CN108743967B - 含有免疫球蛋白fc区作为载体的药物组合物 - Google Patents
含有免疫球蛋白fc区作为载体的药物组合物 Download PDFInfo
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Abstract
本发明公开了免疫球蛋白Fc片段的新用途,更具体而言,本发明公开了含有免疫球蛋白Fc片段作为载体的药物组合物。包含免疫球蛋白Fc片段作为载体的药物组合物可以在保持该药物相对高水平的体内活性的同时显著延长药物的血清半衰期。此外,当所说药物是多肽药物时,与免疫球蛋白Fc片段和靶蛋白的融合蛋白相比,所说的药物组合物引起免疫应答的风险较低,因此可用于开发各种多肽药物的长效制剂。
Description
本申请是2004年11月13日提交的同名发明专利申请 200480001775.5的分案申请201210190634.9的再次分案申请。
技术领域
本发明涉及免疫球蛋白Fc片段的新用途。更具体而言,本发明涉及含有免疫球蛋白Fc片段作为载体的药物组合物,以及延长与免疫球蛋白Fc片段连接的药物的体内作用持续时间的方法。
发明背景
从前,许多药理学家和化学家致力于从化学上改变和/或修饰天然存在的生理学活性分子的体内活性。这些努力主要集中于提高或延长生理学活性物质的某些体内活性、减轻其毒性、消除或减少其副作用或改变其特异的生理学活性。当生理学活性物质被化学修饰时,在许多情况下它会丧失自身的某些或大部分生理学活性。不过,在某些情况下,修饰可导致生理学活性的提高或改变。在这点上,许多研究已集中于能实现所需生理学活性的化学修饰上,且大部分这样的研究涉及将生理学活性物质(药物)与生理学可接受载体共价键合。
例如,国际专利申请WO 01/93911应用了具有数种酸成分作为药物载体的聚合物。国际专利申请公开WO 03/00778公布了含阴离子基团的两性嵌段共聚物,当将它用作阳离子药物的药物载体时,它可提高药物的稳定性。欧洲专利EP0 681 481描述了一种以环式糊精和酸作为载体改进碱性药物特性的方法。另一方面,疏水药物在体内具有低稳定性,这主要是由于它们的低水溶性造成的。为了改进疏水药物的低水溶性,国际专利申请WO 04/064731利用了脂类作为载体。不过,迄今为止,还未有关于将免疫球蛋白Fc片段作为药物载体的报道。
通常,因为多肽由于其低稳定性而相对容易变性、在血液中被蛋白水解酶降解并容易通过肾脏或肝脏清除掉,所以蛋白质药物,包括以多肽作为药物有效成分的药物需要频繁的施用于患者以维持所需要的血液水平浓度和效价。不过,这样频繁施用蛋白质药物,尤其是通过注射施加会引起患者的痛苦。为了解决这些问题,已进行了许多努力来提高蛋白质药物的血清稳定性并长时期保持药物在血液中的高水平,从而使药物的药效最大化。因此,具有相同活性的药物组合物需要提高蛋白质药物的稳定性并保持足够水平的效价而不会引起患者的免疫应答。
为了稳定蛋白质、预防酶促降解和被肾脏清除,常规上使用了诸如聚乙二醇(下文中简称为“PEG”)等具有高溶解性的聚合物来化学修饰蛋白质药物的表面。通过与靶蛋白质的特定区域或各种区域结合,PEG稳定了蛋白质并防止其水解,同时不会造成严重的副作用(Sada等,J.Fermentation Bioengineering 71:137-139,1991)。不过,尽管它能增强蛋白质的稳定性,PEG偶联也存在一些问题,诸如大大降低了生理学“活性”蛋白质的效价。此外,随着PEG分子量的提高,其产量会有所降低,这是由于该蛋白质的反应性有所降低所致。
最近,已经公开了聚合物-蛋白质药物缀合物。例如,如美国专利5,738,846中所述,可通过在PEG两端连接同样的蛋白质药物来制备缀合物,以提高蛋白质药物的活性。此外,如国际专利申请公开 WO 92/16221中所述,也可以将两种不同的蛋白质药物连接于PEG的两端,得到具有两种不同活性的缀合物。不过,以上方法在维持蛋白质药物的活性上不是非常成功。
另一方面,Kinstler等报道了一种通过将粒细胞-集落刺激因子 (G-CSF)与人白蛋白而相偶联而制备的融合蛋白显示出改良的稳定性(Kinstler等,PharmaceuticalResearch 12(12):1883-1888,1995)。不过,在这篇文章中,由于与单纯施用天然G-CSF相比,具有 G-CSF-PEG-白蛋白结构的经修饰药物只显示出体内停留时间提高了大约4倍和血清半衰期的稍微延长,所以它尚未作为蛋白质药物的长效制剂而被工业化生产。
改进生理学活性蛋白质体内稳定性的一种备选方法是通过用遗传重组技术将生理学活性蛋白质的基因与编码具高血清稳定性的蛋白质的基因相连接并培养用所述重组基因转染的细胞以产生融合蛋白。例如,可通过用遗传重组将白蛋白或其片段与感兴趣的生理学活性蛋白质缀合来制备融合蛋白,所述白蛋白是已知在增强蛋白质稳定性上最有效的一种蛋白质(国际专利申请WO 93/15199和WO 93/15200,欧洲专利公开413,622)。由人类基因组科技公司(Human Genome Science Company)研发并以商品名‘AlbuferonTM’被销售的α干扰素和白蛋白的融合蛋白,在猴子中的半衰期从5小时延长到了93 小时,但已知成问题的是它将体内活性降低到低于未修饰的α干扰素的5%(Osborn等,J.Phar.Exp.Ther.303(2):540-548,2002)。
重组DNA技术被用于将蛋白质药物与免疫球蛋白Fc片段融合。例如,干扰素(韩国专利公开申请文件2003-9464)和白介素-4受体、白介素-7受体或促红细胞生成素(EPO)受体(韩国专利登记号249572)先前以与免疫球蛋白Fc片段融合的形式表达于哺乳动物中。国际专利申请公开WO 01/03737描述了一种包含通过肽键与免疫球蛋白Fc片段连接的细胞因子或生长因子的融合蛋白。此外,美国专利5,116,964 公布了通过遗传重组与免疫球蛋白Fc片段的氨基或羧基末端融合的蛋白质。美国专利5,349,053公布了含有通过肽键与免疫球蛋白Fc片段融合的IL-2的融合蛋白。通过遗传重组制备的Fc融合蛋白的其它例子包括β干扰素或其衍生物与免疫球蛋白Fc片段的融合蛋白(国际专利申请WO 00/23472),和IL-5受体与免疫球蛋白Fc片段的融合蛋白(美国专利5,712,121)、α干扰素和免疫球蛋白G4的Fc片段的融合蛋白(美国专利5,723,125),以及CD4蛋白质和免疫球蛋白G2的Fc 片段的融合蛋白(美国专利6,451,313)。
涉及免疫球蛋白Fc片段的氨基酸残基修饰的技术同样也是已知的。例如,美国专利NO.5,605,690公布了一种TNFR-IgG1Fc融合蛋白,它是通过遗传重组利用在补体结合区或受体结合区中有氨基酸变化的IgG1Fc片段制备的。此外,用修饰过的免疫球蛋白Fc片段通过遗传重组制备融合蛋白的其它方法参阅美国专利6,277,375、 6,410,008和6,444,792。
这样的通过遗传重组产生的Fc融合蛋白具有以下缺点:蛋白质融合只发生在免疫球蛋白Fc片段的特定区域,即在氨基或羧基末端;只能产生均二聚体形式而不产生单体形式;融合只可能发生在糖基化蛋白质之间或非糖基化(aglycosylated)蛋白质之间,而不可能形成由糖基化蛋白质和非糖基化蛋白质组成的融合蛋白。此外,融合所建立的新氨基酸序列可能引发免疫应答,而接头区可能变得对蛋白水解的降解作用敏感。
为了解决这些问题,本申请的发明者经过研究得知,当药物以与IgG Fc片段连接的形式被施用时,所述药物在展示体内活性最小程度的降低的同时提高了在体内的稳定性。
发明内容
因此本发明的一个目的是提供包含免疫球蛋白Fc片段作为载体的药物组合物。
本发明的另一目的是提供用于通过包含免疫球蛋白Fc片段作为载体而改善药物的体内作用持续时间的方法。
附图简述
本发明的以上及其它目的、特性和其它优势可通过以下详述并结合附图而更清楚地加以理解,其中:
图1显示了用木瓜蛋白酶切割免疫球蛋白获得的免疫球蛋白 Fc片段的层析结果;
图2显示了纯化的免疫球蛋白Fc片段的SDS-PAGE结果(M: 分子量标准,泳道1:IgG,泳道2:Fc);
图3显示了IFNα-PEG-Fc(A),17Ser-G-CSF-PEG-Fc(B)与 EPO-PEG-Fc(C)缀合物的SDS-PAGE结果,它们是通过偶联反应产生的(M:分子量标准,泳道1:Fc,泳道2:生理学活性蛋白质,泳道3:生理学活性蛋白质-PEG-Fc缀合物);
图4显示了偶联反应后纯化的IFNα-PEG-Fc缀合物大小排阻层析的结果;
图5显示了EPO-PEG-Fc缀合物的MALDI-TOF质谱的结果;
图6a和6b分别显示了天然免疫球蛋白Fc和去糖基化免疫球蛋白Fc(DG Fc)的MALDI-TOF质谱和SDS-PAGE分析结果;
图7显示了IFNα-PEG-Fc缀合物和IFNα-PEG-DG Fc缀合物的MALDI-TOF质谱结果;
图8a至8c显示了IFNα-PEG-Fc、IFNα-PEG-DG Fc和 IFNα-PEG-重组AG Fc衍生物缀合体的反相HPLC结果;
图9是显示天然IFNα、IFNα-40K PEG复合物、IFNα-PEG- 白蛋白缀合物和IFNα-PEG-Fc缀合物的药物代谢动力学分析结果的曲线图;
图10是显示天然EPO、高度糖基化的EPO、EPO-PEG-Fc缀合物和EPO-PEG-AG Fc缀合物的药物代谢动力学分析结果的曲线图;
图11是显示IFNα-PEG-Fc、IFNα-PEG-DG Fc和IFNα-PEG- 重组体AG Fc缀合物的药物代谢动力学分析结果的曲线图;
图12是显示Fab′、Fab′-S-40K PEG复合物、Fab′-N-PEG-N-Fc 缀合物和Fab′-S-PEG-N-Fc缀合物的药物代谢动力学的曲线图;
图13是显示Fab′、Fab′-S-40K PEG复合物和 Fab′-N-PEG-N-Fc缀合物和Fab′-S-PEG-N-Fc缀合物体内活性的曲线图;
图14是显示人IgG亚型与C1q补体结合亲和力的比较结果的曲线图;以及
图15是显示糖基化Fc、酶促去糖基化DG Fc和其中载体为大肠杆菌所产生的AG Fc的干扰素-PEG-载体缀合物与C1q补体结合亲和力的比较结果的示意图。
实施本发明的最佳方式
一方面,本发明涉及包含免疫球蛋白Fc片段作为载体的药物组合物。
术语“载体”用于此处时指与药物连接的物质,它通常通过与药物结合而提高、降低或消除药物的生理学活性。不过,就本发明的目的而言,载体用于本发明中以使与载体连接的目的药物的生理学活性的降低最小化,同时增强了药物的体内稳定性。
为了完成本发明的目的,本发明应用了免疫球蛋白Fc片段作为载体。
免疫球蛋白Fc片段就用作药物载体而言是安全的,因为它是在体内可新陈代谢的生物可降解多肽。此外,免疫球蛋白Fc片段与整个免疫球蛋白分子相比具有相对较低的分子量,因此有利于目的缀合物的制备、纯化和生产。另外,由于免疫球蛋白Fc片段不包含Fab片段(后者的氨基酸序列在抗体亚型之间有所不同,因此具有高度的非同源性),它可能极大的提高了物质的均一性并且具有较低的抗原性。
术语“免疫球蛋白Fc片段”用于此处时是指含有免疫球蛋白重链恒定区2(CH2)和重链恒定区3(CH3)且不含免疫球蛋白重链和轻链可变区、重链恒定区1(CH1)和轻链恒定区1(CL1)的蛋白质。它可能进一步包含位于重链恒定区的铰链区域。此外,除了重链和轻链可变区之外,本发明的免疫球蛋白Fc片段可能包含重链恒定区1(CH1) 和/或轻链恒定区1(CL1)的一部分或全部。同样,只要它具有与天然蛋白质基本上相似或优于天然蛋白质的生理学功能,IgG Fc片段就可以是在CH2和/或CH3氨基酸序列的相对较长部分中有缺失的片段。即,本发明的免疫球蛋白Fc片段可能包含1)CH1结构域、CH2 结构域、CH3结构域和CH4结构域,2)CH1结构域和CH2结构域, 3)CH1结构域和CH3结构域,4)CH2结构域和CH3结构域,5)一个或多个结构域和免疫球蛋白铰链区(或铰链区的一部分)的组合,和 6)重链恒定区和轻链恒定区各个结构域的二聚体。
