71刺激人血中来自红系祖细胞的红系集落形成。本发明所包括的抗体可识别EPO受体上一个可由Mab71或Mab73识别的表位。优选该抗体是单克隆抗体并且可以是人源化的或人的抗体。还包括产生本发明的抗体的杂交瘤细胞系。
还提供了检测生物样品中EPO受体的方法和试剂盒,其中的方法和试剂盆包括本发明的EPO受体抗体。本发明还包括包含EPO受体抗体和药用佐剂的药物组合物。这类组合物可用于治疗具有以低的红细胞水平为特征的疾病的患者。
附图说明
图1示检测Mab71与所示活度的合成肽结合的ELISA分析的结果。这些肽对应于所示的人EPO受体的氨基酸残基。残基1是裂解去除先导序列之后分泌的EPOR中发现的氨基末端的脯氨酸。
图2示不同量的rHuEPO蛋白和纯化的Mab71和73对UT7-EPO细胞3H胸腺嘧啶核苷掺入的影响。
图3示不同量的rHuEPO蛋白,Mab71,Mab73或抗EPO(MabF12)的非中和对照Mab对125I EPO与OCIM1细胞表面的EPO受体结合的抑制作用。
图4示单克隆抗体71和73以及来自Mab71和73的单克隆抗体片段(Fabs)的纯化制品的考马斯染色SDS凝胶。将样品在还原(加2-巯基乙醇)或非还原(减2-巯基乙醇)条件下电泳。
图5示不同量的纯化rHuEPO蛋白,Mab71或Fab71对UT7-EPO细胞3H胸腺嘧啶核苷的掺入的影响。
图6示不同量的纯化Mab71或Fab71对UT7-EPO细胞的3H胸腺嘧啶核苷的掺入的影响,而且还向其中加入了30munits/ml的重组人EPO(rHuEPO)。
图7示来自外周血的纯化的CD34+细胞的图片,其中这些细胞在EPO或Mab71存在下在无血清生长条件下在甲基纤维素中生长了21天。图示用500munits/ml EPO(A),25munits/ml EPO(B),或2.1微克/ml Mab71(C)培养的细胞。
图8示不同量的rHuEPO,Mab71和抗Her2/neu的对照单克隆抗体对红系前体的红系的克隆的形成的影响,克隆条件是在软琼脂中在无血清生长条件下形成的。
发明详述
已通过用纯化的可溶性人EPO受体免疫小鼠产生了识别促红细胞生成素受体的单克隆抗体(Mab)。如实施例1和2所述表达和纯化可溶性人EPO受体。在这些在酶联免疫吸附测试(ELISA)中与可溶性人EPO受体反应的Mab中,对其96个Mab做了进一步筛选。对这些Mab做BIA core分析以检测其与EPO受体的结合(实施例4A)以及用FACS(实施例4C)检测了其与转化CHO细胞表面EPO受体的结合。这些筛选的结果如表1所示。尽管如BIAcore分析所确定的有一些抗体结合EPO受体,但用FACS扫描确定在所测试的96个抗体中只有5个与转化CHO细胞表面表达的EPO受体结合。测定了24个ELISA测定中阳性的(包括这五个FACS扫描阳性的抗体)抗体对UT7-EPO细胞的增殖刺激作用。奇怪地是,发现两个抗体,称为Mab71和Mab73,在无EPO的条件下可刺激UT7-EPO细胞系3H胸腺嘧啶核苷的掺入(Komatsu等.血液82,456(1993))(实施例8A)。UT7-EPO细胞系的生长需要在培养基中含EPO。因此,对UT7-EPO细胞生长的刺激作用可能是由于Mab71和Mab73激活了EPO受体。如图2所示,在Mab71存在时UT7-EPO细胞的应答比Mab73存在时强。另外发现Mab71刺激来自人红系前体细胞(参见实施例9)红系集落的形成。这是第一个抗体可刺激红系前体细胞形成红系集落的例证。
本发明提供了一种可激活促红细胞生成素受体的抗体或其片段。如本文所用,术语“EPO受体的激活”意指EPO受体经历了一个或多个可将信号向受体携带细胞的内部转导的分子过程,其中该信号最终导致一种或多种细胞生理学的变化。对EPO受体激活的细胞应答一般是受体携带细胞增殖或分化的改变。受体携带细胞通常是红系祖细胞。现在,对EPO受体引起的信号转导的分子事件还知之甚少。但是,如背景技术中所提示的,一些证据提示EPO受体的二聚体化可能是至少一个活化所需要的事件。本说明书也提供了对该思路的支持。如图5所示,当用称为Fab71的Fab片段替代时,Mab71对UT7-EPO细胞3H胸腺嘧啶核苷掺入的刺激消失了。
因此,用对应的单价片段代替完整的双价抗体使增殖应答消失。另外,高浓度Mab71抑制EPO受体的激活。这两个观察都支持了EPO受体活化的二聚体化模型。已表明Mab71可与人EPO-R的49-78残基的一个合成肽相互作用(参见实施例6)。因此当EPO-R的这个区域被交联剂如Mab71结合时可导致EPO-R的活化。应该认为通过与残基49-78结合而将两个EPO-R交联的分子也在本发明的范围之内。这些分子可以是抗体或其它具有将两个EPO受体交联特征的二价分子实体,其中该二价分子实体可通过与残基49-78之间的结合而导致EPO受体的二聚体化和激活。
本发明的EPO受体优选哺乳动物EPO受体。并且,在一个更具体的优选实施方案中是人EPO受体。应该认为人EPO受体的类似物也在本发明的范围之内。这类类似物可通过对人EPO受体序列氨基酸的插入,缺失,延伸或取代而构建得到。EPO-R类似物的实例和Yoshimura等的美国专利5,292,654中已有描述,其中对EPOR氨基酸序列129位半胱氨酸残基的取代得到了组成性活化的EPOR。总的说来,EPO-R类似物在激活必需的抗体结合结构域之外的其它区域发生氨基酸变化,其中所说的类似物保留了可被本发明的抗体识别的人EPO受体的二级和三级结构。已经表明Mab71与XEPO-R 49-78残基的一个合成肽相互作用。因此,这些在位点49-78之外的其它位点氨基酸残基发生改变并保留人EPO受体二级和三级结构的EPO-R类似物可能可被Mab71识别。本文所用的人EPOR多肽的氨基酸残基的编码从位点1的脯氨酸开始,该位点是切去25个残基的信号肽之后的氨基末端残基。
本发明的抗体与受体活化中的EPO受体的一个表位结合。在一个实施方案中,这些抗体识别EPO受体一个可被Mab71识别的表位或一个可被Mab73识别的表位。Mab71识别一个包括人EPO-R 49-78氨基酸残基的合成肽。因此,看来Mab71可识别全部或部分由该序列定义的EPO-R的一个表位。如本文所用,术语“表位”指EPO-R的结合抗体的区域,其中结合防止了第二个抗体与EPO-R的联系。
本发明还提供多克隆抗体和单克隆抗体以及其片段。抗体片段包括这些可激活EPO受体的片段。还包括人源化的抗体,特别是用重组方法生产的抗体,其中人序列包含了激活EPO受体的抗体的部分或全部。人源化抗体包括嵌合的或CDR移植的抗体(美国专利4,816,567和5,225,539)。还包括在遗传改变的小鼠(genetically-altered)中产生完全是人的抗EPO受体的抗体(参见PCT申请93/12227)。本发明的抗体还具有与之粘连的可检测的标记。这类标记可以是荧光的(例如异硫氰基荧光素,FITC),酶的(辣根过氧化物酶),亲和性的(如生物素)或同位素的标记(例如,125I)。
本发明还包括产生激活EPO受体的杂交瘤细胞系。在一个实施方案中,该杂交瘤细胞系产生一个单克隆抗体,该抗体可识别可被Mab71或Mab73识别的EPO受体上的一个表位。产生抗人EPO-R的单克隆抗体的杂交瘤细胞系的制备如实施例3中所描述。产生Mab71的杂交瘤细胞系已在1994年7月26日保藏在美国典型培养物保藏中心,Rockville,MD,保藏号HB11689。产生Mab73的杂交瘤细胞系已在1994年7月26日保藏在Rockville MD,美国典型培养物保藏中心,保藏号HB11690。
