CN114395041A - 一种制备抗il-12和/或il-23单克隆抗体的方法 - Google Patents
一种制备抗il-12和/或il-23单克隆抗体的方法 Download PDFInfo
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Abstract
本发明涉及一种制备抗IL‑12和/或IL‑23单克隆抗体，特别是乌司奴单抗的方法。相对于SP2/0细胞及CHO‑K1细胞表达的抗体，采用本发明的方法得到的抗体，产量更高，筛选步骤简单，并且生物学活性更高。
Description
相关申请的交叉引用
本申请要求2021年2月9日向中国国家知识产权局提交的专利申请No.202110174184.3，发明名称为“一种制备抗IL-12和/或IL-23单克隆抗体的方法”的优先权，将其内容整体并入本文作为参考。
技术领域
本发明涉及一种制备抗IL-12和/或IL-23单克隆抗体，特别是乌司奴单抗的方法。
背景技术
白细胞介素是由多种细胞产生并作用于多种细胞的一类细胞因子。白细胞介素-12(IL-12)是具有广泛生物学活性的细胞因子，主要由B细胞和巨噬细胞产生，其分子是一种异二聚体，由p35和p40两个亚基组成。白细胞介素-23(IL-23)的结构与IL-12有同源性，以异二聚体的形式存在，α链为p19，含189个氨基酸，其β链与IL-12的p40相同。其功能为活化经刺激的T细胞，促进其增殖和产生干扰素γ，并诱导记忆性T细胞的增殖。IL-12和IL-23在免疫细胞的成熟、活化、增殖和免疫调节等一系列过程中均发挥重要作用，此外它们还参与机体的多种生理及病理反应。
由于IL-12和IL-23涉及各种病症，已经设计了几种治疗策略来抑制IL-12和/或IL-23活性。其中，强生公司的药物乌司奴单抗(Ustekinumab)于2009年被美国FDA批准上市，用于成人中、重度斑块型银屑病患者的治疗。乌司奴单抗是靶向IL-12和IL-23的共同亚单位p40的IgG1抗体，是一种全人源单克隆抗体，可以抑制IL-12和IL-23的生物活性，从而阻止初始T细胞向Th1、Th17细胞分化，进而抑制炎症反应的发展。在美国和欧洲乌司奴单抗均已被批准用于治疗斑块型银屑病、银屑病性关节炎和克罗恩病，在中国乌司奴单抗已被批准用于治疗中重度斑块型银屑病和成人克罗恩病适应症。
强生公司的乌司奴单抗是使用SP2/0细胞表达的抗体，利用SP2/0这种小鼠骨髓瘤细胞表达的抗体产量较低，免疫原性较强(由于含有NGNA唾液酸化和α-1,3半乳糖化)。因此，需要开发新的表达抗体的方法，在保证抗体生物活性的前提下，进一步提高产量，降低免疫原性。
发明内容
本发明第一方面提供了一种制备抗IL-12和/或IL-23单克隆抗体或其抗原结合片段的方法，包括使用DHFR基因敲除或GS基因敲除的CHO细胞表达所述单克隆抗体或所述抗原结合片段，所述单克隆抗体或所述抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的VH-CDR1、VH-CDR2和VH-CDR3；所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ IDNO:5和SEQ ID NO:6所示的VL-CDR1、VL-CDR2和VL-CDR3。
在一个实施方案中，所述抗IL-12和/或IL-23单克隆抗体或其抗原结合片段包含SEQ ID NO:7所示的重链可变区和SEQ ID NO:8所示的轻链可变区。
在一个实施方案中，所述单克隆抗体是乌司奴单克隆抗体。
发明人经过大量实验，意外地发现，使用DHFR基因敲除的CHO细胞或GS基因敲除的CHO细胞来表达所述单克隆抗体，相对于SP2/0细胞及CHO-K1细胞表达的抗体，具有预料不到的更高的生物学活性，并且同时兼具有较高的产量。
在一个实施方案中，本发明提供了一种制备抗IL-12和/或IL-23单克隆抗体或其抗原结合片段的方法，所述方法包括以下步骤：
a)将抗IL-12和/或IL-23单克隆抗体或其抗原结合片段编码序列克隆至表达载体中，得到重组表达载体，优选地，所述表达载体含有选择标记基因，更优选地，所述选择标记基因是DHFR基因或GS基因；
b)将步骤a)获得的重组表达载体转入DHFR基因敲除的CHO细胞或GS基因敲除的CHO细胞，得到基因工程改造的CHO细胞；
c)培养步骤b)获得的基因工程改造的CHO细胞，选择表达所述抗体的单克隆细胞株；
d)培养c)步骤获得的表达所述抗体的单克隆细胞株，收获包含所述抗体的细胞培养液，分离纯化，获得所述单克隆抗体。
在一个实施方案中，所述方法包括以下步骤：
a)将所述抗IL-12和/或IL-23单克隆抗体或其抗原结合片段的编码序列克隆至表达载体中，得到重组表达载体，优选地，所述表达载体含有选择标记基因，更优选地，所述选择标记基因是DHFR基因或GS基因；
b)将步骤a)获得的重组表达载体转入DHFR基因敲除的CHO细胞或GS基因敲除的CHO细胞，得到基因工程改造的CHO细胞；
c)培养步骤b)获得的基因工程改造的CHO细胞，选择高效表达所述抗体的单克隆细胞株；
d)培养c)步骤获得的高效表达所述抗体的单克隆细胞株，收获包含所述抗体的细胞培养液，分离纯化，获得所述单克隆抗体。
在一个实施方案中，所述DHFR基因敲除的CHO细胞是CHO-DG44细胞。
在一个实施方案中，所述GS基因敲除的CHO细胞通过采用锌指核酸酶(ZFN)技术或利用CRISPR方法敲除GS基因而获得。
在一个实施方案中，所述GS基因敲除的CHO细胞是
-GS细胞。
在一个实施方案中，所述方法包括以下步骤：
a)将所述抗IL-12和/或IL-23单克隆抗体或其结合片段的编码序列克隆至表达载体中，得到重组表达载体，所述表达载体含有GS基因；
b)将步骤a)获得的重组表达载体转入
-GS细胞，得到基因工程改造的
-GS细胞；
c)将步骤b)获得的基因工程改造的
-GS细胞在无谷氨酰胺的培养基中进行培养，选择表达所述抗体的单克隆细胞株；
d)培养c)步骤得到的表达所述抗体的单克隆细胞株，收获包含所述抗体的细胞培养液，分离纯化，获得所述单克隆抗体。
在一个实施方案中，所述方法步骤d中所述的细胞培养过程采用补料-分批培养模式。
本发明第二方面提供一种抗IL-12和/或IL-23单克隆抗体或其抗原结合片段，通过本发明第一方面所述的方法制备。
本发明第三方面提供一种药物组合物，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体、蔗糖、L-组氨酸、L-组氨酸盐酸盐一水合物和聚山梨酯80。
在一个实施方案中，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体90±9mg/mL、蔗糖76±7.6mg/mL、L-组氨酸0.5±0.3mg/mL、L-组氨酸盐酸盐一水合物1±0.5mg/mL和聚山梨酯80 0.04±0.004mg/mL。
在一个实施方案中，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体90mg/mL、蔗糖76mg/mL、L-组氨酸0.636mg/mL、L-组氨酸盐酸盐一水合物1.237mg/mL和聚山梨酯800.04mg/mL。