本发明的Fc片段包括天然氨基酸序列和其序列衍生物(突变体)。氨基酸序列衍生物是由于一个或多个氨基酸残基的缺失、插入、非保守性或保守性取代或它们的联合而不同于天然氨基酸序列的序列。例如,在IgG Fc中,已知对于结合重要的、位于位点214 至238、297至299、318至322或327至331处的氨基酸残基可用作修饰的适当靶目标。此外,其它各种衍生物也是有可能的,包括其中能形成二硫键的区域被删除,或者天然Fc形式N末端的某些氨基酸残基被除去或在此添加了甲硫氨酸残基的衍生物。此外,为了去除效应器功能,缺失可以发生在补体结合位点,诸如C1q结合位点和ADCC位点。制备这样的免疫球蛋白Fc片段序列衍生物的技术参阅国际专利申请WO 97/34631和WO 96/32478。
蛋白质和肽中通常不改变该蛋白质或肽活性的氨基酸替换是本领域所已知的(H.Neurath,R.L.Hill,蛋白质The Proteins, Academic Press,New York,1979)。最通常发生的替换是Ala/Ser、 Val/Ile、Asp/Glu、Thr/Ser、Ala/Gly、Ala/Thr、Ser/Asn、Ala/Val、Ser/Gly、Thy/Phe、Ala/Pro、Lys/Arg、Asp/Asn、Leu/Ile、Leu/Val、 Ala/Glu和Asp/Gly,包括两种方向上的替换。
此外,如果需要,Fc片段可以通过磷酸化、硫酸化、丙烯酸化、糖基化、甲基化、法呢基化、乙酰化、酰胺化等等作用被修饰。
上述Fc衍生物是具有与本发明Fc片段同样的生物学活性或提高了结构稳定性(例如抗热、pH等等)的衍生物。
此外,这些Fc片段可获自从人和包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠在内的其它动物中分离的天然形式,或可以是重组体或它们的衍生物,获自被转化的动物细胞或微生物。在此,它们可通过从人或动物体中分离完整的免疫球蛋白并用蛋白水解酶加以处理而从天然免疫球蛋白获得。木瓜蛋白酶将天然的免疫球蛋白消化成Fab和Fc片段,而胃蛋白酶处理则导致了pF′c和F (ab′)2片段的产生。可对这些片段进行例如大小排阻层析,以分离 Fc或pF′c。
优选的,人源Fc片段是获自微生物的重组免疫球蛋白Fc片段。
此外,本发明的免疫球蛋白Fc片段可以是具有天然糖链、与天然形式相比糖链增加或与天然形式相比糖链减少的形式,或者可以是去糖基化的形式。免疫球蛋白Fc糖链的增加、减少或去除可以通过本领域的通用方法完成,诸如化学法、酶促法和利用微生物的遗传工程方法。将糖链从Fc片段中去除导致了它与第一补体成分 C1的C1q部分的结合亲和力明显降低和抗体依赖性细胞介导的细胞毒性(ADCC)或补体依赖性细胞毒性(CDC)的降低或丧失,从而不会在体内引起不必要的免疫应答。在这点上,去糖基化或未糖基化形式的免疫球蛋白Fc片段可能更适于本发明作为药物载体的目的。
用于此处时,术语“去糖基化”指从Fc片段中酶促除去了糖基部分,而术语“未糖基化”指Fc片段以非糖基化的形式由原核生物,优选大肠杆菌所产生。
另一方面,免疫球蛋白Fc片段可来自人类或其它动物,包括牛、山羊、猪、小鼠、兔子、仓鼠、大鼠和豚鼠,优选人类。此外,免疫球蛋白Fc片段可以是来自IgG、IgA、IgD、IgE和IgM的Fc 片段,或者通过它们的组合或杂合制备而来的Fc片段。优选Fc片段来自IgG或IgM,它们是人类血液中最丰富的蛋白质之一,最优选来自IgG(已知它能延长配体结合蛋白质的半寿期)。
另一方面,术语“组合”用于此处时是指编码同一来源的单链免疫球蛋白Fc片段的多肽与不同来源的单链多肽连接形成二聚体或多聚体。即,二聚体或多聚体可以由IgG1Fc、IgG2Fc、IgG3Fc和 IgG4Fc片段中的两种或多种片段所形成。
术语“杂合体”用于此处时指编码不同来源的两种或多种免疫球蛋白Fc片段的序列存在于单链免疫球蛋白Fc片段中。在本发明中,各种类型的杂合体都是有可能的。即,结构域杂合体可以由IgG1 Fc、IgG2Fc、IgG3Fc和IgG4Fc的CH1、CH2、CH3和CH4中的一种至四种结构域组成,且可能包含铰链区。
另一方面,IgG被划分为IgG1、IgG2、IgG3和IgG4亚型,本发明包括它们的组合和杂合体。优选IgG2和IgG4亚型,最优选 IgG4的Fc片段,它几乎不具有诸如CDC(补体依赖型细胞毒性) 等效应器功能(参阅图14和15)。
即,作为本发明的药物载体,最优选的免疫球蛋白Fc片段是人IgG4来源的非糖基化Fc片段。人源Fc片段比非人源Fc片段更为优选,它可能在人体内作为抗原起作用,并引起不期望有的免疫应答,诸如产生抗该抗原的新抗体。
如上所述制备的本发明的免疫球蛋白Fc片段可用作药物载体并与药物形成缀合物。
术语“药物缀合物”或“缀合物”用于此处时指一种或多种药物与一种或多种免疫球蛋白Fc片段连接。
术语“药物”用于此处时指在施用于人类或动物时展现出治疗活性的物质,例如,但不局限于:多肽、化合物、提取物和核酸。优选多肽药物。
术语“生理学活性多肽”、“生理学活性蛋白质”、“活性多肽”、“多肽药物”和“蛋白质药物”用于此处时在其意义上是可互换的,其特征在于,它们都处于展示各种体内生理学功能的生理学活性状态。
多肽药物具有不能长期保持生理学作用的缺点,这是由于其易被体内存在的蛋白水解酶变性或降解的特性造成。不过,当多肽药物与本发明的免疫球蛋白Fc片段相缀合而形成缀合物时,该药物的结构稳定性得以提高,降解半寿期也延长了。同样,与Fc片段缀合的多肽与其它已知的多肽药物制剂相比,其生理学活性的降低要小得多。因此,与常规多肽药物的体内生物利用率相比,依照本发明的多肽和免疫球蛋白Fc片段的缀合物的特征为具有显著提高的体内生物利用率。这一点也通过本发明的实施方案进行了清楚的阐述。即,与它们单独缀合PEG或缀合PEG和白蛋白二者的常规形式相比,当与本发明的免疫球蛋白Fc片段连接时,IFNα、G-CSF、hGH 和其它蛋白质药物呈现出体内生物利用率提高了大约2至6倍(表8、 9和10)。
另一方面,蛋白质和本发明免疫球蛋白Fc片段之间的连接的特征在于,它不是通过常规的重组方法形成的融合体。通过重组方法获得并用作药物的免疫球蛋白Fc片段与活性多肽的融合形式是以如下方式得到的,即,将多肽与Fc片段的N末端或C末端连接,并由此从编码该融合形式的核苷酸序列表达和折叠成单一多肽。
这导致了所产生的融合蛋白的活性显著降低,因为蛋白质作为生理学功能性物质的活性是由蛋白质的构象决定的。因此,当多肽药物通过重组方法与Fc融合时,即使融合蛋白的结构稳定性提高了,它对其体内生物利用率也没有影响。同样,由于这样的融合蛋白常常错误折叠并以包涵体形式表达,融合方法在蛋白质产生和分离产率方面是不经济的。此外,当多肽的活性形式是糖基化形式时,多肽应在真核细胞中表达。这种情况下,Fc也被糖基化,而此糖基化可能引起体内不适当的免疫应答。
也就是说,只有本发明有可能产生糖基化活性多肽和非糖基化免疫球蛋白Fc片段的缀合物,并克服所有上述问题,包括提高蛋白质产量,因为复合物的两种组分是通过最佳体系单独制备和分离的。
能与本发明的免疫球蛋白Fc片段缀合的蛋白质药物的非局限性例子包括人生长激素、生长激素释放激素、生长激素释放肽、干扰素和干扰素受体(例如干扰素-α、-β和-γ,水溶性I型干扰素受体,等)、粒细胞集落刺激因子(G-CSF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、胰高血糖素样肽(例如GLP-1等)、G-蛋白偶联受体、白介素(例如白介素-1、-2、-3、-4、-5、-6、-7、-8、-9、-10、-11、 -12、-13、-14、-15、-16、-17、-18、-19、-20-、21、-22、-23、-24、 -25、-26、-27、-28、-29、-30等)和白介素受体(例如IL-1受体、IL-4 受体等)、酶(例如葡糖脑苷酯酶、艾杜糖醛酸-2-硫酸酯酶、α-半乳糖苷酶-A、agalsidaseα和β、α-L-iduronidase、丁酰胆碱酯酶、几丁质酶、谷氨酸脱羧酶、imiglucerase、脂酶、尿酸酶、血小板活化因子乙酰水解酶、中性内肽酶、髓过氧化物酶、等)、白介素和细胞因子结合蛋白(例如IL-18bp、TNF结合蛋白,等)、巨噬细胞活化因子、巨噬细胞肽、B细胞因子、T细胞因子、蛋白A、变态反应抑制物、细胞坏死糖蛋白、免疫毒素、淋巴毒素、肿瘤坏死因子、肿瘤抑制物、转移生长因子、α-1抗胰蛋白酶、白蛋白、α-乳清蛋白、载脂蛋白-E、促红细胞生成素、高度糖基化的促红细胞生成素、 angiopoietins、血红蛋白、凝血酶、凝血酶受体激活肽、凝血调节蛋白、因子VII、因子VIIa、因子VIII、因子IX、因子XIII、纤溶酶原活化因子、纤维蛋白结合肽、尿激酶、链激酶、水蛭素、蛋白C、 C反应蛋白、肾素抑制物、胶原酶抑制物、超氧化物歧化酶、leptin、血小板衍生生长因子、上皮生长因子、表皮生长因子、制管张素、血管紧张素、骨生长因子、骨刺激蛋白质、降钙素、胰岛素、心房肽激素(atriopeptin)、软骨诱发因子、依降钙素(elcatonin)、结缔组织活化因子、组织因子途径抑制物、促卵泡激素、黄体生成素、黄体生成素释放激素、神经生长因子(如神经生长因子、睫状神经营养因子、axogenesis因子-1、脑促尿钠排泄肽、神经胶质来源的神经营养因子、netrin、neurophil抑制因子、神经营养因子、neuturin 等)、甲状旁腺激素、松弛素、促胰液素(secretin)、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、缩胆囊素、胰多肽、胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺素、自分泌运动因子(autotaxin)、乳铁蛋白、筒箭毒碱(myostatin)、受体(如TNFR(P75)、TNFR(P55)、IL-1受体、VEGF受体、B细胞活化因子受体,等)、受体拮抗剂(如IL1-Ra等)、细胞表面抗原(如 CD 2、3、4、5、7、11a、11b、18、19、20、23、25、33、38、40、 45、69等)、病毒疫苗抗原、单克隆抗体、多克隆抗体、抗体片段(如 scFv、Fab、Fab′、F(ab′)2和Fd)和病毒衍生疫苗抗原。抗体片段可以是Fab、Fab′、F(ab′)2、Fd或scFv,它能结合特异的抗原,优选Fab′。Fab片段包含轻链的可变区(VL)和恒定区(CL)以及重链的可变区(VH)和第一恒定区(CH1)。Fab′片段不同于Fab片段,它在CH1 结构域的羧基末端添加了包括来自铰链区的一个或多个半胱氨酸残基在内的数个氨基酸残基。Fd片段只包含VH和CH1结构域,而F(ab′) 2片段则作为一对Fab′片段通过二硫键或化学反应产生。scFv(单链 Fv)片段包含通过肽连接物相互连接并因此存在于单一多肽链内的 VL和VH结构域。
具体而言,优选作为生理学活性多肽的是那些施用于个体以治疗或预防疾病时需频繁给药的多肽,它们包括人生长激素、干扰素(干扰素-α、-β、-γ等)、粒细胞集落刺激因子、促红细胞生成素(EPO) 和抗体片段。此外,某些衍生物也包括在本发明生理学活性多肽的范围内,只要与天然形式的生理学活性多肽相比,它们具有基本上相同的或更优越的功能、结构、活性或稳定性即可。在本发明中,最优选的多肽药物是干扰素-α。
除了多肽药物之外,其它药物也在本发明中可供利用。这些药物的非局限性例子包括抗生素,选自以下抗生素的衍生物和混合物:四环素、二甲胺四环素、强力霉素、氧氟沙星、左氧氟沙星、环丙沙星、克拉化霉素、红霉素、氯头孢菌素、氨噻肟头孢菌素、亚胺培南、青霉素、庆大霉素、链霉素、万古霉素等;选自以下抗癌剂的衍生物和混合物的抗癌剂:氨甲喋呤、碳铂、紫杉酚、顺氯氨铂、 5-氟尿嘧啶、阿霉素、依托泊甙、紫杉醇、camtotecin、阿糖胞苷等;抗炎剂,选自以下试剂的衍生物和混合物:消炎痛、布洛芬、酮洛芬、吡氧噻嗪、probiprofen、双氯芬酸等;抗病毒试剂,选自无环鸟苷和robavin的衍生物和混合物;以及抗菌剂,选自以下试剂的衍生物和混合物:酮康唑、依曲康唑、氟康唑、两性霉素B和灰黄霉素。
另一方面,本发明的免疫球蛋白Fc片段能形成通过接头与药物连接的缀合物。
这样的接头包括肽和非肽接头。优选的是非肽接头,更优选的是非肽聚合物。
术语“肽接头”用于此处时指氨基酸,优选1至20个氨基酸,它们通过肽键相互之间线性连接,且可以是糖基化形式的。就本发明的目的而言,优选非糖基化形式的。这样的肽接头优选具有甘氨酸和丝氨酸重复单元的肽,它对于T细胞无免疫学活性。