本发明的抗体可用于检测贫血和其它以EPO-R功能紊乱为特征的疾病。在一个实施方案中,一种对生物样品中可被活化的EPO受体的检测包括如下步骤:(a)将样品与一种激活EPO受体的抗体接触;和(b)检测用该抗体活化的受体。生物样品包括组织样品,完整细胞或其提取物。可将抗体用做诊断试剂盒的一部分以检测生物样品中EPO受体的存在。这类试剂盒应用具有粘附标记的抗体以便于检测。该抗体可用于鉴定正常的或非正常的受体。生物样品中非正常受体的存在可为如先天再生不良性贫血等疾病的征兆,其中认为EPO受体是功能紊乱的。
本发明的抗体可用于治疗以低红细胞水平为特征的疾病。本发明还包括在哺乳动物中调节EPO受体内源活性的方法,优选增加EPO受体活性的方法。一般说来,任何可用促红细胞生成素治疗的疾病,如贫血也可用本发明的抗体来治疗。可以以对所治疗的疾病的特征和严重程度合适的量和用药途径施用该治疗性抗体,并且对本领域的技术人员来说是公知的。优选地,经皮下,肌内或静脉内注射给药。
本发明提供了包含治疗上有效量的可与药用上可接受的佐剂一起活化EPO-R的抗体的药用组合物,其中佐剂选自一种或多种稀释剂,载体,防腐剂,乳化剂,抗氧化剂和/或稳定剂。本文使用的“治疗有效量”指抗体的量对给定的疾病和给药方式是有治疗效果的。在本发明中,治疗效果指由被治疗患者的血细胞比容增加所证实的对红细胞产生的刺激。在优选的实施方案中,抗体是用本领域技术人员已知的方法制备的人源化抗体或人的抗体。药用上可接受的佐剂是本领域技术人员已知的并在Remington药物科学,18版。A.R.Gennaro.编,Easton PA(1990)中有广泛概括。
下面的实施例是为了更全面地介绍本发明而给出的,但不是为了限制发明范围。
实施例1
可溶性人促红细胞生成素受体的制备A.可溶性人促红细胞生成素受体的表达克隆的分离
应用上文Jones等所描述的含人促红细胞生成素受体的克隆,用PCR技术获得了可溶性人促红细胞生成素受体(sHuEPOR)的表达克隆。人促红细胞生成素受体PCR扩增的引物如下:
5′引物
CTC CAA GCT TGC CGT CAC CAT GGA CCA CCT CGG GGC GTC CCT
(SEQ.ID NO:1);和
3′引物
CAG GTC TAG ATT ACT AGG GAT CCA GGT CGC TAG GC
(SEQ.ID NO:2)
PCR反应使用如下物质进行:2.5ng含人EPOR的质粒,5pmol每种上述的寡核苷酸引物,10mM Tris HCl(pH8.3),50mM KCl,1.5mM MgCl2,200μm每种dNTP和1单位Taq聚合酶。在94℃ 30秒,50℃ 1分钟,72℃ 1分钟扩增5个循环,接着94℃ 30秒钟,55℃ 1分钟,72℃ 1分钟扩增20个循环。将DNA通过G-50大小排阻柱而纯化(Boehringer Mannheim Corp.),接着用Hind III和XbaI消化并将其连入已用Hind III和XbaI消化过的表达载体pDSRα2(DeClerck等,生物化学杂志266,3893(1991))。通过DNA序列分析对含所需插入的克隆进行确证。
d40EPOR克隆是用PCR从全长人EPOR克隆制得的(见上文)。在引物内加入终止密码子的结果是d40EPOR的羧基末端是tyr467.PCR扩增用的引物如下:
5′引物
5′-CTC CAA GCT TGC CGT CAC CAT GGA CCA CCT CGG GGC GTC
CCT-3′
(SEQ.ID NO:1);和
3′引物:
5′-AGG TCG ACT ACT AGT AGT CAG TTG AGA-3′
(SEQ.ID NO:3)
PCR扩增使用溶于pfu缓冲液2中的pfu聚合酶(Stratagene,LaJolla,CA)。反应条件是:96°30秒钟;45°1分钟,72°1分钟,1个循环;96℃ 1分钟,55℃ 1分钟,72℃ 2分钟,25个循环。最后在72℃保温5分钟。用琼脂糖凝胶电泳分离反应产物并用基因清洁(Clean)试剂盒分离约1.3Kb的带(BIO 101,Vista,CA)。纯化片段被连入PCR II中(TA克隆试剂盒,Invitrogen,San Diego,CA)。用限制性分析鉴定重组体并测序以确定所需插入的存在。如上所述一个Hind III-Sal I片段被分离并被连入一个预先用Hind III和Sal I消化过的分离的pDSRα2载体中。所获得的载体pDSRαEPORd40用于在CHO细胞中表达。
B.可溶性人EPOR和d40EPOR在CHO细胞中的表达
如上文Jones等所表明的表达质粒pDSRα2-EPOR-X包含编码人EPOR Met 1-pro 249氨基酸序列。质粒pDSRαEPORd40含编码Met 1-Tyr 467的序列。10微克的每种质粒被磷酸钙介导的转染分别导入CHO细胞中(Wigler等,细胞11,233(1977))。根据载体中二氢叶酯还原酶基因的表达对独立的克隆进行了选择。用RNA杂交(Hunt等,实验血液学,19:779(1991))和应用亲和纯化的抗体的Western免疫印迹进行监测。选择了在这些测试中为阳性的细胞系做进一步的扩增。将细胞系用30nM氨甲蝶蛉(Mtx)调书以刺激EPO-R的表达。
在转瓶和中空纤维生物反应器二者中都进行了含可溶性人EPOR的条件培养基的制备。用含2×107细胞的200ml的生长培养基(DMEM:补加有非必需氨基酸(NEAA)的Ham’s 12(1∶1),30nM氨甲蝶蛉和5%胎牛血清(FBS)(来自GIBCO,Grand Island NY的试剂))。在3-4天达到汇合之后,用200ml不含血清的DMEM:Ham’sF12 NEAA,30nM Mtx替换原培养基。在6-7天之后收获条件培养基并用新鲜的无血清培养基替换。并进行第二次和第三次收获。
再生长于补加有5μg/ml庆大霉素的生长培养基(如上)中的5×108个细胞接种细胞药理生物反应器(Cell pharm biorector Cartridge)。将pH值维持在7.3。从接种的第12天开始将细胞慢慢去掉血清以产生无血清条件培养基。从第17天开始收获条件培养基。
实施例2
可溶性人促红细胞生成素受体的纯化
制得了四种不同的可溶性重组人EPOR制品。在第一种制品中,EPOxy活化的Sepharose 6B基本上按生产商的说明与重组人促红细胞生成素(rHuEPO)偶联。向218mg溶于4.5mL 32mM ZnCl2中的rHuEPO中加入7.2g预先用水水合并清洗过的Epoxy活化的Sepharose 6B。将此粘浆滴定到pH10.8并在室温混合过夜。接着经过加入终浓度为1M的乙醇胺并在室温混合4小时以封闭任何反应性基团。后续步骤在8°±2°进行。将偶联的树脂(Epoxy-EPO)装入一个柱子中并用0.5M NaCl(0.1M HOAc pH4和0.5M NaCl/0.1M硼酸盐pH8交替循环清洗。用140mM NaCl/10mM Tris pH7.6(TBS)平衡柱子。向其中装上1560mL用转瓶制得的由表达可溶性EPO-R(sHuEPO-R)的CHO细胞制得的条件培养基。在上样结束后,用300mM NaCl/10mM Tris pH7.6洗柱并接着用1M NaCl/3M尿素/10mM Tris pH7.6洗脱结合的sHuEPO-R。用该缓冲液洗脱时有两个UV280的吸收峰。当含sHuEPOR的第二个峰被洗脱下来时,收集该峰并用H2O稀释20倍。然后将稀释的收集液上样到一个1mL的预装柱的Mono Q柱(Pharmacia)中并用10mM Tris pH7.6的NaCl梯度洗脱。洗脱下来一个单一峰,收集该峰,等份分装并冷冻保存在-80℃。