本发明第四方面提供一种药物组合物，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体、L-组氨酸、L-组氨酸盐酸盐一水合物、聚山梨酯80、蔗糖、EDTA二钠盐二水合物和L-蛋氨酸。
在一个实施方案中，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体5±0.5mg/mL、L-组氨酸0.77±0.077mg/mL、L-组氨酸盐酸盐一水合物1.03±0.10mg/mL、聚山梨酯80 0.4±0.04mg/mL、蔗糖85±8.5mg/mL、EDTA二钠盐二水合物0.02±0.002mg/ml和L-蛋氨酸0.4±0.04mg/ml。
在一个实施方案中，所述药物组合物包含本发明第一方面所述方法制备得到的单克隆抗体5mg/mL、L-组氨酸0.77mg/mL、L-组氨酸盐酸盐一水合物1.03mg/mL、聚山梨酯800.4mg/mL、蔗糖85mg/mL、EDTA二钠盐二水合物0.02mg/ml和L-蛋氨酸0.4mg/ml。
本发明第五方面提供本发明第一方面所述方法制备得到的抗IL-12和/或IL-23单克隆抗体或其抗原结合片段或本发明第三或第四方面所述的药物组合物在制备用于治疗IL-12和/或IL-23相关的疾病或病症的药物中的用途。
在一个实施方案中，所述疾病或病症选自斑块型银屑病、银屑病性关节炎、克罗恩病、系统性红斑狼疮、溃疡性结肠炎、多发性硬化症、结节病、中轴型脊柱关节炎和强直性脊柱炎。优选地，所述疾病或病症选自斑块型银屑病、银屑病性关节炎和克罗恩病。
本发明具有以下有益效果：
1.发明人意外地发现，本发明的方法生产的单克隆抗体对IL-12和/或IL-23的抑制活性高于原研药20％以上。采用本发明的技术手段，可以显著的提高单克隆抗体对IL-12和/或IL-23的抑制活性。
2.使用本发明的方法生产的单克隆抗体，产量可以高达1.5g/L左右。
具体实施方式
白细胞介素-12(也称为IL-12、细胞毒性淋巴细胞成熟因子或自然杀伤细胞刺激因子)是由一个p35和一个p40亚基构成的异二聚体，很多研究结果表明，IL-12在促进Th1免疫应答及促进宿主对细胞内病原体的防御发挥关键作用。此外，IL-12在驱动Th1应答的过程中可以促进一些自身免疫性和炎症性疾病的发生。白细胞介素-23(IL-23)是由IL-12p40亚单位和其自身p19亚单位组成的异二聚体，其受体系统与IL-12R(β1和β2)共享了IL-12Rβ1亚单位，另外其特异性的受体亚单位为IL-23R。
IL-12和IL-23的生物效应彼此不同。IL-12是由激活的炎性细胞(单核细胞、巨噬细胞、嗜中性粒细胞、小胶质细胞、树突细胞)分泌的。在炎症过程中，IL-12诱导NK细胞(自然杀伤细胞)和T细胞产生干扰素-γ(IFN-γ)。然后IL-12可能与IFN-γ联合，诱导T细胞分化成Th1细胞。这种应答刺激了细胞免疫系统并最大化了巨噬细胞对病原体的杀灭效应和CD8+T细胞的增殖(Trinchieri 2003Nat Rev Immunol 3 133-46)。IL-12的过度产生与提高的促炎症反应活性和自体免疫典型的组织损伤相关(Leonard等人，1997Crit RevImmunol 17545-53)。IL-12产生的失调涉及以下疾病：银屑病(Yawalkar等人，1998JInvest Dermatol 111 1053-7)、克罗恩病(Neurath等人，1995J Exp Med 182 1281-90；Simpson等A,1998J Exp Med 187 1225-34；Camoglio等人，2002Eur J Immunol 32 261-9)、多发性硬化症(Fassbender等人，1998Neurology 51 753-8；Laman等人，1998JNeuroimmunol 86 30-45)、类风湿性关节炎(Kim等人，2000 Clin Exp Immunol 119 175-81)，此外还有其他的自身免疫疾病。
由于IL-12和IL-23含有共同的亚基，因此难以将疾病状态仅仅归因于一种白细胞介素或另一种的过度产生。然而，研究表明IL-23失调涉及以下疾病：银屑病(Lee等人，2004J Exp Med 199 125-30)、克罗恩病(Neurath 2007Nat Med 13 26-8)和多发性硬化症(Cua等人，2003Nature 421 744-8)，此外还有其他自身免疫疾病。
本发明所述的“抗IL-12和/或IL-23单克隆抗体或其抗原结合片段”是指能特异性的结合IL-12和/或IL-23的抗体或其抗原结合片段，优选地，抗IL-12和/或IL-23单克隆抗体或其抗原结合片段”结合IL-12和/或IL-23，引起IL-12和/或IL-23的生物活性抑制；更优选地，抗IL-12和/或IL-23单克隆抗体或其抗原结合片段为抗IL-12和IL-23单克隆抗体或其抗原结合片段，引起IL-12和/或IL-23的生物活性抑制；更优选地，所述单克隆抗体的重链可变区包含：氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的VH-CDR1、VH-CDR2和VH-CDR3；所述单克隆抗体的轻链可变区包含：氨基酸序列分别如SEQ IDNO:4、SEQ ID NO:5和SEQ ID NO:6所示的VL-CDR1、VL-CDR2和VL-CDR3；更优选地，所述单克隆抗体包含SEQ ID NO:7所示的重链可变区和SEQ ID NO:8所示的轻链可变区；最优选地，所述单克隆抗体是乌司奴单抗。如本发明所用的，术语“抑制”指的是抗体特异性结合细胞因子时，细胞因子的生物活性的降低。可以通过测量本领域熟知的IL-12和/或IL-23生物活性的一个或多个指示物来评估抗体对IL-12和/或IL-23的生物活性的抑制。
术语“乌司奴单抗”(ustekinumab)，是指商品名为
的全人源IgG1к抗体，其可以结合IL-12和IL-23的p40亚基，从而阻断患者体内的炎症反应。乌司奴单抗的重链和轻链序列如本文SEQ ID NO：9和SEQ ID NO：10所示。在欧美乌司奴单抗已经获得治疗中度至重度斑块型银屑病，活动性银屑病，以及中度至重度活动性克罗恩病的上市许可。在中国乌司奴单抗已被批准用于治疗中重度斑块型银屑病和成人克罗恩病适应症
在本发明中，使用“标品”一词来表示与生物相似产品进行比较，标品是已经获得市场认可的产品。在制备的抗体为乌司奴单抗的情况下，标品是已批准的
产品。
本文中使用的术语“CHO细胞”，源自中国仓鼠卵巢的上皮细胞(Tijo和Puck(1958)J.Exp.Med.108：259-271)；术语“基因工程改造的CHO细胞”指通过本领域技术人员已知的任何合适的遗传手段和方法修饰或改变CHO细胞，使得它们表达特定的蛋白。优选地，基因工程改造的CHO细胞可以表达所述单克隆抗体或其抗原结合片段。更优选地，基因工程改造的CHO细胞是指将含有所述单克隆抗体或其抗原结合片段的编码序列的重组表达载体转染至相应的CHO细胞株而得到的。优选地，所述CHO细胞是二氢叶酸还原酶(DHFR)基因敲除或谷氨酰胺合成酶(GS)基因敲除的CHO细胞；优选地，所述DHFR基因敲除的CHO细胞是CHO-DG44细胞。优选地，所述GS基因敲除的CHO细胞通过采用锌指核酸酶(ZFN)技术或利用CRISPR方法敲除GS基因而获得；更优选地，所述GS基因敲除的CHO细胞是
-GS细胞。
