CN101484472B - 改善的sgp130Fc二聚体 - Google Patents
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- CN101484472B CN101484472B CN2007800248791A CN200780024879A CN101484472B CN 101484472 B CN101484472 B CN 101484472B CN 2007800248791 A CN2007800248791 A CN 2007800248791A CN 200780024879 A CN200780024879 A CN 200780024879A CN 101484472 B CN101484472 B CN 101484472B
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Abstract
本发明描述了包含两个可溶gp130分子的多肽二聚体,其中所述分子每一个融合到IgG1蛋白的Fc结构域,并且其中对所述Fc结构域的铰链区进行修饰,得到所述二聚体的有利性质。在特别优选的实施方案中,所述铰链区包含氨基酸序列基序Ala234-Glu235-Gly236-Ala237。此外,本发明描述了包含所述二聚体的药物组合物以及各种医学应用。
Description
本发明涉及一种多肽二聚体,所述多肽二聚体包含两个可溶的gp130分子,每一个gp130分子融合到IgG1蛋白的Fc结构域,其中对所述Fc结构域的铰链区进行修饰,导致所述二聚体的有利性质。本发明还涉及包含所述二聚体的药物组合物和各种医学应用。
多效细胞因子白细胞介素-6(IL-6)表现出广谱生物功能,其中在肝脏中B细胞的刺激和急性期蛋白质合成的诱导通常是显著的。IL-6通过与IL-6特异性表面受体(IL-6R)结合而在靶细胞上发挥其活性。这种受体/配体复合物促进gp130的同型二聚化,gp130是IL-6受体复合物的第二亚基。gp130的二聚化导致IL-6信号的传导。当与IL-6复合时,通过两种机制(可变剪接和释放)产生的可溶形式的IL-6R(sIL-6R)还能够引起gp130的二聚化和信号传导。
由于IL-6R的细胞质部分不有助于信号转导,由gp130同型二聚体导致的信号传导可以由处于与结合的膜或可溶IL-6R的复合物中的IL-6所诱导。然而,sIL-6R的存在导致IL-6反应细胞对所述配体的致敏。此外,当存在于培养基中的sIL-6R被连续去除时,严格IL-6依赖型杂交瘤细胞不响应于非常低量的IL-6进行增殖。
最初被描述为白细胞介素-6的信号转导物,gp130是许多细胞因子如IL-6、IL-11、白血病抑制因子(LIF)、制癌蛋白M(OSM)和睫状神经营养因子(CNTF)共享的转导物链。所有这些细胞因子通过二-或三联受体复合物作用,在所述复合物中信号传导由gp130的同型二聚化(对于IL-6)或gp130与LIF-R(对于LIF,CT-1,OSM,CLC和CNTF)或OSM-R(对于OSM)异型二聚化引发。因此,这些细胞因子可以在各种组织中介导相似的生物活性。
尽管可以在几乎所有细胞类型上发现gp130,但是IL-6R表现出更加局限得多的表达。一种细胞类型释放sIL-6R使得只表达gp130的其它细胞响应于IL-6。这种关系叫作反式信号传导。事实上,已经描述了一些需要 sIL-6R和IL-6的复合物并且单独利用IL-6不能观察到的细胞活性。在人血浆中发现高浓度的可溶的gp130蛋白。最近已经描述了设计的细胞因子超-IL-6(super-IL-6)(H-IL-6),其中sIL-6R的C-端通过柔性的肽接头共价融合到成熟IL-6的N-端。如利用复合物IL-6/sIL-6R可见,H-IL-6还在仅表达gp130的细胞上作用。与分离的成分IL-6和sIL-6R相反,100-1000倍更低浓度的这种融合分子足以诱导相当的生物学信号。