在第二个制品中,制备了一个更大的Epoxy-EPO柱。将20.4gEpoxy-活化的Sepharose 6B用H2O水合和洗涤,接着用丙酮洗并最后用50%甲醛水溶液pH10.6洗涤。将729mg溶于15mLH2O中的rHuEPO滴定至pH10.6,将其加入到树脂中并在室温混合过夜。加入终浓度为1M的乙醇胺并在室温混合140分钟以封闭任何残留的反应性基团。后续步骤在8℃±2℃进行。将EPOxy-EPO装入一个柱子中并用3M尿素/750mM NaCl/10mM Tris pH7.6清洗,然后用TBS平衡柱子。将100mL用生物反应器从表达sHuEPOR的CHO细胞制得的条件培养基与2ml Q Sepharose Fast Flow(pharmacia)混合。将其在8℃±2℃不断混合保温30分钟,然后滤过0.45微米的硝酸纤维素瓶盖滤器(Corning)。将滤液上样到Epoxy-EPO柱上,用250mM NaCl/10mMTris pH7.6洗涤,然后用3M尿素/750mM NaCl/10mM Tris pH7.6洗脱。收集洗脱峰并用H2O稀释20倍。然后将稀释的收集液上样到15mL的Q Sepharose Fast Flow上并用10mM Tris pH7.6 NaCl梯度洗脱。将洗脱下来的单一峰收集并等份分装和在-80℃冷冻保藏。
在第三个制品中,使用制品2中所用的同样的Epoxy-EPO柱。将850mL用转瓶制备的来自表达sEPO-R的CHO细胞的条件培养基与1.7mL Q Sepharose Fast Flow混合。其操作方式与制品2中所用的相同。
在第四个制品中,将7.25L生物反应品制备的来自表达sHuEPOR的CHO细胞的条件培养基与110mL Q Sepharose Fast Flow混合。将其不断混合在8℃±2℃保温1小时,然后滤过一个0.45微米的硝酸纤维素瓶盖滤器(bottle top filter)。然后将滤液用7.25L H2O稀释并上样到用20mM Tris pH7.6平衡的770mL的Q Sepharose FastFlow柱上。将该柱用20mM Tris pH7.6的NaCl梯度洗脱。合并含基于SOS-PAGE分析为有效量的sHuEPOR的级分。向收集液中加入固体(NH4)2SO4至终浓度为1.2M然后将其滤过0.45μm的硝酸纤维素瓶盖滤器。将滤液上样到60mL的Pheny Sepharose 6(低Sub,pharmacia)上并用递减梯度的20MM Tris pH7.6的1.2M-0M(NH4)2SO4洗脱。合并主要的洗脱峰并使(NH4)2SO4为2.4M以沉淀sHuEPORt。通过离心,用H2O重悬并用Tris·HCl滴定至pH7.9来收获沉淀的sHuEPOR。将得到的溶液滤过0.45微米的硝酸纤维素滤膜,等份分装并在-80℃冷冻保存。
实施例3
杂交瘤细胞系的制备和筛选A. 酶联免疫吸附测定(EIA)
EIA最初用于确定单个动物的血清(Ab)抗体效价,后来用于对优势杂交瘤的筛选。将平底高结合96孔微量滴定EIA/RIA板(Costar公司,Cambridge,MA)用溶于5μg/ml碳酸盐-碳酸氢盐的缓冲液,pH9.2(0.015M Na2CO3,0.035M NaHCO3)的纯化sHuEPOR包被。向每个孔中加入50μl Ab。然后将板用乙酸酯膜(ICN Biomedicals,Inc.Costa Mesa.CA)覆盖并在振荡平台上在室温保温(RT)2小时或4℃过夜。在第1和2次增强(boost)之后使用第1批sHuEPOR,在第3次增强之后使用第2批sHuEPOR。第3和4批sHuEPOR用于筛选杂交瘤。将板在室温用每孔250μl 5%BSA溶液封闭30分钟,其中该溶液是通过将1份BSA稀释剂/封闭溶液浓溶液(kirkegard和PerryLaboratories;Inc.)与1份去离子水(dH2O)混合而制得的。弃去封闭液,向每孔加入50μl血清2倍稀释液(1∶400到1∶51,200)或杂交瘤组织培养上清。血清稀释剂是1%BSA.(10%BSA稀释剂/封闭溶液浓溶液1∶l0在Dulbecco’s磷酸缓冲盐溶液,D-PBS;GibcoBRL,Grand Island,NY中稀释),而杂交瘤上清未稀释即被测试。在杂交瘤测试中,将一个孔做为结合对照,而第2个孔做为阳性Ab对照。再次将板在室温振荡温育1小时,然后用20×浓溶液(Kirkegaard和Perry Laboratories. Inc.)的dH2O 1×漂洗液制品洗4次。将山羊抗小鼠IgG重和轻链特异的辣根过氧化物酶结合的二抗(BoehringerMannheim Biochemicals,Indianabolis,IN)在1%BSA中1∶1000稀释并在每个孔中保温30分钟。如前述洗板,印迹干燥和加入ABTS过氧化物酶单组分底物(kirkegard and Perry Laboratories,Inc.)。用microplate,EL 310读数仪(Bio-tek Instruments,Inc. Winooski,VT)在405nm读取每个孔的光吸收值。通过将血清稀释度的log10对405nm的光密度做图,然后外推到该血清最大光密度的50%的点而得到血清抗体的半最大效价。如果杂交瘤的光密度值比上述背景大5倍的话,即选为阳性。B.免疫
将10只,4.5周龄的Balb/c小鼠(Charles Rivers Laboratories,Wilmington,MA)用50μg sHuEPOR;第1批;抗原皮下注射(乳化于完全弗氏佐剂中)(CFA;50%体积/体积:Difco Laboratories,Detroit,MI)。将这些动物在4周之后用使用不完全弗氏佐剂(ICFA;Difco Laboratories,Detroit,MI)以类似方式制得的25μg抗原(Ag:第一批)加强接种(SQI)。9天后对小鼠尾部采血并用酶联免疫吸附测定(EIA)测定血清抗体(Ab)效价。如果小鼠的1/2最大效价大于5000,则选该动物用做杂交瘤制备。用于产生感兴趣的杂交体的(#71A和73A)三只动物(7,8和9号)需要分别用12.5μg Ag(第一批)和25μg Ag(第二批)在第5周和再次在第29周时加强接种。这些加强接种以与初次加强接种同样的方式进行;即以50%体积/体积ICFA乳化液的形式进行。在每次加强接种之后9天继续进行血清Ab效价测定。在融合之前这些小鼠的最终效价对7,8和9号动物来说分别是5026,6842,和12,945。C.细胞融合
在最后增强接种之后8周向7,8和9号动物静脉内注射25μgsHuEPOR(第3批)。4天后用二氧化碳杀死小鼠并在无菌条件下将脾脏收集到25ml含200U/ml青霉素G,200μg/ml硫酸链霉素和4mM谷氨酰胺(2×P/S/G DMEM)的Dulbecco’s改良Eagle’s培养基中。去除脾脏多余的脂肪组织,然后用三碟干净的2×P/S/G DMEM漂洗三次。接着将其转入含10ml 2×P/S/G DMEM的无菌胃袋(Stomacherbag)中,并用Stomacher Lab Blender 80(Seward LaboratoryUAC House;London,England)打散成单细胞悬液,将袋中换成新鲜的培养基并继续该操作直到所有的脾脏中的细胞都释放出来为止。将这些脾细胞在225×g每次离心10分钟洗3次。在第一次融合时,使用来自9号动物的脾细胞;在第二次融合时,收集来自7号和9号动物的脾细胞。