本文中，“CHO-DG44细胞”是指双等位二氢叶酸还原酶(DHFR)基因被敲除的CHO细胞，是Thermo-Fisher开发的目前广泛用于重组蛋白表达的哺乳动物细胞表达体系。本文中GS基因敲除的CHO细胞如采用锌指核酸酶(ZFN)技术或利用CRISPR方法敲除GS基因的CHO细胞，例如默克公司的
-GS哺乳动物细胞表达体系，采用
锌指核酸酶(ZFN)技术敲除CHO-K1细胞株的内源谷氨酰胺合成酶(GS)基因,从而使该细胞生长过程必须依赖外源L-谷氨酰胺的添加,形成天然的筛选标记,同时又摆脱了GS筛选系统对L-蛋氨酸砜亚胺(MSX)的依赖。未转入质粒时必须在有谷氨酰胺的培养基中才能存活，但是转入质粒后(质粒上有GS基因)可以在无谷氨酰胺的培养基中存活，因此筛选高表达细胞株时不用添加MSX等抗性筛选物质进行筛选，简化了筛选步骤，使本发明的方法更适用于工业扩大化生产。
发明人经过大量实验，意外地发现，使用DHFR(二氢叶酸还原酶)基因敲除的CHO细胞或GS(谷氨酰胺合成酶)基因敲除的CHO细胞，特别是CHO-DG44细胞或
-GS细胞来表达所述单克隆抗体，相对于SP2/0细胞及CHO-K1细胞表达的所述单克隆抗体，尤其是乌司奴单抗，生物学活性更高，并且产量高，免疫原性低，细胞稳定性好，筛选步骤简单。
本发明的制备所述单克隆抗体的方法包括以下步骤：
a)将所述单克隆抗体编码序列克隆至表达载体中，得到重组表达载体，优选地，所述表达载体含有选择标记基因，更优选地，所述选择标记基因是DHFR基因或GS基因；
b)将步骤a)获得的重组表达载体转入DHFR基因敲除的CHO细胞或GS基因敲除的CHO细胞，得到基因工程改造的CHO细胞；
c)培养步骤b)获得的基因工程改造的CHO细胞，选择表达所述抗体的单克隆细胞株；
d)培养c)步骤获得的表达所述抗体的单克隆细胞株，收获包含所述抗体的细胞培养液，分离纯化，获得所述单克隆抗体。
将抗体编码序列克隆至表达载体中，构建适合于在CHO细胞中表达抗体的重组表达载体的方法是本领域技术人员所熟知的，例如在Makrides et al.(1999)ProteinExpr.Purif.17:183-202和Kaufman(2000)Mol.Biotechnol.16:151-161中都有描述。所述表达载体是指在克隆载体基本骨架的基础上增加表达元件(如启动子、终止子等)，使目的基因能够表达的载体，如重组质粒等。优选地，所述表达载体含有选择标记基因。所述选择标记基因编码如下所述的蛋白：(a)赋予对抗生素或其他毒素的抗性，(b)补充营养缺陷型缺陷，或(c)提供不能从复合培养基中获得的关键营养物。更优选地，所述选择标记基因是二氢叶酸还原酶基因或谷氨酰胺合成酶基因。基因工程改造CHO细胞的方法是本领域技术人员所熟知的方法，特别包括用含有编码抗体的重链和轻链的基因的一个表达载体或含有编码抗体重链基因的第一表达载体和含有编码抗体轻链基因的第二表达载体共同转染CHO细胞。将重组表达载体转入CHO细胞的方法，包括电穿孔法、磷酸钙沉淀法、脂质体转染法、DEAE-葡聚糖转染法，也包括使用商业的转染试剂盒，如Thermo fisher Scientific公司的FreeStyleTMMAX Reagent等。
将获得的基因工程改造的CHO细胞在培养基中进行培养，获得能够高效表达抗体的单克隆细胞株。其中，对于DHFR(二氢叶酸还原酶)基因敲除的CHO细胞，根据转染的重组表达载体上所携带的选择标记基因需在目标培养基中加入相应的抑制剂，如甲氨蝶呤(Methotrexate、MTX)、
等，从而获得能够高效表达抗体的细胞株；对于GS(谷氨酰胺合成酶)基因敲除的CHO细胞，由于未转入质粒时必须在有谷氨酰胺的培养基中才能存活，而转入质粒后(质粒上有GS基因)可在无谷氨酰胺的培养基中存活，因此用无谷氨酰胺的培养基培养来获得高效表达抗体的细胞株，培养基中不用添加蛋氨酸亚氨基代砜(methionine sulfoximine,MSX)，
等抑制剂。然后再进行单克隆筛选，获得表达所述抗体的单克隆细胞株；优选地获得能够高效表达所述抗体的单克隆细胞株。
将获得的基因工程改造的CHO细胞在合适的培养基中培养，生产抗体。本文所描述的“培养基”，指含有哺乳动物细胞生长所需的营养物质的溶液。通常，培养基提供细胞所需的必需和非必需氨基酸、维生素、能量来源、脂类和微量元素。
对基因工程改造的CHO细胞有不同的培养模式，包括分批培养、连续培养和补料分批培养。在本发明中，优选使用补料-分批培养方式来生产抗体。在补料分批培养过程中，先以一定量的培养基开始培养，在后续的培养过程中，为防止营养成分耗尽，补充一种或多种营养成分，在该过程中没有任何产品从细胞培养基中移除。
基因工程改造的CHO细胞生产抗体后，由于从哺乳动物细胞中表达的重组蛋白，特别是抗体，通常在培养过程中被分泌到细胞培养液中，因此，在培养过程结束时，通过将含有抗体的细胞培养液与细胞分离从而收获抗体。细胞分离方法应尽量减少细胞裂解，避免细胞碎片增多以及释放蛋白酶和其他可能影响免疫球蛋白产品质量的分子。通常利用离心和/或过滤的手段获得包含抗体的细胞培养液，重组蛋白分别存在于上清液或滤液中。
在获取含有抗体的细胞培养液后，须从细胞培养液中纯化抗体。重组蛋白的纯化通常通过阴离子交换层析、阳离子交换层析、亲和层析、疏水作用层析、羟基磷灰石色谱等一系列色谱步骤来完成。重组抗体的纯化通常从ProteinA亲和层析开始，然后再进行一个或多个其他的色谱步骤，例如阳离子交换层析。进一步地，纯化步骤可包括超滤，渗滤等。
如前所述，本发明人发现，利用本发明的方法生产的乌司奴单抗比标品
产品具有更高的生物活性。乌司奴单抗的生物学活性表现为与IL-23结合能力、与IL-12结合能力、与FcRn结合能力和/或对IL-12和/或IL-23诱导的基因表达的抑制作用，尤其对IL-12和/或IL-23诱导的基因表达的抑制作用。乌司奴单抗与IL-23、IL-12或FcRn的结合能力可以用但不限于ELISA和生物层干涉法来评估；乌司奴单抗对IL-12诱导的基因表达的抑制作用可以通过对IL-12反应的细胞(如T淋巴细胞)，IL-12和抗体样本来研究，然后检测靶分子的产生，如干扰素-γ(IFN-γ)。靶分子干扰素-γ(IFN-γ)可以通过任何合适的方法(如ELISA)来检测和/或量化。
本发明所述方法生产的乌司奴单抗对产生IFN-γ的抑制作用大于标品对产生IFN-γ的抑制作用，并且本发明方法生产的乌司奴单抗对产生IFN-γ的抑制作用超出标品对产生IFN-γ的抑制作用20％以上。
本发明还涉及一种包含本发明所述方法制备得到的抗IL-12和/或IL-23单克隆抗体或其抗原结合片段的药物组合物。在本发明中药物组合物除包含所述单克隆抗体外，还包含至少一种药学上可接受的赋形剂。药学上可接受的赋形剂是指不影响活性物质如乌司奴单抗的生理活性，能够稳定药物成分和/或提高药物组合物的溶解度或降低其粘度的物质。单克隆抗体的常用药用辅料包括缓冲液、盐、糖或糖醇、氨基酸和表面活性剂。
在一个实施例中，一种药物组合物包含所述单克隆抗体、蔗糖、L-组氨酸、L-组氨酸盐酸盐一水合物和聚山梨酯80；优选地，所述药物组合物包含所述单克隆抗体90±9mg/mL，蔗糖76±7.6mg/mL、L-组氨酸0.5±0.3mg/mL、L-组氨酸盐酸盐一水合物1±0.5mg/mL和聚山梨酯80 0.04±0.004mg/mL。更优选地，所述药物组合物包含所述单克隆抗体90mg/mL，蔗糖76mg/mL、L-组氨酸0.636mg/mL、L-组氨酸盐酸盐一水合物1.237mg/mL和聚山梨酯800.04mg/mL。