术语“非肽聚合物”用于此处时指包含通过除肽键之外的共价键彼此连接的两个或多个重复单元的生物相容性聚合物。非肽聚合物的例子包括聚乙二醇、聚丙二醇、乙二醇和丙二醇的共聚物、聚氧乙基化多元醇、聚乙烯醇、多糖、葡聚糖、聚乙烯醚、诸如PLA(聚乳酸)和PLGA(聚乳酸乙醇酸)等生物可降解性聚合物、脂类聚合物、几丁质和透明质酸。最优选的是聚乙二醇(PEG)。
本发明的缀合物、免疫球蛋白Fc片段-药物或免疫球蛋白Fc 片段-接头-药物以多种摩尔比率制备。即,与单一多肽药物连接的免疫球蛋白Fc片段和/或接头的量是不受限制的。不过,优选的,在本发明的药物缀合物中,药物与免疫球蛋白Fc片段以摩尔比率1:1至10:1相互缀合,优选1:1至2:1。
此外,本发明免疫球蛋白Fc片段、某一接头和某一药物的连接包括除了Fc片段和多肽药物通过遗传重组以融合蛋白形式表达时形成的肽键之外的所有共价键,以及所有类型的非共价键,诸如氢键、离子相互作用、范德华力和疏水相互作用。不过,就药物的生理学活性而言,连接优选通过共价键形成。
此外,本发明的免疫球蛋白Fc片段、某一接头和某一药物可在药物的某一位点相互连接。另一方面,本发明的免疫球蛋白Fc片段和多肽药物可彼此在N末端或C末端连接,优选在游离基团处连接,免疫球蛋白Fc片段和药物之间的共价键尤其容易在氨基末端、赖氨酸残基的氨基、组氨酸残基的氨基或游离的半胱氨酸残基处形成。
另一方面,本发明的免疫球蛋白Fc片段、某一接头和某一药物的连接可能以确定的方向进行。即,接头可连接于免疫球蛋白Fc 片段的N末端、C末端或游离基团,也可连接于蛋白质药物的N末端、C末端或游离基团。当接头是肽接头时,连接可能发生在某一连接位点处。
此外,本发明的缀合物可以用本领域已知的许多偶联剂中的任一种制备。偶联剂的非局限性的例子包括1,1-二(叠氮乙酰基)-2-苯乙烷、戊二醛、N-羟基琥珀酰亚胺酯,诸如与4-叠氮基水杨酸的酯、包括诸如3,3′-二硫代双(琥珀酰亚胺基丙酸酯)等双琥珀酰亚胺酯在内的亚胺酯和诸如双-N-马来酰亚胺-1,8-辛烷等双功能性马来酰亚胺类。
另一方面,包含免疫球蛋白Fc片段作为载体的本发明药物组合物可通过多种途径施用。
术语“施用”用于此处时指通过某一合适方法将预定量的物质引入患者中。本发明的缀合物可通过任何常规途径施用,只要它能到达预期组织即可。各种施用方式都在考虑范围内,包括腹膜内、静脉内、肌肉内、皮下、皮内、口服、局部、鼻内、肺内和直肠内施用,但本发明并不局限于这些例证性的施用方式。不过,由于在口服给药时肽会被消化,口服给药组合物的活性成分应被包裹或特殊配制以保护其在胃中免于降解。优选的,本发明组合物可能以可注射的形式施用。此外,本发明的药物组合物可用能将活性成分转运入靶细胞的设备施用。
含有依照本发明的缀合物的药物组合物可包含药物可接受载体。对口服给药而言,药物可接受载体可包括粘合剂、润滑剂、崩解剂、赋形剂、增溶剂、分散剂、稳定剂、悬浮剂、着色剂和芳香剂。对可注射制剂而言,药物可接受载体可包括缓冲剂、保存剂(preserving agent)、止痛剂、增溶剂、等渗剂和稳定剂。对于局部施用的制剂而言,药物可接受载体可包括基质、赋形剂、润滑剂和保存剂。本发明的药物组合物可与前述药物可接受载体组合配制成各种剂型。例如,就口服而言,药物组合物可以配制成片剂、锭剂、胶囊、酏剂、悬浮剂、糖浆或干胶片。就可注射的制剂而言,药物组合物可配制成单位剂型,诸如多剂量容器或单剂量形式的安瓿。药物组合物也可配制成溶液、悬浮液、片剂、胶囊和长效制剂。
另一方面,适于药物制剂的载体、赋形剂和稀释液的例子包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯树胶、藻酸盐(酯)、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、微晶纤维素、聚乙烯吡咯烷酮、水、甲基羟基苯甲酸盐(酯)、丙基羟基苯甲酸盐(酯)、滑石、硬脂酸镁和矿物油。此外,药物组合物可进一步包含填充剂、抗凝剂、润滑剂、保湿剂、芳香剂、乳化剂和防腐剂。
与作为载体的本发明免疫球蛋白Fc片段组合的药物真正剂量可能由数种相关因素决定,包括待治疗疾病的类型、施用途径、患者年龄、性别、体重和病情的严重程度以及作为活性组分的药物的类型。由于本发明的药物组合物在体内具有很长的作用持续时间,所以它具有极大的降低治疗药物施用频率的优势。
在另一方面,本发明的另一目的是提供通过包含免疫球蛋白Fc 片段而延长了药物的体内持续时间的方法。
在本发明的另一实施方案中,生理学活性多肽-PEG-免疫球蛋白 Fc片段缀合物具有比多肽-PEG复合物或多肽-PEG-白蛋白缀合物高得多的稳定性。药物代谢动力学分析显示,与天然IFNα相比,当与40-kDa PEG连接时(IFNα-40K PEG复合物),IFNα的血清半寿期提高了大约20倍,而在IFNα-PEG-白蛋白缀合物中则提高了大约10倍。相反,依照本发明的IFNα-PEG-Fc缀合物显示出半寿期显著增加大约50倍(见表3)。此外,同样的结果可在其它靶蛋白、人生长激素(hGH)、粒细胞集落刺激因子(G-CSF)和其衍生物(17S-G-CSF) 以及促红细胞生成素(EPO)中观察到。与蛋白质的天然形式以及缀合了PEG或PEG-白蛋白的形式相比,依照本发明的蛋白质缀合物(其中各包含与PEG-Fc连接的靶蛋白)显示出平均停留时间(mean residence time,MRT)和血清半寿期延长了大约10倍(见表4至7)。
此外,当PEG-Fc复合物与接近Fab′C末端或Fab′的N末端的-SH基团连接时,产生的Fab’-PEG-Fc缀合物呈现出比40K PEG-Fab’复合物长2到3倍的血清半寿期(见图12)。
此外,当蛋白质缀合物是用去糖基化免疫球蛋白Fc(DG Fc)(其中糖基部分已被去除)和重组的非糖基化免疫球蛋白Fc(AG Fc)衍生物制备的时候,它们的血浆半寿期和体外活性维持在与用天然Fc 制备的蛋白质缀合物相似的水平(见表3和图8和11)。
因此,由于本发明的蛋白质缀合物具有延长的血清半寿期和平均停留时间(MRT),当它们被应用于包括人生长激素、干扰素、促红细胞生产素、集落刺激因子或其衍生物和抗体衍生物在内的各种生理学活性多肽时,它们可用于开发多种生理学活性多肽的长效制剂。
从以下实施例中可以更好的理解本发明,这些实施例的提出只是为了举例说明,而不应被认为是对本发明的限制。
实施例1:IFNα-PEG-免疫球蛋白Fc片段缀合物的制备I
<步骤1>用免疫球蛋白制备免疫球蛋白Fc片段
免疫球蛋白Fc片段制备如下。将溶于10mM磷酸盐缓冲液中的200mg 150-kDa免疫球蛋白G(IgG)(Green Cross,Korea)用2mg 蛋白水解酶——木瓜蛋白酶(Sigma)在37℃温和搅拌处理2小时。酶反应后,对由此再生的免疫球蛋白Fc片段依次用Superdex柱、蛋白A柱和阳离子交换柱进行层析纯化。具体的说,就是将反应液加到用10mM磷酸钠缓冲液(PBS,pH7.3)平衡过的Superdex 200柱 (Pharmacia)上,将柱子用同一缓冲液以流速1ml/分钟洗脱。与免疫球蛋白Fc片段相比具有相对较高分子量的未反应免疫球蛋白分子 (IgG)和F(ab′)2由于其洗脱早于Ig Fc片段的特性而被除去。分子量近似于Ig Fc片段的Fab片段通过蛋白A柱层析除去(图1)。将洗脱自Superdex 200柱的含Ig Fc片段的所得级分以5ml/分钟的流速加到用20mM磷酸盐缓冲液(pH 7.0)平衡的蛋白A柱(Pharmacia) 上,用同一缓冲液洗柱,以去除未结合在柱上的蛋白质。然后,用 100mM柠檬酸钠缓冲液(pH 3.0)洗脱蛋白A柱,以获得高纯度的免疫球蛋白Fc片段。最后用阳离子交换柱(polyCAT,PolyLC Company)纯化从蛋白A柱收集的Fc级分,其中上样了Fc级分的该柱子用10 mM乙酸盐缓冲液(pH 4.5)中的0.15-0.4M NaCl线性梯度洗脱,从而提供了高纯度的Fc级分。用12%SDS-PAGE分析该高纯度的Fc 级分(图2中的泳道2)。
<步骤2>IFNα-PEG复合物的制备
将两端具有醛反应性基团的3.4-kDa聚乙二醇ALD-PEG-ALD (Shearwater)与以5mg/ml的量溶于100mM磷酸盐缓冲液中的人干扰素α-2b(hIFNα-2b,MW:20kDa)以IFNα:PEG摩尔比率1:1、 1:2.5、1:5、1:10和1:20混合。向此混合物中加入终浓度20mM的还原剂氰基硼氢化钠(NaCNBH3,Sigma)并在温和搅拌下在4℃反应 3小时,使PEG与干扰素α的氨基末端连接。为了得到PEG和干扰素α的1:1复合物,将反应混合物用SuperdexR柱(Pharmacia)进行大小排阻层析。用10mM磷酸钾缓冲液(pH 6.0)作为洗脱缓冲液将 IFNα-PEG复合物从柱上洗脱下来,而未与PEG连接的干扰素α、未反应的PEG以及PEG与两个干扰素α分子连接的二聚体副产物则被去除。将纯化的IFNα-PEG复合物浓缩至5mg/ml。通过此实验,发现具有最高反应性且产生最小量诸如二聚体等副产物的 IFNα:PEG最佳反应摩尔比率是1:2.5至1:5。
<步骤3>IFNα-PEG-Fc缀合物的制备
为了将上述步骤2中纯化的IFNα-PEG复合物与免疫球蛋白 Fc片段的N末端连接,将以上步骤1中制备的免疫球蛋白Fc片段 (约53kDa)溶于10mM磷酸盐缓冲液中并以IFNα-PEG复合物:Fc 的摩尔比率1:1、1:2、1:4和1:8与IFNα-PEG复合物混合。在将反应溶液的磷酸盐缓冲液浓度调节到100mM之后,将还原剂 NaCNBH3加入反应液中,终浓度20mM,在温和搅拌下于4℃反应 20小时。通过此实验,发现具有最高反应性且产生最少量的诸如二聚体等副产物的IFNα-PEG复合物:Fc的最佳反应摩尔比率是1:2。
<步骤4>IFNα-PEG-Fc缀合物的分离和纯化
在以上步骤3的反应之后,对反应混合物进行Superdex大小排阻层析,以去除未反应的物质和副产物,并纯化所产生的 IFNα-PEG-Fc蛋白缀合物。将反应混合物浓缩并加到Superdex柱上之后,使10mM磷酸盐缓冲液(pH 7.3)以2.5ml/分钟的流速通过柱子,以去除未结合的Fc和未反应的物质,随后进行柱子洗脱,收集 IFNα-PEG-Fc蛋白质缀合物部分。由于所收集到的IFNα-PEG-Fc 蛋白质缀合物级分包含小量的杂质、未反应的Fc和干扰素α二聚体,进行阳离子交换层析以去除杂质。将IFNα-PEG-Fc蛋白质缀合物级分加到用10mM乙酸钠(pH 4.5)平衡的PolyCAT LP柱(PolyLC)上,并用1M NaCl在10mM乙酸钠缓冲液(pH 4.5)中的0-0.5M NaCl 线性梯度洗脱柱子。最后,用阴离子交换柱纯化IFNα-PEG-Fc蛋白质缀合物。将IFNα-PEG-Fc蛋白质缀合物部分加到用10mM Tris-HCl(pH 7.5)平衡的PolyWAX LP柱(PolyLC)上,然后用1M NaCl在10mM Tris-HCl(pH 7.5)中的0-0.3M NaCl线性梯度洗脱柱子,从而分离高纯度形式的IFNα-PEG-Fc蛋白质缀合物。
实施例2:IFNα-PEG-Fc蛋白质缀合物的制备II
<步骤1>Fc-PEG复合物的制备
将两端具有醛反应性基团的3.4-kDa聚乙二醇ALD-PEG-ALD (Shearwater)与以15mg/ml的量溶于100mM磷酸盐缓冲液中的实施例1步骤1中所制备的免疫球蛋白Fc片段按Fc:PEG摩尔比率1:1、 1:2.5、1:5、1:10和1:20混合。在此混合物中加入终浓度20mM的还原剂氰基硼氢化钠(NaCNBH3,Sigma),并在温和搅拌下使之于 4℃反应3小时。为了得到PEG和Fc的1:1复合物,将反应混合物用SuperdexR柱(Pharmacia)进行大小排阻层析。用10mM磷酸钾缓冲液(pH 6.0)作为洗脱液将Fc-PEG复合物从柱上洗脱下来,而未与 PEG连接的免疫球蛋白Fc片段、未反应的PEG以及PEG与两个免疫球蛋白Fc片段分子连接的二聚体副产物则被去除。将纯化的 Fc-PEG复合物浓缩至大约15mg/ml。通过此实验,发现具有最高反应性且产生最小量诸如二聚体等副产物的Fc:PEG最佳反应摩尔比率是1:3至1:10。
<步骤2>Fc-PEG复合体和干扰素α的缀合物的形成和纯化