同时,将对数生长期的生长在完全培基(DMEM,10%胎牛血清,2mM谷氨酰胺,0.1mM非必需氨基酸,1mM丙酮酸钠,和10mMHepes缓冲剂,Gibco,Laboratories,Inc. Grand Island,NY)中的小鼠骨髓瘤细胞Sp2/0-Ag14(来自美国典型培养物保藏中心,Rockville,MD,保藏号CRL 1581)以同样方式洗三次。从该骨髓瘤群体中的4×107细胞(融合1)或8×107细胞(融合2)与脾细胞悬液混合,并再次沉淀。将吸取自细胞沉淀的培养基和对融合1为2ml而对融合2为3.5ml的聚乙二醇(PEG 1500分子量;Boehringer.Mannheim Biochemicals,Indianapolis,IN)在一分钟中轻轻混合。之后,缓慢加入等体积的2×P/S/G DMEM。将细胞在37℃放置2分钟,然后再加入9ml 2×P/S/G DMEM。再次将细胞在37℃置4分钟。最后向细胞悬液中加入30ml 2×P/S/G DMEM并离心沉淀细胞。从沉淀中吸取培养基并将细胞缓慢重悬于约56ml(融合1)或74ml(融合2)含100U/ml青霉素G和100μg/ml硫酸链霉素的完全培养基中。通过用5ml移液头(pipette)的简单滴入而将细胞分散到10块96孔平底组织培养板中(Becton Dickinson Labware;Lincoln park,NJ)。将培养板在饱和湿度37℃,5%CO2的条件下培养过夜。第2天向每个孔中加入等体积的选择培养基。选择培养基是指在完全培养基中含有0.1mM次黄嘌呤,4×10-4mM氨基喋呤,和1.6×10-2mM胸腺嘧啶核苷。将融合培养板培养7到10天,其中换了两次培养基;在每次改变流体之后都加入HAT选择培养基。从每个含杂交体的孔中取组织培养上清并用EIA检测特定的抗sHuEPOR的抗体的反应性。对96孔中EIA阳性的进行进一步的筛选。D.斑点印迹
对还原sHuEPOR(第4批)的点印迹被用作EIA阳性杂交瘤的第二级的筛选方法。根据说明书安装Dot Blot SF MicrotitrationApparatus(Bio-Rad Laboratories,Inc.,Richmond,CA),并应用硝酸纤维素膜(9×12cm;Bio-Rad Laboratories,Inc.;Richmond,CA)。首次在含2-巯基乙醇(5%体积/体积Bio-RadLaboratoris Inc.;Richmond CA)的Tris缓冲盐溶液的还原条件下煮沸5分钟制备抗原。向每个孔中上样25ng sHuEPOR(第4批)并抽吸通过硝酸纤维素膜以便结合。向各孔中加入250μl Blotto-Tween溶液(封闭溶液;2%wt/vol脱脂干奶粉,50mM Tris,pH7.5,25mM NaCl,0.1mM EDTA,0.9%vol/vol Tween 20,0.01%vol/vol消泡剂A)并在室温温育30分钟。吸去封闭液并重复该操作以确保膜上非特异性位点的完全封闭。接着用含0.1%vol/vol聚氧乙烯山梨聚糖单月桂酸酯(Tween-20;Bio-Rad Laboratories,Inc.;Richmond,CA)洗膜3次。接着向每个孔中加入95μl EIA阳性杂交瘤条件培养基并在室温温育45分钟。用TBS-Tween(20mM Tris,pH7.5,50mMNaCl,0.02%vol/vol Tween 20)每次250μl洗涤各孔3次并用TBS-Tween(20mM Tris,pH7.5,0.5M NaCl,0.09%vol/volTween 20)每孔250μl洗2次,每次洗后将加入液吸走。将100μl羊抗小鼠IgG,重链和轻链特异的HRP结合的二抗(在TBS-Tween中1∶1000稀释;Boehringer Mannheim Riochemicals;Indianapolis,IN)在室温对每个孔温育45分钟。如上所述洗膜,从印迹装置中取下膜,浸入预备好的增强的化学发光试剂(Enhanced ChemiumescentReagent)(ECL试剂;Amersham Life. Sciences,Corporation;Arlington Heights,IL),并用X-OMAT AR胶片曝光(Kodar.Scientfic Imaging,Rochester New York)。十五秒钟之后,从胶片盒中取出胶片并显影。对每个杂交瘤上清以点的强度为依据,从3+到0对每个孔进行评分。
实施例4
抗EPOR抗体与EPOR的结合A.BIAcore分析抗体与EPO-R的结合
以表面胞质团共振(SPR)(Fiagerstam等,分子识别杂志,3,208(1990);Malmoory等,Scord.J.Immunol. 35,643(1992))为基础的实时生物特异性的相互作用分析(BIA,Pharmacia·Biosensor.AB,Uppsala,Sweden)用于筛选ELISA阳性的单克隆抗体。
如实施例1和2所述制备的可溶性HuEPOR通过伯氨基团与传感器片(Chip)CM5共价偶联。在HBS中以5μl/min的流速进行固定化反应(10mM HEPES pH7.4,150mM NaCl,3.4mM EDTA,0.05% BIAcore表面活性剂P-20)。传感器片的羧化基质首先被注入40μl 1∶1的EDC(溶于水的400mM N-乙基-(二甲氨基-丙基)碳化二亚胺(pharmacia Biosensor AB))和NHS(100mM溶于水的N-羟基琥珀酰亚胺;pharmacia Biosensor AB)的混合物。注射入65μl可溶性EPO-R以固定到传感器片上。注入50μl乙醇胺(Pharmacia Biosensor AB)以灭活传感器片上过量的反应性基团。
每个分析循环包括:注入20μl杂交瘤上清,接着注入10μl 10mMHCl以再生传感器片。以共振单位(RU)计量SPR反应。对大多数蛋白来说,1000RU相应于大约1ng/mm2的表面浓度。对96个孔中EIA阳性者的筛选结果如表1所示。在这些试验中,背景一般是约20RU。与EPOR的结合典型地是在50RU或以上。
表1
单克隆抗体
抗 体(1) | BIACORE(2) | (3)竞争基团 | (4)平均荧光 | EPO活性的抑制(5) | 对UT7-EPO细胞的刺激(6) |
123456789101112131415161718192021222324252627282930313233343536373839404142434445 | 988765139894629694153149987298746974984268842111270161815253634161357415222361313 | ANTNTNTNTNTCNTNTNTNTCBNTNTNTNTNTNTNTNTNTNTNTNTNTNTNTNTANTNTNTNTANTNTNTBNTNTNTNTNTNT | --------------------------------------------- | -NTNTNTNT-NTNTNTNTNTNTNTNTNTNTNT-NTNTNTNT-NTNTNTNTNTNT--NTNTNTNTNT-NT-NTNTNTNTNTNT | -NTNTNTNT-NTNTNTNTNTNTNTNTNTNTNT-NTNTNTNT-NTNTNTNTNTNT-NTNTNTNTNTNT-NT-NTNTNTNTNTNT |