为了治疗各种疾病或病症,对依赖于可溶IL-6R的IL-6反应的特异性阻断可能是需要的。这样的疾病包括骨吸收、高钙血症、恶病质、肿瘤或其它类型的癌症(例如,结肠癌、多发性骨髓瘤、淋巴瘤、白血病、霍奇金病)、自体免疫病(例如,多发性硬化(MS)或1型糖尿病)、炎性或特应性疾病(例如,局限性回肠炎、溃疡性结肠炎、类风湿性关节炎、青少年类风湿性关节炎、哮喘、银屑病、结节病、红斑狼疮或葡萄膜炎)、感染(例如,细菌、病毒、真菌、或其它病原体感染)、以及内分泌紊乱和代谢病或分解代谢疾病(例如,2型糖尿病、肥胖症、高血糖症或高胆固醇血症)。发现例如sgp130二聚体或sgp130Fc二聚体对治疗应用是有效的。
本发明潜在的技术问题是提供改善的sgp130Fc二聚体。
所述技术问题的解决通过提供权利要求中表征的实施方案而实现。在导致本发明的实验过程中,发现sgp130Fc融合蛋白的生物活性、可纯化性和稳定性显著地取决于sgp130和Fc部分之间的铰链区的氨基酸组成。将人IgG1-Fc的氨基酸234,235和237(按照EU编号)突变,以减少Fc受体与该基序的结合(Duncan等,自然(Nature)(1988),332:563-564;Canfield和Morrison,实验医学杂志(J.Exp.Med.)(1991),173:1483-1491;Wines等,免疫学杂志(J.Immunol.)(2000),164:5313-5318;Sondermann等,自然(Nature)(2000),406:267)。出乎意料地,通过用谷氨酸(Glu,E)或天冬氨酸(Asp,D)替换野生型序列Leu234-Leu235-Gly236-Gly237的Leu235,并且由此用强亲水性(带电荷的)氨基酸破坏该疏水性基序,可能提高sgp130Fc融合蛋白的生物活性和稳定性。在位置234和237的突变增加这种作用。最有效的突变体特征为序列Ala234-Glu235-Gly236-Ala237。
附图简述
图1:sgp130Fc的铰链区突变蛋白
人IgG1-Fc的下游铰链区(lower hinge region)通过位点定向诱变进行修饰。理想的序列,如在本实验中确定,为“AEGA”(其结合在化合物CR5/18中)。
缩写和符号:aa,氨基酸(s);C,形成Fc融合蛋白的二聚化所需要的两个二硫键的半胱氨酸;X,丙氨酸(Ala,A)或苯丙氨酸(Phe,F);Z,谷氨酸(Glu,E)或天冬氨酸(Asp,D)。
图2:野生型sgp130Fc和CR5/18的大小排阻层析洗脱曲线
与野生型sgp130Fc相比,CR5/18表现出显著减少量的聚集物(aggregate),并且因此显示更高产率的未污染产物。
图3:由MTS细胞存活测定确定的CR5/18或野生型sgp130Fc对IL-6/sIL-6R-诱导的BAF3/gp130细胞的增殖的抑制
在阻断由100ng/mL IL-6和50ng/mL sIL-6R引发的增殖中,CR5/18比野生型(wt)sgp130Fc生物活性显著更高。这由CR5/18的IC50(1)反映出来,其约为sgp130Fc的IC50(2)的一半。
缩写和符号:IC50,具有50%抑制效率的浓度;IL-6,白细胞介素-6;I/R,IL-6加sIL-6R;MTS,被代谢活性细胞转化成在490nm处吸收的可溶的甲 产物的底物;OD,在490nm处的光密度;sIL-6R,可溶的白细胞介素-6受体。
因此,本发明涉及能够抑制激动复合物IL-6/sIL-6R的活性并且包含两个单体的多肽-二聚体,其中每个单体包含与IgG蛋白的Fc结构域融合的可溶的gp130分子或其变体或片段,并且其中至少所述Fc结构域的铰链区的氨基酸残基Leu235被至少一个亲水性氨基酸残基替换。优选的亲水性氨基酸残基是Glu和Asp。
当用于本文时,术语“可溶的”是指缺少细胞内结构域并且优选缺少跨膜结构域的gp130分子。