为了将上述步骤1中纯化的Fc-PEG复合物与IFNα的N末端连接,将Fc-PEG复合物与溶于10mM磷酸盐缓冲液的IFNα以 Fc-PEG复合物:IFNα的摩尔比率1:1、1:1.5、1:3和1:6混合。在将反应溶液中的磷酸盐缓冲液浓度调节到100mM之后,将还原剂 NaCNBH3加入反应液中,终浓度20mM,使之在温和搅拌下于4℃反应20小时。反应完成后,依照实施例1步骤4中同样的纯化方法将未反应的物质和副产物去除,从而分离高纯度形式的 Fc-PEG-IFNα蛋白质缀合物。
实施例3:hGH-PEG-Fc缀合物的制备
依照与实施例1相同的方法制备和纯化hGH-PEG-Fc缀合物,不同之处在于使用除干扰素α之外的药物-人生长激素(hGH,MW: 22kDa),并且hGH:PEG的摩尔比率是1:5。
实施例4:(G-CSF)-PEG-Fc缀合物的制备
依照与实施例1相同的方法制备和纯化(G-CSF)-PEG-Fc缀合物,不同之处在于使用干扰素α之外的药物-人粒细胞集落刺激因子(G-CSF),并且G-CSF:PEG的摩尔比率是1:5。
另一方面,利用在天然G-CSF的第17位氨基酸残基处具有丝氨酸取代的G-CSF衍生物17S-G-CSF,依照上述相同的方法制备和纯化17S-G-CSF-PEG-Fc蛋白质缀合物。
实施例5:EPO-PEG-Fc缀合物的制备
依照与实施例1相同的方法制备和纯化EPO-PEG-Fc缀合物,不同之处在于使用了干扰素α之外的药物-人促红细胞生成素 (EPO),并且EPO:PEG的摩尔比率是1:5。
实施例6:用具有不同反应性基团的PEG制备蛋白质缀合物
用两端具有丙酸琥珀酰亚胺酯(SPA)反应性基团的PEG制备 IFNα-PEG-Fc蛋白质缀合物,步骤如下。将3.4-kDa聚乙二醇 SPA-PEG-SPA(Shearwater)与溶于100mM磷酸盐缓冲液中的 10mg干扰素α以IFNα:PEG摩尔比率1:1、1:2.5、1:5、1:10和1:20 混合。然后使混合物在温和搅拌下于室温反应2小时。为了获得PEG 和干扰素α的1:1复合物(IFNα-PEG复合物)(其中PEG选择性的与干扰素α的赖氨酸残基的氨基连接),对反应混合物进行Superdex大小排阻层析。用10mM磷酸钾缓冲液(pH 6.0)作为洗脱缓冲液从柱上洗脱IFNα-PEG复合物,未连接PEG的干扰素α、未反应的PEG 以及其中两个干扰素α分子与PEG两端连接的二聚体副产物被去除。为了将IFNα-PEG复合物与免疫球蛋白Fc的赖氨酸残基的氨基连接,将纯化的IFNα-PEG复合物浓缩至约5mg/ml,并依照实施例 1步骤3和4中相同的方法制备和纯化IFNα-PEG-Fc缀合物。通过此实验,发现具有最高反应性且产生最少量诸如二聚体等副产物的最佳IFNα:PEG反应摩尔比例是1:2.5至1:5。
另一方面,依照上述相同方法,利用在两端都具有N-羟基琥珀酰亚胺(NHS)反应性基团的PEG即NHS-PEG-NHS(Shearwater) 或两端都具有丁醛反应性基团的PEG即BUA-PEG-BUA (Shearwater)制备另一种IFNα-PEG-Fc缀合物。
实施例7:用具有不同分子量的PEG制备蛋白质缀合物
用两端具有醛反应性基团的10-kDa聚乙二醇即 ALD-PEG-ALD(Shearwater)制备IFNα-10K PEG复合物。依照实施例1步骤2中相同的方法制备和纯化此复合物。通过此实验,发现具有最高反应性且产生最少量诸如二聚体等副产物的最佳 IFNα:10-kDa PEG反应摩尔比率是1:2.5至1:5。将纯化的IFNα-10K PEG复合物浓缩至大约5mg/ml,并用此浓度依照实施例1步骤3 和4中相同的方法制备和纯化IFNα-10K PEG-Fc缀合物。
实施例8:Fab’-S-PEG-N-Fc缀合物(-SH基团)的制备
<步骤1>Fab’的表达和纯化
将表达抗肿瘤坏死因子αFab’的大肠杆菌转化株 BL21/poDLHF(保藏号:KCCM-10511)在100ml LB培养基中振荡培养过夜,接种于5-L的发酵罐(Marubishi)中,并在30℃以500rpm 转数和20vvm空气流速进行培养。为了补充发酵期间细菌生长所需的不足营养物,根据细菌的发酵状态在培养物中补充葡萄糖和酵母提取物。当培养物达到OD600值为80-100时,在培养物中加入诱导剂IPTG以诱导蛋白质的表达。将培养物进一步培养40到45小时,直至600nm的OD值提高到120至140。将由此获得的发酵液以 20,000×g离心30分钟。收集上清,弃沉淀。
对上清液进行以下三步柱层析以纯化抗肿瘤坏死因子αFab’。将上清液加到用20mM磷酸盐缓冲液(pH 7.0)平衡的HiTrap蛋白G 柱(5ml,Pharmacia)上,并用100mM甘氨酸(pH 3.0)洗脱柱子。然后将收集到的Fab’级分加到用10mM磷酸钠缓冲液(PBS,pH 7.3)平衡的Superdex 200柱(Pharmacia)上,并用相同的缓冲液洗脱此柱子。最后,将第二步的Fab’级分加到polyCAT 21×250柱(PolyLC) 上,并用10mM乙酸盐缓冲液(pH 4.5)中的0.15-0.4M NaCl线性梯度洗脱柱子,从而提供高纯度的抗肿瘤坏死因子αFab’级分。
<步骤2>Fc-PEG复合物的制备和纯化
为了将PEG接头连接到免疫球蛋白Fc的N-末端,将依照实施例1步骤1中同一种方法制备的免疫球蛋白Fc溶于100mM磷酸盐缓冲液(pH 6.0)中,终浓度5mg/ml,并以Fc:PEG摩尔比率1:10 与NHS-PEG-MAL(3.4kDa,Shearwater)混合,随后在温和搅拌下于 4℃保温12小时。
反应完成后,用20mM磷酸钠缓冲液(pH 6.0)置换反应缓冲液以去除未结合的NHS-PEG-MAL。然后,将反应混合物加到polyCAT 柱(PolyLC)上。用20mM磷酸钠缓冲液(pH 6.0)中的0.15-0.5M线性NaCl梯度洗脱柱子。在洗脱过程中,免疫球蛋白Fc-PEG复合物早于未反应的免疫球蛋白Fc而洗脱下来,而未反应的Ig Fc稍后被洗脱,从而除去了未反应的Ig Fc分子。
<步骤3>Fab’-S-PEG-N-Fc缀合物(-SH基团)的制备和纯化
为了将免疫球蛋白Fc-PEG复合物连接至Fab’的半胱氨酸基团处,将上述步骤1中纯化的Fab’以2mg/ml的浓度溶于100mM磷酸钠缓冲液(pH 7.3)中,并与上述步骤2中制备的免疫球蛋白Fc-PEG 复合物以Fab’:复合物摩尔比例1:5混合。将反应混合物浓缩至终浓度50mg/ml,并在温和搅拌下于4℃保温24小时。
反应完成后,将反应混合物加到用10mM磷酸钠缓冲液(pH 7.3)平衡的Superdex200柱(Pharmacia)上,并用同样的缓冲液以1 ml/分钟的流速洗脱柱子。被偶联的Fab’-S-PEG-N-Fc缀合物由于其高分子量而相对较早地洗脱下来,而未反应的免疫球蛋白Fc-PEG复合物和Fab’则在稍后被洗脱下来,从而除去了未反应的分子。为了完全除去未反应的免疫球蛋白Fc-PEG,将收集的 Fab’-S-PEG-N-Fc缀合物级分再加到polyCAT 21×250柱(PolyLC) 上,并用20mM磷酸钠缓冲液(pH 6.0)中的0.15-0.5M线性NaCl 梯度洗脱柱子,由此得到含有与接近Fab’的C末端的-SH基团连接的Fc-PEG复合物的纯Fab’-S-PEG-N-Fc缀合物。
实施例9:Fab’-N-PEG-N-Fc缀合物(N-末端)的制备
<步骤1>Fab’-PEG复合物(N-末端)的制备和纯化
将实施例8步骤1中纯化的40mg Fab’以5mg/ml的浓度溶于 100mM磷酸钠缓冲液(pH 6.0)中,以Fab’:PEG摩尔比率1:5与丁基ALD-PEG-丁基ALD(3.4kDa,Nektar)混合。将还原剂NaCNBH3加到反应混合物中,终浓度20mM,然后在温和搅拌下使反应混合物在4℃反应2小时。
在反应完成后,用20mM磷酸钠缓冲液(pH 6.0)置换反应缓冲液。然后,将反应混合物加到polyCAT柱(PolyLC)上。用20mM乙酸盐缓冲液(pH 4.5)中的0.15-0.4M线性NaCl梯度洗脱柱子。在柱洗脱过程中,含有与Fab’的N末端连接的PEG的Fab’-PEG复合物早于未反应的Fab’被洗脱下来,而未反应的Fab’稍后被洗脱下来,从而除去了未反应的Fab’分子。
<步骤2>Fab’-N-PEG-N-Fc缀合物的制备和纯化
为了将以上步骤1中纯化的Fab’-PEG复合物与免疫球蛋白Fc 的N末端连接,将Fab’-PEG复合物以10mg/ml的浓度溶于100mM 磷酸钠缓冲液(pH 6.0)中,并与溶于相同缓冲液中的免疫球蛋白Fc 以Fab’-PEG复合物:Fc摩尔比率1:5混合。在反应混合物浓缩至蛋白质终浓度50mg/ml后,将还原剂NaCNBH3加入反应混合物中,终浓度为20mM,然后在温和搅拌下使反应混合物在4℃反应24 小时。
反应完成后,将反应混合物加到用10mM磷酸钠缓冲液(pH 7.3)平衡的Superdex200柱(Pharmacia)上,并用相同缓冲液以1ml/ 分钟的流速洗脱柱子。被偶联的Fab’-N-PEG-N-Fc缀合物由于其高分子量而被相对较早地洗脱下来,而未反应的免疫球蛋白Fc和Fab’-PEG复合物则在稍后被洗脱下来,从而除去了未反应的分子。为了完全除去未反应的免疫球蛋白Fc分子,将收集的 Fab’-N-PEG-N-Fc缀合物级分再加到polyCAT 21×250柱(PolyLC) 上,并用20mM磷酸钠缓冲液(pH 6.0)中的0.15-0.5M线性NaCl 梯度洗脱柱子,由此得到含有与Fab’的N末端连接的免疫球蛋白Fc -PEG复合物的纯Fab’-N-PEG-N-Fc缀合物。
实施例10:去糖基化免疫球蛋白Fc的制备和纯化
将根据实施例1中的相同方法制备的200mg免疫球蛋白Fc 以浓度2mg/ml溶于100mM磷酸盐缓冲液(pH 7.5)中,并与300U/mg 去糖基化酶PNGase F(NEB)混合。使反应混合物在温和搅拌下于 37℃反应24小时。然后,为了纯化去糖基化的免疫球蛋白Fc,将反应混合物加到SP Sepharose FF柱(Pharmacia)上,然后用1M NaCl在10mM乙酸盐缓冲液(pH4.5)中的0.1-0.6M线性NaCl梯度洗脱柱子。天然免疫球蛋白Fc较早地洗脱下来,而去糖基化免疫球蛋白Fc(DG Fc)则稍后被洗脱下来。
实施例11:IFNα-PEG-DG Fc缀合物的制备
为了将实施例10中制备的去糖基化免疫球蛋白Fc与实施例1 步骤2中纯化的IFNα-PEG复合物连接,将IFNα-PEG复合物与溶于10mM磷酸盐缓冲液中的DG Fc以IFNα-PEG复合物:DG Fc摩尔比例1:1、1:2、1:4和1:8混合。在将反应溶液的磷酸盐缓冲液浓度调节到100mM后,将还原剂NaCNBH3以20mM的终浓度加到反应溶液中,并在温和搅拌下使其在4℃反应20小时。通过此实验,发现具有最高反应性且产生最少量诸如二聚体等副产物的最佳反应摩尔比例IFNα-PEG复合物:DG Fc是1:2。
在偶联反应后,将反应混合物用SuperdexR柱(Pharmacia)进行大小排阻层析,以去除未反应的物质和副产物,并纯化 IFNα-PEG-DG Fc蛋白质缀合物。在将反应混合物加到柱子上后,使磷酸盐缓冲液(pH 7.3)以2.5ml/分钟的流速通过柱子,以去除未结合的DG Fc和未反应的物质,随后柱洗脱,以收集IFNα-PEG-DG Fc 蛋白质缀合物级分。由于所收集到的IFNα-PEG-DG Fc蛋白质缀合物级分包含小量的杂质、未反应的DG Fc和IFNα-PEG复合物,进行阳离子交换层析以去除杂质。将IFNα-PEG-DG Fc蛋白质缀合物级分加到用10mM乙酸钠(pH 4.5)平衡的PolyCAT LP柱(PolyLC) 上,并用1M NaCl在10mM乙酸钠缓冲液(pH4.5)中的0-0.6M NaCl 线性梯度洗脱柱子。最后,用阴离子交换柱纯化IFNα-PEG-DG Fc 蛋白质缀合物。将IFNα-PEG-Fc蛋白质缀合物级分加到用10mM Tris-HCl(pH 7.5)平衡的PolyWAX LP柱(PolyLC)上,然后用1M NaCl在10mM Tris-HCl(pH 7.5)中的0-0.3M NaCl线性梯度洗脱柱子,从而分离高纯度形式的IFNα-PEG-DG Fc蛋白质缀合物。
实施例12:重组非糖基化免疫球蛋白Fc衍生物的制备和纯化
<IgG4 Fc衍生物1表达载体的制备>