表1(续)
抗 体(1) | BIACORE(2) | 竞争基团(3) | 平均(4)荧光 | EPO活性的抑制(5) | 对UT7-EPO细胞(6)刺激 |
4647484950515253545556575859606162636465666768697071727374757677787980818282B838485868788899091 | 710569345316130133411101510104881415391222-59751000495877789158411903544089476434119811-44-13102551185944-140 | NTNTNTNTCNTNTANTNTNTNTNTNTNTNTABCNTCACAACBCABNTCANTNTNTNTNTCNTNTCNTNTNTNTNT | ------------14.99------------23.55-13.7118.53--------NT----12.81---- | NTNTNTNT-NTNTNTNTNTNTNT-NTNTNT-NTNT+/-NT-NT--+(7)---NTNT-NTNTNTNTNTNT-NTNTNT-+/-+/-NT- | NTNTNTNT-NTNTNTNTNTNTNT?NTNTNT-NTNT?NT?NT-?+++-+-NTNT-NTNTNTNTNTNT-NTNTNT---NT- |
表1(续)
抗 体(1) | BIACORE(2) | 竞争基团(3) | 平均(4)荧光 | EPO活性的抑制(5) | 对UT7-EPO细胞的(6)刺激 |
9293949596 | -3254177 | NTNTNTANT | ----- | NTNTNTNTNT | NTNTNTNTNT |
分泌所述抗体的杂交瘤的条件培养基用所述的测定检测。含所有全部抗体的上清在ELISA分析中表明为阳性信号。+++,++,+示阳性反应,其中+++示具有最大效果的反应。“-”示该反应比对照培养基的反应小或相等。NT示样品未被测试。?示样品不能被确定其应答程度。(1)抗体1-61来自7和8号小鼠。抗体62-96来自9号小鼠。(2)用结合了sHuEPOR的biacore片作为测定Mab的反应设备。(3)对BIACORE的竞争是抗sHuEPOR IG2的。将与传感器片结合的sHuEPOR用IG2温育,然后测定与未用IG2预处理的EPOR相比其与Mab的结合效果。其结合被完全封闭(80%-100%)的是A。50-80%的结合被封闭的是C。其结合之少于50%被封闭的抗体为B。(4)那些给于细胞平均荧光大于对照(12.73)的抗体的值被示为“-”指平均荧光强度小于或等于对照的抗体。(5)对UT7-EPO细胞3H胸腺嘧啶核苷掺入的抑制。将30munits EPO和各种量的抗体与细胞温育。在细胞培养过夜后对其用3H胸腺嘧啶核苷进行脉冲标记并进行计数测定。阳性反应被定义为随着抗体量的增加而逐渐减小的反应。(6)对UT7-EPO细胞3H掺入的刺激。用各种量的抗体温育细胞。在培养过夜后,对细胞进行3H胸腺嘧啶核苷脉冲标记并进行计数测定。阳性反应被定义为随抗体量的增加而逐渐增加掺入的反应。(7)抑制比激活所需要的浓度高。B.表位竞争分析
可注射入65μl杂交瘤上清IG2使固定有sHuEPOR的传感器片饱和。IG2是用实施例3所描述的方法得到的抗sHuEPOR的单克隆抗体。每个分析循环包括注入20μl被或未被注入的65μl IG2饱和的杂交瘤上清。在RU中IG2饱和后注射20μl的结合信号对在RU中只有20μl注射的结合信号的比率被定义为IG2的封闭百分比(%)。这些有80-100%封闭的抗体被称为A组,有小于50%的封闭的称为B组,而有50-80%封闭的为C组。结果如表1所示。C.荧光激活的细胞分选(FACS)分析抗体与转染的CHO细胞上d40EPOR的结合
用FACS分析测定了抗EPOR杂交瘤上清民与pDSRαEPORd40转染的CHO细胞表面的EPO受体的结合。如实施例1所述的构建了用编码d40EPO受体的DNA转染的CHO细胞。将CHO/EPOR细胞从组织培养碟中刮下来并以单细胞重悬在PBS/0.5%BSA中,接着以约3×105/孔接种到96孔圆底培养板中。然后将板在1000×g离心5分钟。离心之后,去除PBS/BSA上清并将各个沉淀细胞团或备用对照培养基或者一种EPOR杂交瘤上清重悬。将细胞在4℃温育1小时。温育之后,用PBS/BSA洗细胞并接着将细胞重悬于异硫氰基荧光素(FITC)标记的羊抗小鼠单克隆抗体(Southern Biotech,Birmingham Ala.)的溶液中。再次将细胞在4℃温育1小时,洗涤并用FACS分析。在所测试的96个上清中,有5个的平均细胞荧光大于对照培养基(参见表1)。Mab71的荧光水平最高,接下来是Mab74,58,73和87。没有其它的测试上清的荧光比对照值高。
实施例5
抗EPOR抗体和Fab片段的纯化A.腹水制备
在注射细胞系之前7到10天对大于5周龄的Balb/c小鼠(CharlesRivers Laboratories,Wilmington,MA)用2,4,10,14-十五碳烷(Pristane;Sigma,St.Louis,MO)激活。每个小鼠单次腹膜内注射0.5ml;对每个细胞系注射10到20只动物并制备其腹水。
获取在完全培养基中生长到汇合的杂交瘤细胞系,用D-PBS洗一次接着用Neubauer血球计数器计数。对每只小鼠腹膜内注入107细胞,并保持在食物和水任取的啮齿动物实验室(Rodent Lab)中,直到产生腹水。监测小鼠最大程度形成腹水,用CO2杀死小鼠,并用18G针头插入盛满液体的腔中收集液体。在225×g离心15分钟或在微量离心机(Eppendorf)中离心3分钟澄清液体。将每份4ml的等份贮藏在-20℃直到用蛋白-A柱层析来纯化。B.单克隆抗体的蛋白质-A纯化:
用蛋白质A柱层析对4ml腹水液体或10ml杂交瘤条件培养基进行纯化。使用了Bio-Rad单克隆抗体纯化系统II(MAPS II;Bio-Rad Laboratories;Richmond,CA)。简而言之,5ml亲和胶(Affi-gel)蛋白质-A悬液被装入一个1×10cm的一次性玻璃柱中。将蛋白质-A凝胶用大约30ml D-PBS洗涤并接着用20ml结合缓冲液(Binding Buffer)(Maps II Binding Buffer;Bio-Rad)流过柱子而制得。然后将用结合缓冲液1∶1稀释的腹水液体或条件培养基加入到柱子顶端并使之流过柱子。在免疫球蛋白与蛋白质-A结合之后,将未结合的部分弃去。接着用30ml结合缓冲液洗去柱子中未结合的蛋白以使其达到在280nm的吸光值小于0.01。然后用大约30ml Bio-Rad洗脱缓冲液洗脱含免疫球蛋白的级分。将该级分对4LD-PBS在4℃缓冲交换过夜。将得到的PBS平衡免疫球蛋白在Centricon Concentrator设备(Amicon Inc.,Beverly,MA)上1700×g离心浓缩。C.抗体结合结构域的片段化
用Pierce Immuno Pure Fab制备试剂盒(Pierce Chemical Company,Rockford,IL)对蛋白质-A纯化的免疫球蛋白进一步片段化为两个组成部分,可结晶部分(Fc)和抗体结合部分(Fab)。将蛋白-A纯化的免疫球蛋白用20mM磷酸盐/10mM EDTA缓冲液在pH7.0透析,并浓缩到大约20mg/ml。对10mg免疫球蛋白进行了片段化处理。用所提供的含42mg半酰氨酸的12ml磷酸缓冲液的消化缓冲液洗涤固定化的木瓜蛋白酶凝胶两次。然后将免疫球蛋白样品加入到凝胶中并在一个振荡摇床上37℃温育过夜。将溶解的Fab从Fc和蛋白质-A纯化的未消化的免疫球蛋白中分离出来。将收集的未结合级分做Fab样品。如上所述将未结合部分用4LD-PBS在4℃透析过夜而浓缩。