在另一个实施例中，一种药物组合物包含所述单克隆抗体、蔗糖、L-组氨酸、L-组氨酸盐酸盐一水合物、EDTA二钠盐二水合物、L-蛋氨酸和聚山梨酯80。优选地，所述药物组合物包含：所述单克隆抗体5±0.5mg/mL，L-组氨酸0.77±0.077mg/mL,L-组氨酸盐酸盐一水合物1.03±0.10mg/mL，聚山梨酯80 0.4±0.04mg/mL，蔗糖85±8.5mg/mL，二水EDTA二钠0.02±0.002mg/ml和L-蛋氨酸0.4±0.04mg/ml。更优选地，所述药物组合物包含：所述单克隆抗体5mg/mL，L-组氨酸0.77mg/mL,L-组氨酸盐酸盐一水合物1.03mg/mL，聚山梨酯800.4mg/mL，蔗糖85mg/mL，二水EDTA二钠0.02mg/ml和L-蛋氨酸0.4mg/ml。
本发明所述的抗IL-12和/或IL-23单克隆抗体或其抗原结合片段或所述的药物组合物可以用于制备治疗IL-12和/或IL-23相关的疾病或病症的药物；优选地，所述疾病或病症选自斑块型银屑病、银屑病性关节炎、系统性红斑狼疮、克罗恩病、溃疡性结肠炎、多发性硬化症、结节病、中轴型脊柱关节炎和强直性脊柱炎。更优选地，所述疾病或病症选自斑块型银屑病、银屑病性关节炎和克罗恩病。
在一个实施例中本发明所述的抗IL-12和/或IL-23单克隆抗体或其抗原结合片段或所述的药物组合物可用于治疗斑块型银屑病，特别是治疗中至重度斑块型银屑病的成年人，对其他系统疗法(包括环孢菌素、甲氨蝶呤(MTX)或PUVA(补骨脂素和紫外线A)没有反应，或有禁忌，或不能耐受的患者，以及治疗12岁及以上青少年中至重度斑块型银屑病，对其他系统疗法或光疗疗法不能充分的控制或无法耐受的患者，以及治疗银屑病性关节炎，尤其是在治疗活动性银屑病时，对以往非生物疾病修饰抗风湿药物(DMARD)的治疗效果一直不够的成年患者。
在另外一个实施例中本发明所述的抗IL-12和/或IL-23单克隆抗体或其抗原结合片段或所述的药物组合物可用于治疗克罗恩病，特别是患有中度至严重活动性克罗恩病，对常规疗法或TNF-а拮抗剂反应不足、失去反应、无法耐受、或有医学禁忌症的成年患者。
本文描述为“包括”或“包含”一个或多个命名的要素或步骤的组合物或方法是开放式的，意味着所述命名的元件或步骤是必要的，但可在所述组合物或方法的范围内添加其他元件或步骤。在本文公开的任何组合物或方法中，任何命名的基本元件或步骤的已知或公开的等同物可取代所述元素或步骤。
具体实施方式
实施例1表达乌司奴单抗的CHO-DG44细胞株构建
载体构建
(1)表达抗体重链蛋白载体的制备
在乌司奴单抗重链的编码序列(SEQ ID No:11)两端设计BamHl和Xhol两个酶切位点，分别与表达载体pCDNA3.1(购自Thermo Fisher Scientific公司)上的BamHl和Xhol酶切位点连接，实现目标蛋白编码DNA片段与表达载体的连接，获得重组质粒DNA。将重组质粒DNA导入大肠杆菌DH5а(购自天根生化科技有限公司)，提取单菌落质粒送去测序，获得测序结果正确的质粒，记作重组质粒pCDNA-HC。
(2)表达抗体轻链蛋白载体的制备
在乌司奴单抗轻链的编码序列(SEQ ID No:12)两端设计了HindIII和Xhol两个酶切位点，分别与表达载体pOptiVEC(购自Thermo Fisher Scientific公司)上的HindIII和Xhol酶切位点连接，实现目标蛋白编码DNA片段与表达载体的连接，获得重组质粒DNA，将重组质粒DNA导入大肠杆菌DH5а(购自天根生化科技有限公司)，提取单菌落质粒送去测序，获得测序结果正确的质粒，记作重组质粒pOptiVec-LC。
表达乌司奴单抗的CHO-DG44细胞株构建
将重组质粒pOptiVec-LC和pCDNA-HC分别线性化后，同时转染至CHO-DG44细胞(购自Thermo Fisher Scientific公司)，培养得到高表达目的蛋白的细胞株。具体步骤如下：
(1)重组质粒转染CHO-DG44细胞
利用重组质粒pOptiVec-LC和pCDNA-HC上PvuI酶切位点将质粒线性化。采用乙醇沉淀法，浓缩酶切后的质粒DNA至浓度为1ug/ul。
取1.5×107个CHO-DG44细胞置换到30mL、预热至37℃的CD DG44培养基(购自Thermo Fisher Scientific公司)中，混合均匀；将60ug质粒DNA(pcDNA-HC与pOptiVec-LC摩尔比＝1:1)和15uL FreeStyleTMMAX Reagent(购自Thermo Fisher Scientific公司)放入1.2mL OptiPROTMSFM培养基(购自Thermo Fisher Scientific公司)并轻轻混匀，室温孵育10min，得到DNA-Reagent复合物。将1.2mL DNA-Reagent复合物加入到125mL CHO-DG44细胞摇瓶中，轻轻晃动摇瓶，将摇瓶放置在摇床中，在37℃、含8％CO2、转速为130-135rpm条件下进行培养。转染48小时后，将细胞传代至次黄嘌呤/胸腺嘧啶(HT)缺陷型CD OptiCHOTM完全培养基(购自Gibco Life Technologies公司)。
(2)筛选稳定转染细胞
第一轮
筛选：对上一步骤得到的细胞系进行计数；在预热的含有500ug/mL
试剂(购自Thermo Fisher Scientific公司)的CD OptiCHOTM完全培养基(购自Gibco Life Technologies公司)中稀释细胞，使细胞的最终密度达5×105个活细胞/mL；将培养瓶置于培养箱中，在37℃，含8％CO2的湿化空气条件下，转速为130-135rpm条件下培养。每2-3天更换适量的新鲜培养基，保持14-21天，直至细胞存活率>90％。
第二轮MTX梯度筛选：对第一轮筛选完毕的细胞进行计数，1000rpm离心5分钟，然后吸弃旧培养基，将细胞接种至含有50nM MTX的CD OptiCHOTM完全培养基(购自Gibco LifeTechnologies公司；含500μg/mL
)，接种密度为3×105细胞/mL。将培养瓶置于培养箱中，在37℃，含8％CO2的湿化空气条件下，转速为130-135rpm条件下培养。每2-3天更换适量的新鲜培养基，在细胞活率大于80％的时候，将细胞接种至含有100nM MTX的CDOptiCHOTM完全培养基(购自Gibco Life Technologies公司；含500μg/mL
)，接种密度为3×105细胞/mL,将培养瓶置于培养箱中，在37℃，含8％CO2的湿化空气条件下，转速为130-135rpm条件下培养。每2-3天更换适量的新鲜培养基，在细胞活率大于80％的时候，将细胞接种至含有500nM MTX的CD OptiCHOTM完全培养基(购自Gibco LifeTechnologies公司；含500μg/mL
)，接种密度为3×105细胞/mL，总共的培养时间20-30天，直至细胞存活率>90％。
(3)单克隆筛选及扩大培养
将上一步骤得到的细胞用克隆培养基EX-CELL CHO Cloning Medium(购自SigmaAldrich公司)梯度稀释至1000个活细胞/mL，接种至96孔板中，每孔0.8个细胞。将接种的96孔板放置于37℃、含有5％CO2的空气条件下的培养箱中静置孵育培养板10-14天，在本实施例中是11天，使用显微镜检查孔中单克隆集落的生长情况。