为了制备人免疫球蛋白IgG4重链恒定区,先制备了在天然铰链区氨基末端具有9个氨基酸缺失的第一种衍生物(IgG4delta-Cys) 和由于铰链区所有12个氨基酸都被缺失而缺少铰链区的第二种衍生物(IgG4单体)。利用了本发明的发明者在本发明前研发的含大肠杆菌分泌序列的表达载体pT14S1SH-4T20V22Q(Korean Pat.No. 38061)。
为了得到人免疫球蛋白IgG4重链恒定区,用分离自人血液细胞的RNA作为模板进行RT-PCR,步骤如下。首先,用Qiamp RNA 血液试剂盒(Qiagen)从大约6ml血液中分离总RNA,以此总RNA为模板用One-Step RT-PCR试剂盒(Qiagen)进行基因扩增。在此PCR 中,使用了SEQ ID Nos.1和2代表的一对合成引物以及SEQ ID Nos. 2和3代表的另一对合成引物。SEQ ID NO.1是起始自IgG4铰链区如下12个氨基酸残基中的第10个残基丝氨酸的核苷酸序列。SEQ ID NO.3被设计成编码以丙氨酸作为第一个氨基酸残基的CH2结构域的核苷酸序列。SEQ ID NO.2被设计成具有含终止密码的BamHI 识别位点。
为了将各个已扩增的IgG4恒定区片段克隆入含大肠杆菌分泌序列衍生物的表达载体中,利用了本发明的发明者在本发明之前研发的pT14S1SH-4T20V22Q(KoreanPat.No.38061)。此表达载体包含具有SEQ ID NO.4所示核苷酸序列的热稳定性肠毒素分泌序列衍生物。为了便于克隆,用SEQ ID Nos.5和6表示的一对引物,通过定位诱变将StuI识别位点插入pT14S1SH-4T20V22Q质粒的大肠杆菌热稳定性肠毒素分泌序列衍生物的一端,以引发诱变,从而在编码分泌序列最后一个氨基酸残基的核苷酸序列处引入成功插入了StuI位点。通过DNA测序发现成功插入了StuI位点。所产生的含StuI位点的pT14S1SH-4T20V22Q质粒被称为pmSTII。用StuI和 BamHI处理pmSTII质粒并进行琼脂糖凝胶电泳,纯化含大肠杆菌热稳定性肠毒素分泌序列衍生物的大片段(4.7kb)。然后,用BamHI 消化已扩增的基因片段,并与线性化的表达载体连接,由此产生了 pSTIIdCG4Fc和pSTIIG4Mo。
将最终的表达载体单独转化入大肠杆菌BL21(DE3)中,所产生的转化体被称为“BL21/pSTIIdCG4Fc(HM10932)”和“BL21/pSTIIdCG4Mo(HM10933)”,它们在2004年9月15日保藏于韩国微生物保藏中心(KCCM),指定的保藏号分别是KCCM-10597 和KCCM-10598。其后,当培养物达到OD600值为80时,将诱导剂 IPTG加到培养物中以诱导蛋白质表达。将培养物进一步培养40到 45小时,直至600nm的OD值增加到100至120。破碎自发酵液收集的大肠杆菌细胞,对产生的细胞裂解物进行两步柱层析,以纯化存在于大肠杆菌胞质中的重组免疫球蛋白恒定区衍生物。
用PBS平衡5ml蛋白-A亲和柱(Pharmacia),将细胞裂解物以5ml/分钟的流速加到柱子上。未被结合的蛋白质用PBS洗出,而已结合的蛋白质则用100mM柠檬酸盐(pH 3.0)洗脱。对收集到的级分通过HiPrep 26/10脱盐柱(Pharmacia)用10mM Tris缓冲液(pH 8.0)脱盐。然后,用50ml Q HP 26/10柱(Pharmacia)进行二次阴离子交换柱层析。将初步纯化的重组非糖基化免疫球蛋白Fc级分加到 Q-Sepharose HP 26/10柱上,用10mM Tris缓冲液(pH8.0)中的0-0.2 M NaCl线性梯度洗脱柱子,从而得到高纯度的重组非糖基化免疫球蛋白Fc(AG Fc)衍生物、IgG4delta-Cys和高纯度的IgG4单体级分。
实施例13:IFNα-PEG复合物和重组AG Fc衍生物的缀合体的制备
依照与实施例1和11中相同的方法,将IFNα-PEG复合物与实施例12中作为AG Fc衍生物制备的IgG4delta-Cys的N末端连接。偶联反应后,将未反应的物质和副产物从反应混合物中去除,由此产生的IFNα-PEG-AG Fc蛋白质缀合物(I)首先用50ml Q HP 26/10柱(Pharmacia)纯化,然后进一步用polyCAT 21.5×250柱 (polyLC)通过高压液相层析试验进行纯化,由此高度纯化所述缀合物。用10mM Tris缓冲液(pH 8.0)通过HiPrep 26/10脱盐柱 (Pharmacia)对偶联反应溶液进行脱盐。然后,将反应溶液以流速8ml/ 分钟加到50ml QHP 26/10柱(Pharmacia)上,并用0-0.2M的线性 NaCl梯度洗脱此柱子,以获得预期的级分。将收集到的级分以流速 15ml/分钟再加到用10mM乙酸盐缓冲液(pH 5.2)平衡的polyCAT21.5×250柱上,用0.1-0.3M的线性NaCl梯度洗脱此柱子,由此提供高度纯化的级分。依照与上述相同的方法,用实施例12中制备的另一种AG Fc衍生物IgG4单体制备另一种IFNα-PEG-AG Fc蛋白质缀合物(II)。
实施例14:EPO-PEG-重组AG Fc衍生物缀合体的制备
依照与实施例13中相同的方法,通过将AG Fc衍生物IgG4 delta-Cys与EPO-PEG复合物连接而制备EPO-PEG-重组AG Fc衍生物缀合体。
比较实施例1:IFNα-40K PEG复合物的制备
将5mg干扰素α溶于100mM磷酸盐缓冲液中,达到终体积5ml,并与40-kDa活化的甲氧基-PEG-醛(Shearwater)混合, IFNα:40-kDa PEG的摩尔比例是1:4。向此混合物中,加入终浓度 20mM的还原剂NaCNBH3,并在温和搅拌下使其在4℃反应18小时。为了灭活不与IFNα反应的PEG,将乙醇胺以50mM的终浓度加到反应混合物中。
用Sephadex G-25柱(Pharmacia)除去未反应的PEG,并用另一种缓冲液置换原缓冲液。首先,用两倍柱体积(CV)的10mM Tris-HCl缓冲液(pH 7.5)平衡此柱子,然后加反应混合物上样。通过用UV分光光度计测定260nm处的吸收值来检测流过液。当柱子用同一种缓冲液洗脱时,因在N末端加入PEG而被修饰具有较高分子量的干扰素α较早被洗脱下来,而未反应的PEG则较晚被洗脱下来,从而可以只分离IFNα-40K PEG。
进行以下层析以进一步从收集的级分中纯化IFNα-40K PEG 复合物。用10mMTris-HCl(pH 7.5)平衡3ml PolyWAX LP柱 (PolyLC)。将收集到的含有IFNα-40K PEG复合物的级分以流速1ml/ 分钟加样到柱子上,用15ml平衡缓冲液洗脱柱子。然后,用30ml 1MNaCl以0-100%的线性NaCl梯度洗脱柱子,由此顺序洗脱与三-、二-和单-PEG缀合的干扰素α。为了进一步纯化单-PEG-缀合的干扰素α,对收集到的含有单-PEG-缀合干扰素α的级分进行大小排阻层析。将级分浓缩并加样到用10mM磷酸钠缓冲液(pH 7.0)平衡的Superdex200柱(Pharmacia)上,用相同缓冲液以流速1ml/分钟洗脱柱子。三和二-PEG-缀合的干扰素α分子由于其早于单-PEG-缀合的干扰素α被洗脱下来的特性而被去除,从而以高纯度形式分离出单-PEG-缀合的干扰素α。
依照上述同一种方法,将40-kDa PEG与人生长激素、粒细胞集落刺激因子(G-CSF)和G-CSF衍生物的N末端缀合,从而得到 hGH-40K PEG、G-CSF-40K PEG和40K PEG-17S-G-CSF衍生物复合物。
比较实施例2:IFNα-PEG-白蛋白缀合物的制备
为了将实施例1步骤2中纯化的IFNα-PEG复合物与白蛋白的 N末端连接,将IFNα-PEG复合物与溶于10mM磷酸盐缓冲液中的人血清白蛋白(HSA,约67kDa,Green Cross)以IFNα-PEG复合物: 白蛋白摩尔比例1:1、1:2、1:4和1:8混合。将反应溶液中的磷酸盐缓冲液浓度调节到100mM后,将还原剂NaCNBH3以终浓度20mM 加到反应液中,并在温和搅拌下使其在4℃反应20小时。通过此实验,发现具有最高反应性且产生最少量诸如二聚体等副产物的最佳反应摩尔比例IFNα-PEG复合物:白蛋白是1:2。
在偶联反应后,将反应混合物用SuperdexR柱(Pharmacia)进行大小排阻层析,以去除未反应的物质和副产物,并纯化所产生的 IFNα-PEG-白蛋白蛋白质缀合物。在将反应混合物浓缩并加样到柱子上后,使10mM乙酸钠缓冲液以2.5ml/分钟的流速通过柱子,以去除未结合的白蛋白和未反应的物质,随后进行柱洗脱,以仅仅纯化IFNα-PEG-白蛋白蛋白质缀合物。由于所收集到的IFNα-PEG-白蛋白蛋白质缀合物级分包含小量的杂质、未反应的白蛋白和干扰素α二聚体,故进行阳离子交换层析以去除杂质。将IFNα-PEG-白蛋白蛋白质缀合物级分加样到用10mM乙酸钠(pH 4.5)平衡的SP5PW柱 (Waters)上,并用1M NaCl在10mM乙酸钠缓冲液(pH 4.5)中的 0-0.5M NaCl线性梯度洗脱,从而分离高纯度形式的IFNα-PEG-白蛋白蛋白质缀合物。
依照上述同一种方法,将白蛋白与人生长激素、G-CSF和 G-CSF衍生物缀合,从而提供hGH-PEG-白蛋白、G-CSF-PEG-白蛋白和17S-G-CSF-PEG-白蛋白缀合物。
比较实施例3:Fab’-S-40K PEG复合物的制备
通过在活化缓冲液(20mM乙酸钠(pH 4.0),0.2mM DTT)中保温1小时激活实施例8步骤1中纯化的Fab’的游离半胱氨酸残基。用PEG修饰缓冲液置换所述缓冲液后,将50mM磷酸钾(pH 6.5)、马来酰亚胺-PEG(MW:40kDa,Shearwater)以Fab’:40-kDa PEG摩尔比例1:10加到其中,并在温和搅拌下在4℃反应24小时。
反应完成后,将反应液加到用10mM磷酸钠缓冲液(pH 7.3) 平衡的Superdex 200柱(Pharmacia)上,用相同缓冲液以流速1ml/ 分钟洗脱柱子。被Fab’缀合的40-kDa PEG(Fab’-40K PEG)由于其高分子量而相对较早地洗脱下来,而未反应的Fab’则稍后洗脱下来,从而去除了未反应的Fab’。为了完全去除未反应的Fab’,再次将收集到的Fab’-40K PEG复合物级分加样到polyCAT 21×250柱 (PolyLC)上,用20mM磷酸钠缓冲液(pH 4.5)中的0.15-0.5M线性 NaCl梯度洗脱此柱,从而提供了含有与Fab’的-SH基团连接的 40-kDaPEG的纯Fab’-S-40K PEG复合物。
实验实施例1:蛋白质缀合物的鉴定和定量分析
<1-1>蛋白质缀合物的鉴定
用4-20%梯度凝胶和12%凝胶以及ELISA(R&D System)通过非还原性SDS-PAGE分析上述实施例中制备的蛋白质缀合物。
SDS-PAGE分析的结果如图3中所示,其中生理学多肽、非肽聚合物、PEG和免疫球蛋白Fc片段的偶联反应导致成功形成了 IFNα-PEG-Fc缀合物(A)、17Ser-G-CSF-PEG-Fc缀合物(B)和 EPO-PEG-Fc缀合物(C)。
此外,用非还原性12%SDS-PAGE分析实施例10中制备的 DG Fc。如图6b中所示,在与缺乏糖基部分的天然Fc的分子量相应的位置处检测到了DG Fc条带。
<1-2>蛋白质缀合物的定量分析
用HiLoad 26/60Superdex 75柱(Pharmacia)通过大小排阻层析对以上实施例中制备的蛋白质缀合物进行定量,其中以10mM磷酸钾缓冲液(pH 6.0)作为洗脱缓冲液,并将其中各蛋白质缀合物的峰面积与对照组的峰面积相比较。将先前已定量分析的标准品、IFNα、 hGH、G-CSF、17S-G-CSF、EPO和Fc单独进行大小排阻层析,测定浓度和峰之间的转换因子。对预定量的各蛋白质缀合物进行相同大小排阻层析。通过从由此获得的峰面积中减去相应于免疫球蛋白 Fc片段的峰面积,测定存在于各蛋白质缀合物中的生理学活性蛋白质的数量值。图4显示了已纯化的IFNα-PEG-Fc缀合物的大小排阻层析结果,其中观察到了单一的峰。此结果表明,已纯化的蛋白质缀合物不包含诸如二聚体、三聚体和更高数量单体等多聚体杂质。
当用特异于生理学活性多肽的抗体对与Fc缀合的生理学活性多肽进行定量分析时,抗体与多肽的结合被阻止,导致了数值低于由层析计算的真实值。以IFNα-PEG-Fc缀合物为例,ELISA中得到的ELISA数值相当于真实值的大约30%。
<1-3>蛋白质缀合物的纯度和质量分析