实施例6
EPOR上Mab71表位的作图
制备了包括人EPO受体1-224残基的17-30氨基酸长的重叠合成肽,其中残基1是脯氨酸而224位残基是天冬氨酸。该10个不同的肽在其两个末端各有6个氨基酸的重叠。人EPO-R氨基酸序列之中的肽的序列和其位置如下所示:5E-1 PPPNLPDPKFESKAALLAARGPEELCFTE
(残基1-30)SE-2A LLCFTERLEDLVCFWEEA
(残基25-42)SE-2B CFWEEAASAGVGPGNYSF
(残基37-54)SE-3 PGNYSFSYQLEDEPWKLCRLHQAPTARGAV
(残基49-78)SE-4 TARGAVRFWCSLPTADTSSFVPLELRVTAA
(残基73-102)SE-5 LRVTAASGAPRYHRVIHINEVVLLDAPVGL
(残基97-126)SE-6 DAPVGLVARLADESGHVVLRVLPPPETPMT
(残基121-150)SE-7 PETPMTSHIRYEVDVSAGNGAGSVQRVEIL
(残基145-174)SE-8 QRVEILEGRTECVLSNLRGRTRYTFAVRAR
(残基169-198)SE-9 FAVRARMEAPSFGGFWSAWSEPVSLLTPSDLD
(残基193-224)
聚苯乙烯孔(Costar,Cambridge,MA)被上述的EPO-R肽分别以在碳酸盐-碳酸氢盐缓冲液中的(0.015M Na2CO3 0.035MNaHCO3,pH92)100μg/ml,20μg/ml和0.8μg/ml的浓度包被。将板在室温(RT)温育2小时然后在4℃过夜。在同样条件下对可溶性HuEPOR以10μg/ml,2μg/ml,0.4μg/ml和0.08μg/ml的浓度做为阳性对照进行包被。在用PBS 5% BSA室温封闭30分钟,将该板用如实施例5中所述的纯化的Mab71以溶于1%BSA中的5μg/ml的浓度在室温封闭2小时。在用洗涤缓冲液(kirkegard and Perry Labs,Inc.)洗涤之后,将该板用1∶1000稀释的辣根过氧化物酶(BoehringerMannheim)偶联的羊抗小鼠IgG在室温温育1小时。洗板并用ABTS(Kirkegard and Perry Labs,Inc.)底物溶液显色。在405am进行比色。Mab与合成肽的结合结果如图1所示并且表明与其它被测试的肽相比,Mab71结合显著量的SE-3肽(包含于人EPO-R中的49到78氨基酸残基)。这表明Mab71与包含人EPO-R49到78残基或与之重叠的区域的结合。
实施例7
抗EPOR抗体在细胞增殖测试中的活性
测定如上所述在条件培养基中制备的抗体对UT7-EPO细胞3H胸腺嘧啶核苷的掺入的刺激能力(Komatsu等,上文)。UT7-EPO细胞对EPO应答并在其细胞表面表达人EPO受体。将UT7-EPO细胞在生长培养基中长到约3×105细胞/ml(1×Iscone’s改良Dulbecco’s培养基,其中含L-谷氨酰胺,25mM HEPES缓冲剂,和3024mg/L碳酸氢钠,但是不含α-硫代甘油或β-巯基乙醇(GIBCO)/10%v/v胎牛血清/1%v/v L-谷氨酰胺-青霉素-链霉素溶液(Irvine Scientific)/1 Unit/ml rHuEPOR)。离心收集细胞(大约500×G)并用磷酸缓冲的盐溶液洗两次并在测试培养基(1×RPMI培养基不含L-谷氨酰胺(Gibcc)/1% L-谷氨酰胺/4%胎牛血清)中重悬于5×104细胞/ml,在测试培养中稀释至少5倍的测试样品或EPO标准(rHuEPO)100μl被加入到96孔微量滴定板的孔中。接着加入50μL细胞(5000细胞/孔)并将板培养在饱和湿度的培养箱中在37℃和5%CO2中培养。72小时后,加入50μL 1∶100稀释在测试缓冲液中的甲基-3H-胸腺嘧啶核苷(1mCi/ml;20Ci/mMole)。将细胞再另外在37℃5%CO2中培养4小时。用PHO细胞收获器(Cambridge Technology Inc.)收获到玻璃纤维滤膜(filtermats)中并用去离子水做为洗涤液。最后用2-丙醇洗涤膜然后干燥并在Beckman Model LS 6000 IC闪烁计数仪中计数。
如上所述测试了来自含抗EPOR Mab的组织培养板的条件培养基刺激增殖的能力。测定了几个稀释度的样品。阳性反应的定义是那些在刺激胸腺嘧啶核苷的掺入时为背景水平的至少2倍且当样品稀释时还导致刺激减弱的反应。如表1所示,在24个所测样品中有两个(Mab71和73)为阳性反应。四个样品可能具有弱的刺激活性(表1中?)。其余的样品相对于背景无显著增加。来自用于产生单克隆的多克隆血清也可刺激胸腺嘧啶核苷掺入。这表明该血清中的多克隆抗体也能刺激UT7-EPO细胞的增殖。
还测试了上清对EPO刺激的UT7-EPO细胞的胸腺嘧啶核苷掺入的抑制能力。将细胞用25munits/ml rHuEPO和各种量的含条件培养基的抗体温育。如上所述测定胸腺嘧啶核苷的掺入。结果如表1所示。大多数抗体与对照培养基无显著差异。在表明可抑制胸腺嘧啶核苷的掺入的抗体中,其中两个样品(Mab58和73)显示为确定的抑制作用而三个样品(Mab65,88和89)示可能的抑制作用。Mab73与对照值相比在最高剂量时起抑制作用,但在较低剂量时它却刺激胸腺嘧啶核苷的掺入。
实施例8
抗EPOR抗体和片段对EPOR的活化A. UT7-EPO细胞增殖测定
如实施例5所述对Mab71和73进行纯化。用如实施例7中所述的UT7-EPO胸腺嘧啶核苷的掺入的测定来确定增殖活性。如rHuEPO-样Mab71和73二者都以剂量依赖的方式刺激UT7-EPO细胞的掺入(参见图2)。在高剂量Mab71时活性减弱。对Mab71在1-2μg/ml剂量和对Mab73在>100μg/ml剂量观察到刺激活性峰。一种非中和对照抗体(抗EPO Mab F12)无刺激作用;这表明刺激作用是对EPO受体抗体特异的。B. EPO冷置换测试
EPO受体的抗体可与EPO结合的同样区域结合。为了检测这种可能性,用OCIM1细胞进行了冷置换测试。OCIM1细胞是来自人的并且已知在其细胞表面含EPO受体(Broudy等,美国国家科学院院刊85,6517(1988))。细胞在OCIM1在生长培养基中生长到大约2-5×105细胞/ml(Iscove’s改良Dulbecco’s培养基(IMDM)/10%胎牛血清/10%青-链抗霉剂(fungisone))。离心收集细胞,用结合缓冲液(RPMI1640/1%BSA/25mM HEPES pH7.3)中洗两次;接着以1-2×107细胞/ml重悬于含0.1%叠氮化物和10μg/ml细胞松弛素B的结合缓冲液中。然后用10μL样品和10μL 125I-EPO(Amersham高特异活性,3000Ci/mMole,2μCi/ml)在37℃饱和湿度组织培养箱中温育96孔组织培养板中的细胞。3小时后在滴定试管中对邻苯二甲酸酯油(60∶40(v/v)二丁基/二壬基邻苯二甲酸酯)离心。将含细胞的试管快速在干冰-乙醇浴中冷冻并且将细胞沉淀取下并在LKB 1277伽马捕获仪自动伽马计数仪中计数。