当96孔板中细胞生长到汇合度为80％时，采用ELISA方法检测各个克隆的产量，具体采用双抗体夹心法测定发酵液中乌司奴单抗的含量，包被抗体为鼠源单克隆抗人IgG Fc段抗体(购自Sigma，货号I6260-.5mL)，二抗为HRP标记的鼠源单克隆抗人IgG Fab段抗体(购自金斯瑞生物科技有限公司，货号A01855)，所使用标准品为原研药
稀释最大浓度为1ug/mL，二倍梯度稀释12个标准品。
筛选产量高的前25％克隆进行克隆扩增，无菌收集想要的单克隆细胞，放大至24孔板。3-5天后，使用相同检测方法，选择前10％产量较高的目标克隆转移至6孔板中，继续培养。使用相同的检测方法，选择6孔板中前10％产量的单克隆细胞并将各细胞株单独转入T-25培养瓶中进行扩大培养，当T-25培养瓶中细胞数达到1*107后，将细胞转移至125mL培养瓶。在37℃和含有8％CO2的空气条件下，振荡速度为130-135rpm，采用补料-分批培养模式培养,培养过程中添加补料培养基DynamisTM Medium(购自Thermo Fisher公司)，培养14天后，收集高表达乌司奴单抗的CHO-DG44细胞株的细胞发酵液。
实施例2表达乌司奴单抗的
-GS细胞株构建
载体构建
(1)表达抗体重链蛋白与载体连接
在乌司奴单抗重链的编码序列两端设计BstB I和Pac I两个酶切位点，分别与表达载体pCGS(购自Merck KGaA公司)上的BstB I和Pac I酶切位点连接，实现目标蛋白编码DNA片段与表达载体的连接，获得重组质粒DNA。将重组质粒DNA导入大肠杆菌DH5а(购自天根生化科技有限公司)，涂布在含有100ug/ml氨苄青霉素(终浓度)的LB琼脂培养基(配制每升培养基,950ml去离子水中加入:胰蛋白胨10g，酵母提取物5g，NaCl10g，然后加去离子水至1L)。将获得的克隆在含有100ug/ml氨苄青霉素(终浓度)的LB液体培养基中培养，用质粒提取试剂盒(购自天根生化科技有限公司)提取质粒，进行核酸测序鉴定，选择测序结果正确的质粒，记作重组质粒pCGS-HC。
(2)表达抗体轻链蛋白与载体连接
在乌司奴单抗轻链的编码序列两端设计了HindIII和Xhol两个酶切位点，分别与重组质粒pCGS-HC上的HindIII和Xhol酶切位点连接，实现目标蛋白编码DNA片段与表达载体的连接，获得新的重组质粒DNA。将重组质粒DNA导入大肠杆菌DH5а(购自天根生化科技有限公司)，涂布在含有100ug/ml氨苄青霉素(终浓度)的LB琼脂培养基(配制每升培养基,950ml去离子水中加入:胰蛋白胨10g，酵母提取物5g，NaCl 10g，然后加去离子水至1L)。将获得的克隆在含有100ug/ml氨苄青霉素(终浓度)的LB液体培养基中培养，用质粒提取试剂盒(购自天根生化科技有限公司)提取质粒，进行核酸测序鉴定，选择测序结果正确的质粒，记作重组质粒pCGS-HC-LC。
细胞系筛选
将重组质粒pCGS-HC-LC转染至
-GS细胞(购自Merck KGaA公司)，经过筛选得到高表达目的蛋白的细胞株。具体步骤如下：
(1)重组质粒转染
-GS细胞
a.转染前一天细胞传代至转染时密度为1×106活细胞/mL。
b.细胞计数，每个样品所需总细胞数为6×106，取所需细胞于50mL离心管，1000rpm离心10min。
c.每个样品用600μLEX-CELL CD CHO培养基(购自Sigma Aldrich公司)重悬细胞，加入40μg质粒(质粒浓度>1μg/μL)混匀后，与电转杯一起放入4℃冰箱预冷。
d.将加有质粒的细胞吹打均匀后加入电转杯中，电压300V进行电转，完成后加入37℃预热的含6mM谷氨酰胺的培养基EX-CELL CD CHO Fusion(购自Sigma Aldrich公司)中，放入二氧化碳培养箱静置培养48h。
(2)筛选稳定转染细胞
48h后，将细胞传代至不含谷氨酰胺的培养基EX-CELL CD CHO Fusion(购自SigmaAldrich公司)中。每2天传代一次，每代细胞以“P1”，“P2”...“Pn”加以标注，其中n为细胞代次。每次传代前，取40μL细胞悬浮液用Countstar IC 1000自动细胞计数仪进行细胞计数及细胞存活率分析，测定每一代细胞的活细胞密度和活率。当细胞活率>95％时，完成筛选过程。本过程也可通过筛选细胞池(minipool)的方法完成。
(3)单克隆筛选及扩大培养
将上一步骤得到的细胞接种至96孔板中，每孔0.5–1个细胞。
a.取400mL的完全克隆培养基EX-CELL CHO Cloning Medium(购自Sigma Aldrich公司)，预热至37℃；
b.将待铺板的细胞进行计数，并计算所需细胞个数，即20块96孔板，以每孔0.5个细胞计算，需要960个细胞；
c.使用上述完全克隆培养基连续稀释细胞至最终浓度1000个活细胞/mL，取960uL细胞稀释液至预热好的克隆培养基中；
d.轻轻混匀细胞悬液，转移至无菌加样槽中；使用多通道移液器，在无菌条件下将200μL稀释细胞分配至96孔板中；
e.在37℃、含有5％CO2的空气条件下的培养箱中静置孵育培养板10-14天，在本实施例中是11天，使用显微镜检查孔中单克隆集落的生长情况。
当96孔板中细胞生长到汇合度为80％时，采用实施例1中相同的ELISA方法检测各个克隆的产量，筛选生产率高的前25％克隆进行克隆扩增，无菌收集想要的克隆，轻轻上下吹打并转移每个孔中的全部内容物至24孔板的各个孔中，24孔板的每个孔中含有1mL的新鲜培养基EX-CELL CD CHO Fusion(购自Sigma Aldrich公司)。
3-5天后，使用相同检测方法，选择前10％产率较高的目标克隆转移至6孔板中，无菌收集想要的克隆，并向6孔板的每个孔中加入新鲜培养基EX-CELL CD CHO Fusion，最终每个孔的培养体积均为3mL。
使用相同的检测方法，选择6孔板中前10％产率的单克隆细胞系并将各细胞株单独转入T-25培养瓶中进行扩大培养，当T-25培养瓶中细胞数达到1*107后，将细胞转移至125mL培养瓶。在37℃和含有8％CO2的空气条件下，振荡速度为130-135rpm，采用补料-分批培养模式培养,
培养过程中添加补料培养基EX-CELL Advanced CHO Feed 1(购自Sigma Aldrich公司)，培养14天后，收集高表达乌司奴单抗的
-GS细胞株的细胞发酵液。
实施例3抗体纯化步骤
将收集到的高表达乌司奴单抗的CHO-DG44细胞株和
-GS细胞株的细胞发酵液，分别进行以下操作：
1、离心
将离心机转速设置为3000rpm，离心10min，去除细胞；接着将离心机转速设置为15000rpm，离心20min，去除细胞碎片。本步骤还可以采用深层过滤的方法使细胞发酵液澄清。
2、微滤
使用颇尔公司的除菌级过滤器(孔径0.22μm，材料聚醚砜)对样品进行微滤。
3、洗脱
仪器：AKTA蛋白纯化仪和Mabselect sure LX填料(均购买自GE公司)
试剂：平衡液：150mM NaCl、20mM磷酸缓冲液pH7.4
淋洗液1：1M NaCl 20mM柠檬酸钠pH5.5
淋洗液2：20mM柠檬酸钠pH5.5
洗脱液：20mM柠檬酸钠pH3.0
再生液：0.1M NaOH
步骤：1)使用上述平衡液进行平衡；2)上样；3)淋洗：依次用淋洗液2，淋洗液1，淋洗液2淋洗；4)使用上述洗脱液洗脱；5)使用0.5M氢氧化钠进行原位清洗(CIP)；6)再平衡：使用上述平衡液进行平衡。