对以上实施例中制备的蛋白质缀合物进行大小排阻层析,并检测280nm处的吸收值。结果显示,IFNα-PEG-Fc、hGH-PEG-Fc、 G-CSF-PEG-Fc和17Ser-G-CSF-PEG-Fc缀合物在70至80-kDa大小的物质保持时间处显示出单一峰。
另一方面,进行反相HPLC,以确定实施例1、11和13中制备的蛋白质缀合物IFNα-PEG-Fc、IFNα-PEG-DG Fc和IFNα-PEG- 重组AG Fc衍生物的纯度。利用了反相柱(259VHP54柱,Vydac)。利用配制于0.5%TFA中的40-100%乙腈梯度洗脱柱子,通过检测 280nm处的吸收值分析纯度。结果如图8所示,样品中不含有未结合的干扰素或免疫球蛋白Fc,且发现所有蛋白质缀合物 IFNα-PEG-Fc(A)、IFNα-PEG-DG Fc(B)和IFNα-PEG-重组AG Fc 衍生物(C)的纯度都高于96%。
为了确定已纯化蛋白质缀合物的准确分子量,用高通量 MALDI-TOF质谱仪(Voyager DE-STR,Applied Biosystems)分析各个缀合物的质量。用芥子酸作为基质。将0.5μl各待测样品包被在样品载玻片上并空气干燥,再与等体积的基质溶液混合并空气干燥,放入离子源中。用线性模式TOF分析仪以阳性方式进行检测。离子用通过延迟提取(delayed extraction,DE)操作的分裂提取源加速,在总加速电压约2.5kV处采用750纳秒至1500纳秒的延迟提取时间。
由MALDI-TOF质谱仪观察到的各实施例中制备的Fc蛋白质缀合物的分子量如下表1中所示。图5显示了EPO-PEG-Fc缀合物的MALDI-TOF质谱分析结果,而图7显示了IFNα-PEG-Fc和 IFNα-PEG-DG Fc缀合物的MALDI-TOF质谱分析结果。结果发现, EPO-PEG-Fc蛋白质缀合物纯度高于95%,且分子量非常接近理论分子量。此外,发现EPO与免疫球蛋白Fc片段以1:1的比例偶联。
表1
理论MW(kDa) | 测定MW(kDa) | |
IFNα-PEG-Fc(E.1) | 75.4 | 75.9 |
hGH-PEG-Fc(E.3) | 78.4 | 78.6 |
G-CSF-PEG-Fc(E.4) | 75.3 | 75.9 |
<sup>17</sup>S-G-CSF衍生物-PEG-Fc(E.4) | 75.0 | 75.9 |
EPO-PEG-Fc(E.5) | 91.4 | 91.0 |
此外,当用MALDI-TOF质谱仪测定实施例10中制备的Fc和 DG Fc的分子量时,发现DG Fc是50kDa,这比天然Fc小大约 3-kDa(图6a)。由于这3-kDa分子量相应于糖基部分的理论大小,该结果证实了糖基部分完全被去除了。
下表2显示了实施例11中制备的IFNα-PEG-DG Fc缀合物和实施例13中所制备的IFNα-PEG-重组AG Fc衍生物缀合体(I和II)的MALDI-TOF质谱分析结果。发现IFNα-PEG-DGFc缀合物比 IFNα-PEG-Fc缀合物的75.9kDa小3kDa,而IFNα-PEG-重组AG Fc 衍生物缀合体(I)比IFNα-PEG-Fc缀合物的75.9kDa小大约3-4 kDa。与Fc单体偶联的IFNα-PEG-重组AG Fc衍生物缀合体(II) 显示出分子量降低了24.5kDa,这相应于Fc单体的分子量。
表2
实验实施例2:药物代谢动力学分析I
评估天然形式的生理学活性蛋白质(对照)以及实施例和比较实施例中制备的蛋白质缀合物-40K PEG复合物、-PEG-白蛋白缀合物、-PEG-Fc缀合物、-PEG-DG Fc缀合物和-PEG-重组AG Fc衍生物缀合体在SD大鼠(每组5只大鼠)中的血清稳定性和药物代谢动力学参数。以100μg/kg的剂量单独皮下注射对照以及-40K PEG复合物、-PEG-白蛋白缀合物、-PEG-Fc缀合物、-PEG-DG Fc缀合物和-PEG-重组AG Fc衍生物缀合体(试验组)。皮下注射后,在0.5、1、 2、4、6、12、24、30、48、72和96小时收集对照组的血样,在1、 6、12、24、30、48、72、96、120、240和288小时收集试验组的血样。血样收集在装有抗凝剂肝素的试管中,用Eppendorf高速小型离心机离心5分钟以去除血细胞。用特异于所述生理学活性蛋白质的抗体通过ELISA检测血清蛋白质水平。
天然形式的IFNα、hGH、G-CSF和EPO以及它们的-40K PEG 复合物、它们的-PEG-白蛋白缀合物、它们的-PEG-Fc缀合物以及它们的-PEG-DG Fc缀合物的药物代谢动力学分析结果如下表3至7中所示。在以下的表中,Tmax表示达到最大药物血清浓度所需的时间, T1/2指药物的血清半寿期,而MRT(平均停留时间)指药物分子在身体中停留的平均时间。
表3
干扰素α的药物代谢动力学
表4
人生长因子的药物代谢动力学
表5
G-CSF的药物代谢动力学
表6
17S-G-CSF衍生物的药物代谢动力学
表7
EPO的药物代谢动力学
如表3的数据和图9的药物代谢动力学曲线所示, IFNα-PEG-Fc蛋白质缀合物的血清半寿期是90.4小时,这比天然 IFNα高大约50倍,而比比较实施例1中制备的IFNα-40KPEG的 35.8小时半寿期高大约2.5倍。此外,发现本发明的IFNα-PEG-Fc 蛋白质缀合物在血清半寿期上优于IFNα-PEG-白蛋白,后者的半寿期为17.1小时。
另一方面,如表3和图11中所示,IFNα-PEG-DG Fc缀合物的血清半寿期为71.0小时,这几乎与IFNα-PEG-Fc缀合物相同,表明Fc的去糖基化确实极大影响了IFNα-PEG-DG Fc缀合物的体内稳定性。此外,发现用通过重组方法产生的重组AG Fc衍生物制备的缀合物具有与天然形式衍生的DG Fc相同的效应。不过,与Fc单体偶联的复合物的血清半寿期大约是与正常Fc二聚体偶联的复合物的血清半寿期的一半。
如表4中所示,人生长激素在依照本发明与IgG Fc片段缀合时也显示出了血清半寿期延长。也就是说,与天然形式(1.1小时) 相比,hGH-40K PEG复合物和hGH-PEG-白蛋白缀合物的半寿期稍微延长,分别为7.7小时和5.9小时,而本发明的hGH-PEG-Fc蛋白质缀合物则显示出半寿期明显延长至11.8小时。
正如从表5和6中有关G-CSF和其衍生物的药物代谢动力学数据中显而易见的,G-CSF-PEG-Fc和17S-G-CSF-PEG-Fc缀合物呈现出比-40K PEG复合物和-PEG-白蛋白缀合物长得多的血清半寿期。发现免疫球蛋白Fc片段在血清中延长了天然形式的生理学活性蛋白质的作用持续时间,而它们的具有某些氨基酸残基改变的衍生物也呈现出与天然形式相似水平的这种特性。从这些结果中可很容易的预计本发明的方法会对其它蛋白质及其它们的衍生物具有类似的影响。
如表7和图10中所示,天然糖基化EPO与Fc片段的缀合也导致了血清半寿期的延长。也就是说,EPO在天然形式时血清半寿期为9.4小时,当为了提高血清稳定性而将其高度糖基化时,其血清半寿期延长至18.4小时。EPO-PEG-Fc缀合物(包含依照本发明与免疫球蛋白Fc片段偶联的EPO)显示出血清半寿期显著延长至61.5 小时。此外,当与大肠杆菌来源的重组非糖基化(AG)Fc衍生物缀合时,EPO的血清半寿期则延长至87.9小时,表明Fc片段的非糖基化使得蛋白质缀合物的制备不影响无抗体功能的蛋白质的血清稳定性。
正如从以上结果中显而易见的,依照本发明通过非肽聚合物与免疫球蛋白Fc片段共价键合的蛋白质缀合物呈现出血清半寿期比其天然形式的血清半寿期延长了数倍至数十倍。此外,当免疫球蛋白 Fc由于在大肠杆菌中产生而为非糖基化形式或由于通过酶处理而去糖基化时,它的延长其蛋白质缀合物的血清半寿期的效果保持在相似水平。
特别是,与为了延长蛋白质在血清中的持续作用时间而用PEG 分子中具有最长持续作用时间的40-kDa PEG修饰的蛋白质相比,免疫球蛋白Fc蛋白质缀合物具有更为优越的血清稳定性。此外,与偶联了白蛋白而非免疫球蛋白Fc的蛋白质缀合物相比,本发明的蛋白质缀合物呈现出极高的血清稳定性,表明本发明的蛋白质缀合物在研发长效形式的蛋白质药物中是有效的。这些结果,即与常规PEG -或白蛋白-缀合蛋白质相比,本发明的蛋白质缀合物对包括点突变的集落刺激因子衍生物在内的多种蛋白质的血清稳定性以及 MRT具有极好的影响,表明本发明缀合物的稳定性提高和持续时间延长的效果可适用于其它的生理学活性多肽。
另一方面,当用如上所述的相同方法评估利用非肽聚合物 10-kDa PEG制备的IFNα-10K PEG-Fc蛋白质缀合物(实施例7)的血清半寿期时,显示出其血清半寿期为48.8小时,这稍微短于用 3.4-kDa PEG制备的蛋白质缀合物的血清半寿期(79.7小时)。
此外,蛋白质缀合物的血清半寿期随着非肽聚合物PEG的分子量的增加而降低。这些结果表明,提高蛋白质缀合物的血清稳定性并延长其持续时间的主要因素是被缀合的免疫球蛋白Fc片段,而不是非肽聚合物。
即使当PEG的反应性基团被除醛基外的反应性基团置换时,含PEG的蛋白质缀合物仍然显示了与偶联了具有醛反应性基团的 PEG的那些缀合物相似模式的表观分子量和血清半寿期。
实验实施例3:药物代谢动力学分析II
为了测定实施例8和9中制备的Fab’-S-PEG-N-Fc和 Fab’-N-PEG-N-Fc缀合物以及比较实施例3中制备的Fab’-S-40K PEG复合物的血清半寿期,依照实验实施例2中相同的方法以Fab’作为对照进行了缀合物和复合物的药物代谢动力学分析。结果如图 12中所示。
如图12中所示,Fab’-S-PEG-N-Fc和Fab’-N-PEG-N-Fc缀合物呈现出血清半寿期与Fab’或Fab’-S-40K PEG复合物相比延长了两或三倍。
实验实施例4:蛋白质缀合物细胞内活性的评估
<4-1>IFNα蛋白质缀合物细胞内活性的比较
为了比较IFNα蛋白质缀合物的细胞内活性,用感染了疱疹性口腔炎病毒的MadinDarby牛肾(MDBK)细胞(ATCC CCL-22)通过细胞培养物生物测定法评估IFNα-PEG-Fc(实施例1)、 IFNα-PEG-DG Fc(实施例11)、IFNα-PEG-重组AG Fc衍生物(实施例13)、IFNα-40KPEG(比较实施例1)和IFNα-PEG-白蛋白(比较实施例2)的抗病毒活性。以可获自生物学标准品和对照国家研究院 (NIBSC)的未PEG化干扰素α-2b作为标准物质。
在补充了10%FBS和1%青霉素/链霉素的MEM(最低基本培养基,JBI)中于37℃,5%CO2的条件下培养MDBK细胞。用培养基将待分析样品和标准物质稀释至预定浓度,将100-μ1等分试样加入96孔板的各孔中。分离所培养的细胞,将其加入以100μ1的体积含有样品的平板中,在37℃和5%CO2的条件下培养约1小时。然后,将5-7×103PFU的疱疹性口腔炎病毒(VSV)50μl加入平板的各孔中,细胞在37℃、5%CO2的条件下进一步培养大约16-20小时。将不含样品或标准物质而只包含病毒的孔用作阴性对照,而只含有细胞的孔则用作阳性对照。
去除培养基后,将100μl中性红溶液加入平板中对存活的细胞染色,随后在37℃、5%CO2的条件下保温2小时。去除上清液后,将100μl 100%乙醇和1%乙酸的1:1混合物加入平板的每个孔中。充分混合以溶解自被染色细胞洗脱的所有中性红结晶后,检测540 nm的吸收值。将阴性对照用作空白,计算ED50值(造成50%细胞生长抑制的剂量),其中阳性对照的细胞生长被设定为100%。
表8
如表8中所示,IFNα-40K PEG活性降低至天然IFNα的4.8%。尤其是,当PEG部分的大小增加时,蛋白质缀合物具有提高的血清稳定性,但活性逐渐降低。据报道,干扰素α在用12-kDa PEG修饰时具有25%的体外活性,而当用40-kDa PEG修饰时则具有大约 7%的活性(P.Bailon等,Bioconjugate Chem.12:195-202,2001)。也就是说,由于随着PEG部分分子量的增加,蛋白质缀合物的半寿期变长,但生物学活性急速降低,所以需要研发具有较长半寿期和较强活性的蛋白质缀合物。此外,IFNα-PEG-白蛋白缀合物展示了弱的活性,与天然IFNα相比大约为5.2%。相反,本发明的 IFNα-PEG-Fc和IFNα-PEG-DG Fc缀合物呈现了显著提高的相对活性,与天然IFNα相比大约为28.1%和25.7%。此外,IFNα与重组 AG Fc衍生物的缀合导致了活性的相似提高。从这些结果预计,与免疫球蛋白Fc片段缀合的干扰素α具有显著延长的血清半寿期和极大提高的体内药效。