图3示冷置换试验的结果。当加入的未标记的rHuEPO增加时,被从细胞上EPO受体置换的125I-EPO的量也增加。同样的方式,如实施例5所述纯化的Mab71随抗体的量增加时,它置换的125I-EPO的量也增加。相反Mab73在最高剂量时有置换的迹象,但非中和抗rHuEPOMab(F12)无显著置换作用。这些结果Mab F12不干扰EPO与其受体的结合,但Mab71和73却干扰。该结果还表明Mab71通过在EPO结合位点上或其邻近的结合而与EPO受体结合并活化之。C. Mab71和Fab71活性的比较
如实施例5所述制备了Mab71的EPO受体片段。如图4所示用SDS凝胶电泳时该制品进行特征鉴定(Laemmli等.自然.227,680(1970))。将样品在含或不含0.7M 2-巯基乙醇的2%SDS的样品缓冲液中煮沸,将还原(2-巯基乙醇)和非还原(不含2-巯基乙醇)的蛋白区别开,然后在12.5%丙烯酰胺SDS凝胶上电泳。将凝胶用考马斯兰染色以显示蛋白。通过与蛋白质标准的迁移率进行比较而估测其分子大小。当在还原条件下电泳时,Mab71和73被分离为轻链和重链。重链大约为52KDa。73的轻链(28KDa)比Mab71的轻链(28.5KDa)稍微小些。Fab片段也有两条链:Fab71的28.3和27.3KDa,Fab73的27.5和26.5KDa。当这些Fab片段在非还原条件下电泳时,Fab71和73的大小分别是48和47KDa。这表明Fab是单价的,该复合物含轻,重链各一条。相反在非还原SDS凝胶中Mab71和73的迁移率为大约200KDa。这表明Mab是二价的,有重链的轻链各两条。
为了看单价的Fab71片段是否可活化EPO受体,如实施例7所述用Mab71和Fab71培养UT7-EPO细胞并测定了胸腺嘧啶核苷的掺入。如图5所示,rHuEPO和Mab71都可刺激胸腺嘧啶核苷的掺入。但是单价的Fab71片段却不能。得到的抗非相关受体(Her2/neu)的对照单克隆抗体也不刺激胸腺嘧啶核苷的掺入。这表明为了激活受体这些抗体必须是双价的。D.在rHuEPO的存在下Mab71和Fab71对胸腺嘧啶核苷掺入的刺激作用
Mab71抑制EPO与EPO受体的结合的事实表明该抗体在EPO的存在下不能活化EPO受体。为了检测这种可能性,将UT7-EPO细胞30munits/ml rHuEPO和各种不同量的纯化的Mab71,Fab71或Mab对照(抗Her2/neu)温育。如上所述测定了胸腺嘧啶核苷的掺入。如图6所示,在高剂量时,Mab71和Fab71二者都可以抑制胸腺嘧啶核苷的掺入。但是Mab71在大约30和3000μg/ml之间时刺激胸腺嘧啶核苷掺入的水平比rHuEPO单独的水平要高。Fab71和对照抗体无此作用。这表明Mab71和rHuEPO在EPO受体活化时具有增加作用。
实施例9
抗EPOR抗体对红系集落形成的刺激作用
为了看是否纯化的Mab71可刺激来自外周血的前体细胞红系细胞的形成,而进行了BFUe测试。为了纯化红系细胞前体细胞,根据标准方法对正常人供体进行了淋巴细胞分层(Lymphopheresed)。用250mlHank’s平衡盐溶液(HBSS)洗涤淋巴分层细胞(250ml)。将细胞重悬于HBSS中并在一个密度梯度上(Ficoll-papue)在500×g离心30分钟进行分离。将从该梯度收获的低密度细胞(LD)用500ml HBSS洗涤并以5×108细胞/ml的浓度在添加有0.5%牛血清白蛋白和5mMEDTA PBS中重悬。用Miltenyi Biotech GmbH制造的CD34祖细胞分离试剂盒(QBend/10)对CD细胞做进一步纯化。简而言之,将细胞用抗CD34单克隆抗体标记,并根据标准方法接着对其与磁性小球结合。然后将标记细胞通过预先填装的MiniMacs分离柱,然后洗柱并从柱上将CD34+细胞洗脱下来。重复该操作一次以得到较高纯度的CD34+细胞。根据Iscove等(细胞生理杂志83,309(1974))所述和下面的改良对纯化的CD34+细胞进行体外测试。培养用培养基获自GibcoBRL.(人骨髓细胞增殖试剂盒,Grand Island,NY)。为了在35×100mm的组织培养板接种出1ml样品的重复样,在17×100的无菌聚苯乙烯管制备了过量的3ml。向每支管中加入2.5ml干细胞生长培养基,0.1ml CD34+细胞(以90,000细胞/ml重悬),0.015ml干细胞因子(20μg/ml),和等于0.385ml的样品与干细胞稀释培养基的组合。将试管旋涡振荡开放置以去除气泡。将所含物质用带有17×1-1/2针头的注射器等份分装。将培养板在37℃10%CO2的饱和湿度的组织培养箱中培养。在21天后得到了红系集落(颜色为橙色到红色)。在缺少EPO或Mab71的板中未见到红系集落。rHuEPO(30munits/板)在每个板上有多于400个的集落。Mab71也产生红系集落。峰活性在2-6μg/ml,这结果表明Mab71刺激红系集落的形成。
还测试了使用不含血清的甲基纤维素时纯化的Mab71形成红系集落的能力。如上所述分离CD34+细胞并用在共同未决和共同所有的美国专利,流水号08/079,719中所描述的无血清生长培养基培养,并有下面的修改,本文引入做为参考。测试试管的建立不用细胞外基质分子,氢化皮质酮,和生长因子EGF,FGF和PDGF。如上所述制备3ml样品以便在培养板上接种出重复的1ml的样品。向试管中加入各0.03ml 100×贮液(2-巯基乙醇,核苷酸(nudeosides),胆固醇,丙酮酸钠,人转铁蛋白,人胰岛素),0.4ml去离子化的BSA(15%),0.015ml SCF(20μg/ml),0.1ml CD34+细胞(以300,000细胞/ml重悬),1.080ml甲基纤维素(2.3%)和其中样品不超过150ml的1.195ml的样品和IMDM的组合物。如上所述培养培养板并在21天后计数集落。在EPO或Mab71但不缺少这两种因子的条件下观察到了红系集落。图7系红系集落类型的一个实例。用25munits rHuEPO培养的集落看起来与用2.1μg/ml纯化Mab71培养的相似。高剂量的rHuEPO给出较多的集落。剂量反应曲线如图8所示。Mab71的峰活性在1-5μg/ml之间。较低和较高的剂量都得到较少的集落。抗Her2/neu的对照单克隆抗体在该剂量范围内不产生任何集落。该结果表明,Mab将刺激红系前体细胞红系集落的形成并且不需要额外的血清。因此Mab71可刺激红系前体细胞分化成红系细胞。
* * *
尽管本发明以优选实施方案的形式进行的描述,应该理解对本领域的技术人员来说可进行各种变化和修饰。因此,希望所附的权利要求书覆盖所要求的本发明范围之内的这些变化。
序列表
(1)一般信息:
(i)申请人:Amgen Inc.
(ii)发明题目:活化促红细胞生成素受体的抗体
(iii)序列数:3
(iv)通信地址:
(A)收信人:Amgen Inc.
(B)街道:1840 Dehavilland Drive
(C)城市:Thousand Oaks
(D)州:加利福尼亚
(E)国家:美利坚合众国
(F)邮区:91320-1789
(v)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOC/MS-DOC
(D)软件:Patent In Release #1.0,Version #1.25
(vi)现今申请数据:
(A)申请号:
(B)提交日:
(C)分类:
(viii)委托/代理人信息
(A)姓名:Winter,Robert B.