使用CHO-DG44细胞株生产乌司奴单抗最终得到纯度为97％的乌司奴单抗产物，产量为1.5g/L左右。
使用
-GS细胞株的细胞生产乌司奴单抗最终得到纯度为98％的乌司奴单抗产物，产量为1.5g/L左右。
实施例4CHO-DG44细胞表达的乌司奴单抗对IL-12抑制活性
T细胞的分离和活化
将购买自雷德倍尔公司的外周血单个核细胞(PBMC)按照EasySep Human T CellIsolation Kit说明书操作，用磁珠分选试剂(STEM cell)去除非T细胞，分选出总CD3+T细胞。T细胞按照浓度1x106/mL培养在12-well板中，培养基为含有10％FBS(胎牛血清)(GibcoLife Technologies公司)的1640培养基(购自Sigma Aldrich公司)或者无血清培养基TexMACX medium(购自Miltenyi Biotec公司)，在培养T细胞时，加入刺激剂anti-CD3/CD28抗体(购自Thermo Fisher Scientific公司)，同时加入IL-2(30u/mL)(购自Thermo FisherScientific公司)。
将1.0×106ml-1T细胞用磁珠预刺激，使其产生γ-干扰素(γ-IFN)，刺激2天后，第3天通过离心换液把细胞浓度调为0.5×106ml-1(换液至TexMACX medium中)。
IL-12 ELISA
使用人IFN-γELISA试剂盒(购自上海依科赛公司)，从已平衡至室温的密封袋中取出试验所需板条，留空白孔。将上一步得到的T细胞调节成0.5×105/孔，再分别将IL-12、本发明的乌司奴单抗样品(以下简称为“样品”)和标品按照表1所示的量加入相应孔中(100ul/孔)，按照表1设计进行细胞活性实验。用封板胶纸封住反应孔，37℃孵箱孵育90分钟。洗板5次，除空白孔外，加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，37℃孵箱孵育60分钟。洗板5次，除空白孔外，加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔，37℃孵箱，避光孵育30分钟。洗板5次，加入显色底物(包括空白孔)100ul/孔，37℃孵箱，避光孵育15分钟。加入终止液(包括空白孔)100ul/孔，混匀后即刻测量OD450nm值。
表1乌司奴单抗样品的细胞活性实验及结果
抑制率＝(Abs(IL-12平均值)–Abs(抗体))/(Abs(IL-12平均值)–Abs(细胞上清液平均值))
根据表1的数据结果和抑制率计算公式得到CHO-DG44表达的乌司奴单抗样品对IL-12活性的抑制率为43.19％，标品对IL-12活性的抑制率为34.9％，CHO-DG44表达的乌司奴单抗样品比乌司奴单抗标品对IL-12抑制活性高23.753％。
实施例5
-GS细胞表达的乌司奴单抗对IL-12抑制活性
T细胞的分离和活化
将购买自雷德倍尔公司的外周血单个核细胞(PBMC)按照EasySep Human T CellIsolation Kit说明书操作，用磁珠分选试剂(STEM cell)去除非T细胞，分选出总CD3+T细胞。T细胞按照浓度1x106/mL培养在12-well板，培养基为X-vivo(购自Lonza公司)，在培养T细胞时，加入刺激剂anti-CD3/CD28抗体(购自Thermo Fisher Scientific公司)，同时加入IL-2(30u/mL)(购自Thermo Fisher Scientific公司)。
调节T细胞浓度为1×106个/mL。将1.0×106ml-1T细胞用磁珠预刺激，使其活化，产生γ-干扰素(γ-IFN)，刺激2天后，第3天通过离心换液把细胞浓度调为0.3×106ml-1。
IL-12ELISA
本步骤使用人IFN-γELISA试剂盒(购自上海依科赛公司)，从已平衡至室温的密封袋中取出试验所需板条96孔板，将IL-12按表2所示的量加入96孔板中(50uL/孔)，再将本发明的乌司奴单抗样品(以下简称为“样品”)和标品按照表2所示的量加入相应孔中(50ul/孔)，培养箱孵育1h后，将上一步得到的T细胞离心去除原培养基，用X-vivo调节成0.3×105/孔，再分别加入相应孔中(100uL/孔)。用封板胶纸封住反应孔，37℃孵箱孵育90分钟。洗板5次，加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，37℃孵箱孵育60分钟。洗板5次，加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔，37℃孵箱，避光孵育30分钟。洗板5次，加入显色底物100ul/孔，37℃孵育箱，避光孵育15分钟。加入终止液100ul/孔，混匀后即刻测量OD450nm值。
表2乌司奴单抗样品的细胞活性实验及结果
根据表2中的数据，利用如下抑制率计算公式，计算得到如表3各个抗体的抑制率。
抑制率＝(Abs(IL-12)–Abs(标品))/(Abs(IL-12)–Abs(细胞上清液))
表3实验结果处理
| 抗体 | 0.5ug标品 | 0.5ug样品 | 1ug标品 | 1ug样品 |
| 抑制率(％) | 9.52 | 11.67 | 27.64 | 37.98 |
计算可得，使用0.5ug乌司奴单抗进行上述实验时，
-GS表达的乌司奴单抗样品比乌司奴单抗标品对IL-12抑制活性高22.58％，使用1ug乌司奴单抗进行上述实验时，
-GS表达的乌司奴单抗样品比乌司奴单抗标品对IL-12抑制活性高37.41％。
通过实施例4和实施例5实验结果，可以看出本发明的乌司奴单抗样品比乌司奴单抗标品对IL-12的抑制活性高出20％以上。
如无特别注明，本发明具体实施例中所用原料和试剂均可通过市售获得。
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。
序列表
<110> 甘李药业股份有限公司
<120> 一种制备抗IL-12和/或IL-23单克隆抗体的方法
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Thr Tyr Trp Leu Gly
1 5
<210> 2
<211> 17
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe Gln
1 5 10 15
Gly
<210> 3
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe
1 5 10
<210> 4
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala
1 5 10
<210> 5
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ala Ala Ser Ser Leu Gln Ser
1 5
<210> 6
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr
1 5
<210> 7
<211> 119
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile
35 40 45
Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 8
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 9
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Thr Tyr
20 25 30
Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Asp Trp Ile
35 40 45
Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr
65 70 75 80
Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ser Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 10
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 11
<211> 1413
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gccaccatgg agttcggcct gtcctgggtg ttcctggtgg ccctgttcag gggcgtgcag 60
tgtgaggtgc agctggtgca gtccggcgcc gaggtgaaga agcctggcga gtccctgaag 120
atcagctgca agggctccgg ctactccttc accacatact ggctgggctg ggtgaggcag 180
atgcccggca agggcctgga ttggatcggc atcatgagcc ccgtggatag cgatattagg 240
tacagcccta gcttccaggg ccaggtgaca atgtccgtgg ataagagcat cacaaccgcc 300
tacctgcagt ggaacagcct gaaggccagc gacaccgcca tgtactactg cgccaggaga 360
agacccggcc agggctactt cgacttctgg ggccagggca ccctggtgac agtgagcagc 420
agcagcacca agggccctag cgtgttcccc ctggccccct ctagcaagtc cacctccggc 480
ggcaccgccg ctctgggatg tctggtgaag gactacttcc ccgagcccgt gaccgtgagc 540
tggaactccg gcgccctgac atccggcgtg cacacattcc ccgccgtgct gcagtccagc 600
ggcctgtact ccctgagctc cgtggtgaca gtgccctcca gctccctggg cacccagacc 660
tacatctgca acgtgaacca caagcctagc aacacaaagg tggacaagag ggtggagccc 720
aagagctgcg acaagacaca cacctgccct ccctgtcctg cccctgagct gctgggcgga 780
ccttccgtgt tcctgttccc ccctaagccc aaggacacac tgatgatctc cagaacaccc 840
gaggtgacat gtgtggtggt ggatgtgagc cacgaggacc ccgaggtgaa gttcaactgg 900
tacgtggacg gcgtggaggt gcacaacgcc aagacaaagc ctcgggagga gcagtacaac 960
tccacataca gagtggtgtc cgtgctgacc gtgctgcacc aggattggct gaacggcaag 1020
gagtacaagt gcaaggtgag caacaaggcc ctgcccgccc ctatcgagaa gaccatctcc 1080
aaggccaagg gccagcccag ggagccccag gtttacacac tgccccccag cagagatgag 1140
ctgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgatatt 1200
gccgtggagt gggagagcaa cggccagccc gagaacaact acaagaccac accccccgtg 1260
ctggatagcg atggcagctt cttcctgtac agcaagctga ccgtggataa gtccagatgg 1320
cagcagggca acgtgttcag ctgttccgtg atgcacgagg ccctgcacaa ccactacaca 1380
cagaagagcc tgtccctgag ccctggcaag tga 1413
<210> 12
<211> 717
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gccaccatgg acatgagggt gcctgcccag ctgctgggcc tgctgctgct gtggctgagc 60
ggcgctagat gcgatattca gatgacacag tcccctagca gcctgagcgc cagcgtgggc 120
gatagagtga ccatcacatg cagggccagc cagggcatct ccagctggct ggcctggtac 180
cagcagaagc ctgagaaggc ccctaagtcc ctgatctacg ccgcctccag cctgcagagc 240
ggcgtgccta gcaggttctc cggcagcggc agcggcacag atttcaccct gacaatcagc 300
tccctgcagc ccgaggactt cgccacctac tactgccagc agtacaacat ctacccctac 360
acattcggcc agggcaccaa gctggagatc aagaggacag tggccgcccc cagcgtgttc 420
atcttccctc ctagcgacga gcagctgaag agcggcacag cctccgtggt gtgtctgctg 480
aacaacttct accctcggga ggccaaggtg cagtggaagg tggacaacgc cctgcagtcc 540
ggcaactccc aggagagcgt gaccgagcag gatagcaagg actccaccta ctccctgagc 600
tccacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaggtg 660
acccaccagg gcctgtccag ccccgtgacc aagagcttca acagaggcga gtgctga 717
Claims (15)