<4-2>人生长激素蛋白质缀合物细胞内活性的比较
为了比较人生长激素蛋白质缀合物的细胞内活性,比较了 hGH-PEG-Fc、hGH-40KPEG和hGH-PEG-白蛋白的细胞内活性。
用大鼠结节淋巴瘤细胞系Nb2(欧洲细胞培养物保藏中心 (ECACC)#97041101)通过体外试验检测hGH缀合物的细胞内活性,所说细胞系显示了人生长激素依赖型有丝分裂。
将Nb2细胞培养于补充了10%FBS(胎牛血清)、0.075% NaCO3、0.05mM 2-巯基乙醇和2mM谷氨酰胺的Fisher’s培养基中,并进一步在不含10%FBS的相似培养基中培养24小时。然后,对所培养的细胞进行计数,将大约2×104个细胞的等分试样加至96 孔板的各孔中。将hGH-PEG-Fc、hGH-40K PEG、hGH-PEG-白蛋白、可获自生物学标准品和对照国家研究院(NIBSC)作为对照的标准品和天然人生长激素(HM-hGH)稀释,并以多种浓度加入各孔中,随后在37℃5%CO2的条件下保温48小时。之后,为了通过测定各孔中的细胞数目来检测细胞增殖活性,将25μl Cell Titer 96 Aqueous One Solution Reagent(Promega)加入各孔中,将细胞进一步培养4 小时。检测490nm处的吸收值,计算各样品的效价。结果如下表9 中所示。
表9
如表9中所示,同样以人生长激素为例,与40-kDa PEG的缀合(hGH-40K PEG)导致了活性降低至大约为天然形式的7.6%,而 hGH-PEG-白蛋白缀合物显示出大约为天然hGH的5.2%的低体外活性。不过,本发明的hGH-PEG-Fc缀合物显著提高了相对活性,达到天然hGH的28%以上。从这些结果预计,与免疫球蛋白Fc片段连接的人生长激素具有显著延长的血清半寿期和极大提高的体内药效。此外,我们认为本发明免疫球蛋白Fc蛋白质缀合物活性的提高是由于血清稳定性的提高,而与受体的结合亲和力的保留则归因于免疫球蛋白Fc或非肽聚合物形成的空间。这些效果预计可适用于与其它生理学活性蛋白质偶联的免疫球蛋白Fc蛋白质缀合物。
<4-3>G-CSF蛋白质缀合物的细胞内活性比较
为了比较具有G-CSF衍生物的蛋白质缀合物的细胞内活性,比较了天然G-CSF(Filgrastim,Jeil Pharm.Co.,Ltd.)、17Ser-G-CSF 衍生物、20K PEG-G-CSF(Neulasta)、40K PEG-17S-G-CSF、17Ser-G-CSF-PEG-白蛋白和17S-G-CSF-PEG-Fc的细胞内活性。
首先,将人骨髓细胞系HL-60(ATCC CCL-240,前髓细胞性白血病患者/36岁白种女性)培养于补充了10%FBS的RPMI 1640培养基中。将培养的细胞以大约2.2×105个细胞/ml的密度悬浮,并在其中加入DMSO(二甲亚砜,培养级,Sigma)至终浓度1.25%(v/v)。然后,将90μl细胞悬浮液接种于96孔板(Corning/低蒸发96孔板) 的各孔中,从而密度大约为每孔2×104个细胞,在37℃含5%CO2的培养箱中培养大约72小时。
将蛋白质浓度用G-CSF ELISA试剂盒(R&D systems)测定过的各样品用RPMI 1640稀释至同一浓度10μg/ml,再进一步用RPMI 1640稀释19次,每次稀释2倍。将连续的两倍稀释液10μl独立加到含HL-60细胞的各孔中,从而各样品浓度起始为1μg/ml。然后,将细胞在37℃培养箱培养72小时。
用Cell Titer 96TM(Cat.NO.G4100,Promega)检测HL-60细胞的增殖,通过测量670nm的吸收值确定增加的细胞数。
表10
如表10中所示,与具有氨基酸取代的G-CSF衍生物17Ser-G-CSF的免疫球蛋白Fc蛋白质缀合物同样呈现出了与天然 G-CSF-偶联蛋白质缀合物相似的效果。先前报道了17Ser-G-CSF-PEG与未PEG化的17Ser-G-CSF(韩国专利公开文件 2004-83268)相比具有相对较长的血清半寿期,但活性有所降低。尤其是,当PEG部分的大小增大时,蛋白质缀合物的血清稳定性增加,但活性逐渐降低。17Ser-G-CSF-40K PEG显示出非常低的活性,与天然形式相比低于大约10%。也就是说,由于蛋白质缀合物随着PEG 部分的分子量增加而具有较长的血清半寿期但活性急速降低,所以需要研发一种具有长血清半寿期和强活性的蛋白质缀合物。17Ser-G-CSF-PEG-白蛋白也显示出与天然G-CSF相比大约为其 23%的低活性。相反,17Ser-G-CSF-PEG-Fc则大大提高了相对活性,其相对活性大于天然G-CSF的51%。从这些结果中,预计与免疫球蛋白Fc片段连接的17Ser-G-CSF具有显著增加的血清半寿期,且极大提高了其体内药效。
<4-4>Fab’缀合物的细胞毒性中和测定
用实施例8和实施例9中制备的Fab’-S-PEG-N-Fc和 Fab’-N-PEG-N-Fc缀合物以及比较实施例3中制备的Fab’-S-40K PEG复合物进行体外活性测定。通过基于测量TNFα-介导的细胞毒性的细胞毒性测定,评估Fab′缀合物以确定其是否在小鼠成纤维细胞系L929(ATCC CRL-2148)中中和了TNFα-诱导的凋亡。
按两倍系列稀释Fab’-S-PEG-N-Fc和Fab’-N-PEG-N-Fc缀合物以及Fab’-S-40KPEG复合物,将100-μl等分溶液加到96孔板的孔中。将rhTNF-α(R&D systems)和放线菌素D(Sigma)作为RNA合成抑制剂分别以终浓度10ng/ml和1μg/ml加入各孔中,在37℃含 5%CO2的培养箱中保温30分钟,再转移到微量滴定板上供检测。将L929细胞以5×104个细胞/50μl培养基的密度加入各孔中并在 37℃含5%CO2的培养箱中培养24小时。去除培养基后,将以5mg/ml浓度溶于PBS中的50μl MTT(Sigma)加入各孔中,细胞进一步在37℃含5%CO2的培养箱中培养大约4小时。将150μl DMSO 加入各孔中,通过测定540nm的吸收值确定细胞毒性中和的程度。作为对照,使用了实施例8步骤1中纯化的Fab′。
如图13中所示,所有用于此试验的蛋白质缀合物都具有与 Fab′相似的效价。这些结果表明,当将免疫球蛋白Fc通过PEG连接至Fab′的N末端或C末端附近的游离半胱氨酸来制备蛋白质缀合物时,Fab′呈现出显著增高的血清半寿期和高体内活性。
<4-5>补体依赖型细胞毒性(CDC)测定
为了确定在大肠杆菌转化体表达并纯化的实施例中制备的衍生物和相应于免疫球蛋白恒定区的蛋白质是否结合人C1q,进行了如下的酶联免疫吸附测定(ELISA)。在试验组中,使用了HM10932 和HM10927转化体产生的免疫球蛋白恒定区以及以上实施例中制备的衍生物,所说的两个转化体于2004年9月15日保藏于韩国微生物保藏中心(KCCM),保藏号为KCCM-10597、KCCM-10588。作为标准参照,使用了糖基化的免疫球蛋白(IVIG-球蛋白S,Green Cross PBM)和数种作为治疗抗体的商品化抗体。试验和标准样品以浓度1μg/ml配制于10mM碳酸盐缓冲液(pH 9.6)中。样品以每孔 200ng的量等分至96孔板(Nunc)中,并将平板在4℃包被过夜。然后,将各孔用PBS-T(137mM NaCl,2mM KCl,10mM Na2HPO4,2 mMKH2PO4,0.05%Tween 20)漂洗三次,用250μl封闭缓冲液(溶于 PBS-T中的1%牛血清白蛋白)室温封闭1小时,再用同样的PBS-T 漂洗三次。将标准和试验样品在PBS-T中稀释至预定浓度,并添加到抗体包被的孔中,将平板在室温保温1小时,并用PBS-T洗三次。之后,将2μg/ml C1q(R&D Systems)加入平板中,并于室温反应2 小时,然后将平板用PBS-T洗6次。将200μl 1:1000稀释于封闭缓冲液中的人抗人C1q抗体-过氧化物酶缀合物(Biogenesis,USA)加入各孔中,并于室温反应1小时。用PBS-T将各孔漂洗三次后,混合等体积的显色试剂A和B(显色剂A:稳定的过氧化物和显色剂B:稳定的色素原;DY 999,R&D Systems),将200μl混合物加到各孔中,随后保温30分钟。然后,在各孔中加入50μl反应终止液,2M硫酸。用微量滴定板读数仪(Molecular Device)对平板读数。在波长450nm 处测量标准和试验样品的吸收值,结果分别参见图14和15。
在相互比较免疫球蛋白亚型之间其免疫球蛋白Fc片段中的补体活性时,发现人免疫球蛋白IgG1(Fitzgerald)具有最高的C1q结合亲和力,其次是IgG2(Fitzgerald),然后是IgG4(Fitzgerald),表明在亚型之间补体活性存在差异。此试验中所用的IVIG是IgG亚型的合并物,由于IgG1占了IVIG的大部分,所以IVIG呈现出与纯化的IgG1近乎相同的C1q结合亲和力。与这些标准相比,在由于去糖基化而造成的C1q结合亲和力的改变方面,具有最强的补体活性的IgG1Fc在非糖基化时此活性显著降低。IgG4Fc,已知不会诱发补体的活化,它对C1q几乎没有结合亲和力,表明IgG4Fc可被用作无补体活性的极好重组载体(图14)。
为了确定载体是否在即使与生理学活性肽缀合后仍保持了对 C1q没有结合亲和力的特性,用糖基化的Fc、酶促去糖基化的Fc 和非糖基化重组Fc作为IFNα的载体制备IFNα-Fc缀合物,并评估其对C1q的结合亲和力。发现偶联了糖基化Fc的IFNα缀合物 (IFNα-PEG-Fc:糖基化IgG1Fc)保持了对C1q的高结合亲和力。相反,当将干扰素α与用PNGase F和其他酶去糖基化的Fc偶联时,所产生的缀合物(IFNα-PEG-DGFc:去糖基化IgG1Fc)展示了明显降低的C1q结合亲和力,这与大肠杆菌来源的非糖基化Fc缀合物类似。此外,当以IgG4部分替换偶联了非糖基化IgG1Fc的干扰素α缀合物(IFNα-PEG-AGFcG1:非糖基化IgG1Fc)的IgG1部分时,发现所产生的干扰素缀合物(IFNα-PEG-FcG4衍生物1:非糖基化IgG4Fc) 完全丧失了其与C1q的结合亲和力。当IgG1Fc部分被IgG4Fc单体替换时,所产生的缀合物(IFNα-PEG-FcG4衍生物2:非糖基化 IgG4Fc)。这些结果显示这样的IgG4Fc片段形式可用作不具有抗体片段效应器功能的极好载体(图15)。
工业适用性
如上文所描述的,本发明的药物缀合物极大的提高了药物的血浆半寿期。另一方面,所述蛋白质缀合物克服了常规长效制剂最显著的缺陷,即药物效价降低,从而使血液循环时间和体内活性超过了原先已知最有效的白蛋白。此外,所述蛋白质缀合物没有诱发免疫应答的风险。由于这些优势,该蛋白质缀合物可用于开发蛋白质药物的长效制剂。依照本发明的蛋白质药物长效制剂能减少患者频繁注射的痛苦,在较长时间内保持活性多肽的血清浓度,从而使药效稳定。
此外,本发明制备蛋白质缀合物的方法克服了遗传操作生产融合蛋白质的缺点,包括表达体系难以建立、糖基化作用不同于天然形式、诱发免疫应答和蛋白质融合的方位有限、由于非特异性反应造成的低产率以及用作粘合剂的化合物的毒性等化学偶联的问题,从而可以方便且经济的提供血清半寿期延长并具有高活性的蛋白质药物。
本说明书还包括如下内容:
1.包含免疫球蛋白Fc片段作为载体的药物组合物,其中所述免疫球蛋白Fc片段通过聚乙二醇PEG与生理学活性多肽药物共价连接,其中所述Fc片段包含免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3),且不含免疫球蛋白的重链和轻链可变区、重链恒定区1(CH1)以及轻链恒定区1(CL1)。
2.依照实施方式1的药物组合物,其中所述免疫球蛋白Fc片段是非糖基化的。
3.依照实施方式1的药物组合物,其中所述免疫球蛋白Fc片段由选自CH2、CH3和CH4结构域中的1到4个结构域组成。
4.依照实施方式3的药物组合物,其中所述免疫球蛋白Fc片段还包含铰链区。
5.依照实施方式1的药物组合物,其中所述免疫球蛋白Fc片段选自由来自IgG、IgA、IgD、IgE、IgM的Fc片段以及它们的组合和杂合体组成的组。
6.依照实施方式5的药物组合物,其中所述免疫球蛋白Fc片段选自由来自IgG1、IgG2、IgG3、IgG4的Fc片段以及它们的组合和杂合体组成的组。