(C)参考/档案号:A-307
(2)SEQ ID NO:1:的信息
(i)序列特征:
(A)长度:42碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:1:CTCCAAGCTT GCCGTCACCA TGGACCACCT CGGGGCGTCC CT
(2)SEQ ID NO:2:的信息
(i)序列特征:
(A)长度:35碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:2:CAGGTCTAGA TTACTAGGGA TCCAGGTCGC TAGGC
(2)SEQ ID NO:3:的信息
(i)序列特征:
(A)长度:27碱基对
(B)类型:核酸
(C)链型:单
(D)拓扑学:线性
(ii)分子类型:cDNA
(xi)序列描述:SEQ ID NO:3:AGGTCGACTA CTAGTAGTCA GTTGAGA
Claims (26)
1.一种可活化促红细胞生成素受体的抗体或其片段。
2.权利要求1的抗体,其中的促红细胞生成素受体是一种哺乳动物促红细胞生成素受体。
3.权利要求1的抗体,其中的促红细胞生成素受体是一种人促红细胞生成素受体。
4.权利要求1的抗体,它是一种单克隆抗体。
5.权利要求1的抗体,它是人源化的抗体。
6.权利要求1的抗体,它是一种人抗体。
7.权利要求1的带有可检测标记的抗体。
8.一种能产生权利要求4的单克隆抗体的杂交瘤细胞系。
9.一种可识别促红细胞生成素受体上的一个表位的抗体或其片段,它由杂交瘤细胞系ATCC No.HB11689或ATCC No.HB11690产生的单克隆抗体识别。
10.权利要求9的抗体,它可活化促红细胞生成素受体。
11.权利要求9的抗体,其中促红细胞生成素受体是一种人促红细胞生成素受体。
12.权利要求9的抗体,它是一种单克隆抗体。
13.权利要求9的抗体,它是一种人源化的抗体。
14.权利要求9的带有可检测标记的抗体。
15.一种能产生权利要求12的单克隆抗体的杂交瘤细胞系。
16.一种由杂交瘤细胞系ATCC No.HB11689或ATCC No.HB11690产生的抗体。
17.杂交瘤细胞系ATCC No.HB11689或ATCC No.HB11690。
18.一种在生物样品中检测能被活化的促红细胞生成素受体的方法,该方法包括如下步骤:
(a)将样品与权利要求1或9的抗体接触;
(b)检测抗体对受体的活化,
从而确定能被活化的促红细胞生成素受体的存在。
19.一种包含权利要求1或9的抗体的检测生物样品中可被活化的促红细胞生成素受体的试剂盒。
20.一种在哺乳动物中调节促红细胞生成素受体的内源活性的方法,它包括施用有效量的权利要求1或9的抗体以调节受体的活性。
21.权利要求20的方法,其中对促红细胞生成素受体活性的调节调控了红系祖细胞的增殖或分化。
22.一种在患者中治疗贫血的方法,它包括施用治疗上有效剂量的权利要求1或9的抗体。
23.一种在药用上可接受的佐剂中包含了治疗上有效量的权利要求1或9的抗体的药用组合物。
24.权利要求23的组合物,其中抗体是单克隆抗体。
25.权利要求24的组合物,其中抗体是人源化抗体。
26.权利要求24的组合物,其中抗体是人抗体。
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US6103879A (en) | 1996-06-21 | 2000-08-15 | Axys Pharmaceuticals, Inc. | Bivalent molecules that form an activating complex with an erythropoietin receptor |
-
1994
- 1994-07-26 US US08/280,864 patent/US5885574A/en not_active Expired - Lifetime
-
1995
- 1995-07-24 IL IL11471795A patent/IL114717A0/xx unknown
- 1995-07-26 DE DE69535419T patent/DE69535419T2/de not_active Expired - Fee Related
- 1995-07-26 ZA ZA956215A patent/ZA956215B/xx unknown
- 1995-07-26 AT AT01111554T patent/ATE356148T1/de not_active IP Right Cessation
- 1995-07-26 WO PCT/US1995/009458 patent/WO1996003438A1/en active IP Right Grant
- 1995-07-26 CN CN95195117A patent/CN1158134A/zh active Pending
- 1995-07-26 AT AT95927476T patent/ATE209218T1/de not_active IP Right Cessation
- 1995-07-26 DE DE69524102T patent/DE69524102T2/de not_active Expired - Fee Related
- 1995-07-26 AU AU31499/95A patent/AU697369B2/en not_active Ceased
- 1995-07-26 DK DK01111554T patent/DK1146056T3/da active
- 1995-07-26 PT PT95927476T patent/PT773962E/pt unknown
- 1995-07-26 NZ NZ290689A patent/NZ290689A/en unknown
- 1995-07-26 CA CA002195868A patent/CA2195868A1/en not_active Abandoned
- 1995-07-26 JP JP50596596A patent/JP3225493B2/ja not_active Expired - Fee Related
- 1995-07-26 ES ES95927476T patent/ES2168376T3/es not_active Expired - Lifetime
- 1995-07-26 DK DK95927476T patent/DK0773962T3/da active
- 1995-07-26 ES ES01111554T patent/ES2283355T3/es not_active Expired - Lifetime
- 1995-07-26 PT PT01111554T patent/PT1146056E/pt unknown
- 1995-07-26 SI SI9530563T patent/SI0773962T1/xx unknown
- 1995-07-26 MX MX9700553A patent/MX9700553A/es not_active IP Right Cessation
- 1995-07-26 EP EP01111554A patent/EP1146056B1/en not_active Expired - Lifetime
- 1995-07-26 EP EP95927476A patent/EP0773962B1/en not_active Expired - Lifetime
-
1998
- 1998-06-05 US US09/092,291 patent/US6319499B1/en not_active Expired - Fee Related
-
1999
- 1999-09-27 JP JP11273329A patent/JP2000095800A/ja active Pending
-
2003
- 2003-02-10 US US10/364,276 patent/US7081523B2/en not_active Expired - Fee Related
-
2006
- 2006-04-18 US US11/406,835 patent/US20070014793A1/en not_active Abandoned
-
2007
- 2007-10-30 US US11/981,631 patent/US20080182976A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
DE69524102D1 (de) | 2002-01-03 |
PT1146056E (pt) | 2007-05-31 |
AU3149995A (en) | 1996-02-22 |
US20080182976A1 (en) | 2008-07-31 |
US20030215444A1 (en) | 2003-11-20 |
AU697369B2 (en) | 1998-10-01 |
IL114717A0 (en) | 1995-11-27 |
ZA956215B (en) | 1997-01-03 |
DE69535419D1 (de) | 2007-04-19 |
DK1146056T3 (da) | 2007-07-02 |
JPH09508535A (ja) | 1997-09-02 |
DE69535419T2 (de) | 2007-11-08 |
SI0773962T1 (en) | 2002-06-30 |
NZ290689A (en) | 1998-07-28 |
EP0773962A1 (en) | 1997-05-21 |
ES2283355T3 (es) | 2007-11-01 |
EP1146056B1 (en) | 2007-03-07 |
WO1996003438A1 (en) | 1996-02-08 |
ATE356148T1 (de) | 2007-03-15 |
US6319499B1 (en) | 2001-11-20 |
DK0773962T3 (da) | 2002-05-21 |
ATE209218T1 (de) | 2001-12-15 |
US5885574A (en) | 1999-03-23 |
EP1146056A1 (en) | 2001-10-17 |
US7081523B2 (en) | 2006-07-25 |
MX9700553A (es) | 1997-05-31 |
ES2168376T3 (es) | 2002-06-16 |
US20070014793A1 (en) | 2007-01-18 |
JP2000095800A (ja) | 2000-04-04 |
CA2195868A1 (en) | 1996-02-08 |
DE69524102T2 (de) | 2002-07-04 |
PT773962E (pt) | 2002-05-31 |
EP0773962B1 (en) | 2001-11-21 |
JP3225493B2 (ja) | 2001-11-05 |
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