1.一种制备抗IL-12和/或IL-23单克隆抗体或其抗原结合片段的方法，包括使用DHFR基因敲除或GS基因敲除的CHO细胞表达所述单克隆抗体或所述抗原结合片段，所述单克隆抗体或所述抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的VH-CDR1、VH-CDR2和VH-CDR3；所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的VL-CDR1、VL-CDR2和VL-CDR3。
2.根据权利要求1所述的方法，所述抗IL-12和/或IL-23单克隆抗体或其抗原结合片段包含SEQ ID NO:7所示的重链可变区和SEQ ID NO:8所示的轻链可变区。
3.根据权利要求2所述的方法，所述单克隆抗体是乌司奴单克隆抗体。
4.根据权利要求1-3任一项所述的方法，所述方法包括以下步骤：
a)将所述单克隆抗体编码序列克隆至表达载体中，得到重组表达载体，优选地，所述表达载体含有选择标记基因，更优选地，所述选择标记基因是DHFR基因或GS基因；
b)将步骤a)获得的重组表达载体转入DHFR基因敲除的CHO细胞或GS基因敲除的CHO细胞，得到基因工程改造的CHO细胞；
c)培养步骤b)获得的基因工程改造的CHO细胞，选择表达所述抗体的单克隆细胞株；
d)培养c)步骤获得的表达所述抗体的单克隆细胞株，收获包含所述抗体的细胞培养液，分离纯化，获得所述单克隆抗体。
5.根据权利要求1-4任一项所述的方法，所述DHFR基因敲除的CHO细胞是CHO-DG44细胞。
6.根据权利要求1-4所述的方法，所述GS基因敲除的CHO细胞通过采用锌指核酸酶(ZFN)技术或利用CRISPR方法敲除GS基因而获得；优选，所述GS基因敲除的CHO细胞是
-GS细胞。
7.一种抗IL-12和/或IL-23单克隆抗体或其抗原结合片段，由权利要求1-6中任一项所述的方法制备得到。
8.一种药物组合物，所述药物组合物包含权利要求1-6中任一项所述方法制备得到的单克隆抗体、蔗糖、L-组氨酸、L-组氨酸盐酸盐一水合物和聚山梨酯80。
9.根据权利要求8所述的药物组合物，所述药物组合物包含所述单克隆抗体90±9mg/mL、蔗糖76±7.6mg/mL、L-组氨酸0.5±0.3mg/mL、L-组氨酸盐酸盐一水合物1±0.5mg/mL和聚山梨酯800.04±0.004mg/mL。
10.根据权利要求9所述的药物组合物，所述药物组合物包含所述单克隆抗体90mg/mL、蔗糖76mg/mL、L-组氨酸0.636mg/mL、L-组氨酸盐酸盐一水合物1.237mg/mL和聚山梨酯800.04mg/mL。
11.一种药物组合物，所述药物组合物包含权利要求1-6中任一项所述方法制备得到的单克隆抗体、L-组氨酸、L-组氨酸盐酸盐一水合物、聚山梨酯80、蔗糖、EDTA二钠盐二水合物和L-蛋氨酸。
12.根据权利要求11所述的药物组合物，所述药物组合物包含所述单克隆抗体5±0.5mg/mL、L-组氨酸0.77±0.077mg/mL、L-组氨酸盐酸盐一水合物1.03±0.10mg/mL、聚山梨酯80 0.4±0.04mg/mL、蔗糖85±8.5mg/mL、EDTA二钠盐二水合物0.02±0.002mg/ml和L-蛋氨酸0.4±0.04mg/ml。
13.根据权利要求12所述的药物组合物，所述药物组合物包含所述单克隆抗体5mg/mL、L-组氨酸0.77mg/mL、L-组氨酸盐酸盐一水合物1.03mg/mL、聚山梨酯80 0.4mg/mL、蔗糖85mg/mL、EDTA二钠盐二水合物0.02mg/ml和L-蛋氨酸0.4mg/ml。
14.权利要求1-6中任一项所述方法制备得到的抗IL-12和/或IL-23单克隆抗体或抗原结合片段、或权利要求8-13中任一项所述的药物组合物在制备用于治疗IL-12和/或IL-23相关的疾病或病症的药物中的用途。
15.根据权利要求14所述的用途，所述疾病或病症选自斑块型银屑病、银屑病性关节炎、克罗恩病、系统性红斑狼疮、溃疡性结肠炎、多发性硬化症、结节病、中轴型脊柱关节炎和强直性脊柱炎；优选地，所述疾病或病症选自斑块型银屑病、银屑病性关节炎和克罗恩病。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024092203A1 (en) * | 2022-10-28 | 2024-05-02 | Amgen Inc. | Compositions of anti-interleukin 12/interleukin 23 antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100021911A1 (en) * | 2008-07-23 | 2010-01-28 | Boehringer Ingelheim Pharma Gmbh & Co Kg | Production host cell lines |
| CN102876684A (zh) * | 2012-09-17 | 2013-01-16 | 青岛康立泰药业有限公司 | 人白细胞介素-12的编码基因、真核宿主细胞和表达方法 |
| WO2020183418A1 (en) * | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
-
2022
- 2022-01-29 CN CN202210109484.8A patent/CN114395041B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100021911A1 (en) * | 2008-07-23 | 2010-01-28 | Boehringer Ingelheim Pharma Gmbh & Co Kg | Production host cell lines |
| CN102876684A (zh) * | 2012-09-17 | 2013-01-16 | 青岛康立泰药业有限公司 | 人白细胞介素-12的编码基因、真核宿主细胞和表达方法 |
| WO2020183418A1 (en) * | 2019-03-14 | 2020-09-17 | Janssen Biotech, Inc. | Manufacturing methods for producing anti-il12/il23 antibody compositions |
Non-Patent Citations (2)
| Title |
|---|
| JIANWEI ZHU等: "Mammalian cell protein expression for biopharmaceutical production", BIOTECHNOLOGY ADVANCES * |
| 杨林等: "细胞因子对类风湿性关节炎骨平衡的调节作用", 生命科学 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024092203A1 (en) * | 2022-10-28 | 2024-05-02 | Amgen Inc. | Compositions of anti-interleukin 12/interleukin 23 antibody |
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