7.依照实施方式6的药物组合物,其中所述免疫球蛋白Fc片段是IgG4Fc片段。
8.依照实施方式1的药物组合物,其中所述生理学活性多肽选自:激素、细胞因子、酶、抗体、生长因子、转录调控因子、凝血因子、疫苗、结构蛋白质、配体蛋白质和受体。
9.依照实施方式8的药物组合物,其中所述生理学活性多肽选自:人生长激素、生长激素释放肽、干扰素、干扰素受体、集落刺激因子、胰高血糖素样肽、G蛋白偶联受体、白介素、白介素受体、酶、白介素结合蛋白、细胞因子结合蛋白、巨噬细胞活化因子、 B细胞因子、T细胞因子、蛋白A、变态反应抑制物、细胞坏死糖蛋白、免疫毒素、淋巴毒素、肿瘤坏死因子、肿瘤抑制物、转移生长因子、α-1抗胰蛋白酶、白蛋白、α-乳清蛋白、载脂蛋白-E、促红细胞生成素、高度糖基化的促红细胞生成素、angiopoietins、血红蛋白、凝血酶、凝血酶受体激活肽、凝血调节蛋白、因子VII、因子 VIIa、因子VIII、因子IX、因子XIII、纤溶酶原活化因子、纤维蛋白结合肽、尿激酶、链激酶、水蛭素、蛋白C、C反应蛋白、肾素抑制物、胶原酶抑制物、超氧化物歧化酶、leptin、血小板衍生生长因子、上皮生长因子、表皮生长因子、制管张素、血管紧张素、骨生长因子、骨刺激蛋白质、降钙素、胰岛素、心房肽激素、软骨诱发因子、依降钙素、结缔组织活化因子、组织因子途径抑制物、促卵泡激素、黄体生成素、黄体生成素释放激素、神经生长因子、甲状旁腺素、松弛素、促胰液素、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、缩胆囊素、胰多肽、胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺素、自分泌运动因子、乳铁蛋白、筒箭毒碱、受体、受体拮抗剂、细胞表面抗原、病毒衍生疫苗抗原、单克隆抗体、多克隆抗体和抗体片段。
10.依照实施方式9的药物组合物,其中所述生理学活性多肽选自:人生长激素、集落刺激因子、α干扰素、人促红细胞生成素和 Fab′抗体片段。
11.以免疫球蛋白Fc片段作为载体在制备用于延长药物的体内作用持续时间的药物中的用途,其中所述免疫球蛋白Fc片段通过 PEG与生理学活性多肽药物共价连接,其中所述Fc片段包含免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3),且不含免疫球蛋白的重链和轻链可变区、重链恒定区1(CH1)以及轻链恒定区 1(CL1)。
12.依照实施方式11的用途,其中所述免疫球蛋白Fc片段是非糖基化的。
13.依照实施方式11的用途,其中所述免疫球蛋白Fc片段由选自CH2、CH3和CH4结构域中的1个到4个结构域组成。
14.依照实施方式13的用途,其中所述免疫球蛋白Fc片段中还包含铰链区。
15.依照实施方式11的用途,其中所述免疫球蛋白Fc片段选自由来自IgG、IgA、IgD、IgE、IgM的Fc片段以及它们的组合和杂合体组成的组。
16.依照实施方式15的用途,其中所述免疫球蛋白Fc片段是选自由来自IgG1、IgG2、IgG3、IgG4的Fc片段以及它们的组合和杂合体组成的组。
17.依照实施方式16的用途,其中所述免疫球蛋白Fc片段是 IgG4Fc片段。
18.依照实施方式17的用途,其中所述免疫球蛋白Fc片段是人非糖基化IgG4Fc片段。
19.依照实施方式11的用途,其中所述生理学活性多肽选自:激素、细胞因子、酶、抗体、生长因子、转录调控因子、凝血因子、疫苗、结构蛋白质、配体蛋白质和受体。
20.依照实施方式19的用途,其中所述生理学活性多肽选自:人生长激素、生长激素释放肽、干扰素、干扰素受体、集落刺激因子、胰高血糖素样肽、G蛋白偶联受体、白介素、白介素受体、酶、白介素结合蛋白、细胞因子结合蛋白、巨噬细胞活化因子、B细胞因子、T细胞因子、蛋白A、变态反应抑制物、细胞坏死糖蛋白、免疫毒素、淋巴毒素、肿瘤坏死因子、肿瘤抑制物、转移生长因子、α-1抗胰蛋白酶、白蛋白、α-乳清蛋白、载脂蛋白-E、促红细胞生成素、高度糖基化的促红细胞生成素、angiopoietins、血红蛋白、凝血酶、凝血酶受体激活肽、凝血调节蛋白、因子VII、因子VIIa、因子VIII、因子IX、因子XIII、纤溶酶原活化因子、纤维蛋白结合肽、尿激酶、链激酶、水蛭素、蛋白C、C反应蛋白、肾素抑制物、胶原酶抑制物、超氧化物歧化酶、leptin、血小板衍生生长因子、上皮生长因子、表皮生长因子、制管张素、血管紧张素、骨生长因子、骨刺激蛋白质、降钙素、胰岛素、心房肽激素、软骨诱发因子、依降钙素、结缔组织活化因子、组织因子途径抑制物、促卵泡激素、黄体生成素、黄体生成素释放激素、神经生长因子、甲状旁腺激素、松弛素、促胰液素、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、缩胆囊素、胰多肽、胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺素、自分泌运动因子、乳铁蛋白、筒箭毒碱、受体、受体拮抗剂、细胞表面抗原、病毒衍生疫苗抗原、单克隆抗体、多克隆抗体和抗体片段。
21.依照实施方式20的用途,其中所述生理学活性多肽选自:人生长激素、集落刺激因子、α干扰素、人促红细胞生成素和Fab′抗体片段。
序列表
<110> Hanmi Pharm. Co., Ltd.
<120> 包含免疫球蛋白Fc区作为载体的药物组合物
<150> KR10-2003-0080299
<151> 2003-11-13
<160> 6
<170> KopatentIn 1.71
<210> 1
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 1
cgtcatgccc agcacctgag ttcctggggg gacca 35
<210> 2
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 2
gggggatcct catttaccca gagacaggga gaggctcttc tg 42
<210> 3
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 3
cggcacctga gttcctgggg ggaccatca 29
<210> 4
<211> 69
<212> DNA
<213> 人(Homo sapiens)
<400> 4
atgaaaaaga caatcgcatt tcttcttgca tctatgttcg ttttttctat tgctacaaat 60
gcccaggcg 69
<210> 5
<211> 45
<212> DNA
<213> 人
<400> 5
tctattgcta caaatgccca ggccttccca accattccct tatcc 45
<210> 6
<211> 45
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 6
agataacgat gtttacgggt ccggaagggt tggtaaggga atagg 45
Claims (23)
1.包含免疫球蛋白Fc片段作为载体的药物组合物,
其中所述免疫球蛋白Fc片段通过非肽聚合物与生理学活性多肽药物共价连接,
其中所述Fc片段包含免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3),且不含免疫球蛋白的重链和轻链可变区、重链恒定区1(CH1)以及轻链恒定区1(CL1),
其中所述免疫球蛋白Fc片段与所述药物之间的连接不是通过遗传重组形成的融合体,
其中所述免疫球蛋白Fc片段是非糖基化的,
其中所述非肽聚合物是聚乙二醇,并且
其中所述生理学活性多肽选自:激素、细胞因子及抗体。
2.依照权利要求1的药物组合物,其中所述免疫球蛋白Fc片段由选自CH2、CH3和CH4结构域中的1到4个结构域组成。
3.依照权利要求2的药物组合物,其中所述免疫球蛋白Fc片段还包含铰链区。
4.依照权利要求1的药物组合物,其中所述免疫球蛋白Fc片段选自由来自IgG、IgA、IgD、IgE、IgM的Fc片段以及它们的组合和杂合体组成的组。
5.依照权利要求4的药物组合物,其中所述免疫球蛋白Fc片段选自由来自IgG1、IgG2、IgG3、IgG4的Fc片段以及它们的组合和杂合体组成的组。
6.依照权利要求5的药物组合物,其中所述免疫球蛋白Fc片段是IgG4 Fc片段。
7.依照权利要求1的药物组合物,其中所述非肽聚合物的分子量是3.4kDa或10kDa。
8.依照权利要求1的药物组合物,其中所述生理学活性多肽选自:干扰素及生长因子。
9.依照权利要求1的药物组合物,其中所述生理学活性多肽选自:人生长激素、生长激素释放肽、干扰素、集落刺激因子、胰高血糖素样肽、白介素、肿瘤坏死因子、促红细胞生成素、高度糖基化的促红细胞生成素、血管生成素、血小板衍生生长因子、上皮生长因子、表皮生长因子、骨生长因子、降钙素、胰岛素、心房肽激素、促卵泡激素、黄体生成素、黄体生成素释放激素、神经生长因子、甲状旁腺素、松弛素、促胰液素、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、缩胆囊素、胰多肽、胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺素、单克隆抗体、多克隆抗体和抗体片段。
10.依照权利要求1的药物组合物,其中所述生理学活性多肽选自:B细胞因子和T细胞因子。
11.依照权利要求1的药物组合物,其中所述生理学活性多肽选自:人生长激素、集落刺激因子、α干扰素、人促红细胞生成素和Fab′抗体片段。
12.以免疫球蛋白Fc片段作为载体在制备用于延长药物的体内作用持续时间的药物中的用途,
其中所述免疫球蛋白Fc片段通过非肽聚合物与生理学活性多肽药物共价连接,
其中所述Fc片段包含免疫球蛋白的重链恒定区2(CH2)和重链恒定区3(CH3),且不含免疫球蛋白的重链和轻链可变区、重链恒定区1(CH1)以及轻链恒定区1(CL1),
其中所述免疫球蛋白Fc片段与所述药物之间的连接不是通过遗传重组形成的融合体,
其中所述免疫球蛋白Fc片段是非糖基化的,
其中所述非肽聚合物是聚乙二醇,并且
其中所述生理学活性多肽选自:激素、细胞因子及抗体。
13.依照权利要求12的用途,其中所述免疫球蛋白Fc片段由选自CH2、CH3和CH4结构域中的1个到4个结构域组成。
14.依照权利要求12的用途,其中所述免疫球蛋白Fc片段中还包含铰链区。
15.依照权利要求12的用途,其中所述免疫球蛋白Fc片段选自由来自IgG、IgA、IgD、IgE、IgM的Fc片段以及它们的组合和杂合体组成的组。
16.依照权利要求15的用途,其中所述免疫球蛋白Fc片段是选自由来自IgG1、IgG2、IgG3、IgG4的Fc片段以及它们的组合和杂合体组成的组。
17.依照权利要求16的用途,其中所述免疫球蛋白Fc片段是IgG4 Fc片段。
18.依照权利要求17的用途,其中所述免疫球蛋白Fc片段是人非糖基化IgG4 Fc片段。
19.依照权利要求12的用途,其中所述非肽聚合物的分子量是3.4kDa或10kDa。
20.依照权利要求12的用途,其中所述生理学活性多肽选自:干扰素及生长因子。
21.依照权利要求12的用途,其中所述生理学活性多肽选自:人生长激素、生长激素释放肽、干扰素、集落刺激因子、胰高血糖素样肽、白介素、肿瘤坏死因子、促红细胞生成素、高度糖基化的促红细胞生成素、血管生成素、血小板衍生生长因子、上皮生长因子、表皮生长因子、骨生长因子、降钙素、胰岛素、心房肽激素、促卵泡激素、黄体生成素、黄体生成素释放激素、神经生长因子、甲状旁腺激素、松弛素、促胰液素、生长调节素、胰岛素样生长因子、肾上腺皮质激素、胰高血糖素、缩胆囊素、胰多肽、胃泌素释放肽、促肾上腺皮质激素释放因子、促甲状腺素、单克隆抗体、多克隆抗体和抗体片段。
22.依照权利要求12的用途,其中所述生理学活性多肽选自:B细胞因子和T细胞因子。
23.依照权利要求12的用途,其中所述生理学活性多肽选自:人生长激素、集落刺激因子、α干扰素、人促红细胞生成素和Fab′抗体片段。
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