本发明的二聚体可以使用已知方法加工。所用的结构域可以由gp130的完整的细胞外结构域组成,或者它们可以由其保留抑制激动复合物IL-6/sIL-6R的活性能力的突变体或片段组成。优选的片段是至少由细胞外结构域D1-D3组成的片段。
表述“融合到IgG蛋白的Fc结构域”意指,优选地,所述二聚体的融合配偶体仅由IgG1蛋白的Fc结构域组成。然而,所述Fc部分可以包含来自超过一种IgG同种型的序列,并且选择特别的序列基序以最优化需要的效应子功能在本领域普通技术人员的技术范围内。
在本发明的多肽二聚体的优选实施方案中,铰链区氨基酸残基Leu234被Phe或Ala替换。
在本发明的多肽二聚体的更优选实施方案中,铰链区的氨基酸残基Leu234和/或Gly237被氨基酸残基Ala替换。
在本发明的多肽二聚体的甚至更优选实施方案中,铰链区包含氨基酸序列基序Ala234-Glu235-Gly236-Ala237而不是Leu234-Leu235-Gly236-Gly237。
特别优选的是这样的多肽二聚体,其中铰链区包含氨基酸序列Asp221-Lys222-Thr223-His224-Thr225-Cys226-Pro227-Pro228-Cys229-Pro230-Ala231-Pro 232-Glu233-Ala234-Glu235-Gly236-Ala237-Pro238-Ser239-Val240。
gp130细胞外结构域(sgp130),优选地在C-端,或其变体或片段与Fc部分铰链区的融合,可以是直接的,或者它们可以利用不同长度和氨基酸组合的柔性的多肽接头结构域。这些接头可以完全是人工的(例如,包含2-50个独立地选自由下列各项组成的组的氨基酸残基:甘氨酸、丝氨酸、天冬酰胺、苏氨酸和丙氨酸)或采用天然存在的蛋白。这样的接头可以提高所述二聚体的柔性和结合特性。
另外,本发明的sgp130Fc融合蛋白可以进一步融合到标记上,所述标记诸如聚(His)、Myc、Strep、聚精氨酸、Flag、绿色荧光蛋白(GFP)、TAP、谷胱甘肽S-转移酶(GST)、HA、钙调蛋白-结合肽(CBP)、麦芽糖-结合蛋白(MBP)、V5、HSV、S、疱疹性口腔炎病毒(VSV)、蛋白质C、萤光素酶、Glu-Glu、E、β-GAL、T7或抗体或其它结合分子可用来允许快速纯化、在蛋白质印迹或ELISA中检测、免疫沉淀、或在生物测定中活性消耗/阻断的其它表位。
在本发明的多肽二聚体的更优选实施方案中,在可溶gp130分子或变体或片段和Fc结构域之间***一个或多个N-糖基化位点。具有核心序列Asn-X-Ser或Asn-X-Thr的N-糖基化位点的氨基酸基序取决于该基序在蛋白中的背景,并且可以由本领域技术人员预测和设计,例如,通过使用免 费软件如NetNGlyc(生物学序列分析中心(Center for Biological Sequence Analysis),丹麦科技大学(Technical University of Denmark))进行。对于本发明的sgp130Fc二聚体的优选的N-糖基化接头元件是His-Asn-Leu-Ser-Val-Ile。
本发明的另一个目的是该二聚体的PEG化或其它化学修饰的形式。sgp130分子的PEG化可以,例如,按照关于人IFN-γ,IFN-α,IFN-β,IL-15或IL-2所述的方法(Youngster等,现代药物设计(Curr Pharm Des)(2002),8:2139;Grace等,干扰素细胞因子研究杂志(J Interferon Cytokine Res)(2001),21:1103;Pepinsky等,药学实验治疗杂志(J Pharmacol Exp Ther)(2001),297:1059;Pettit等,生物的化学杂志(J Biol Chem)(1997),272:2312;Goodson等.生物技术纽约(Biotechnology NY)(1990),8:343;Katre;免疫学杂志(J Immunol)(1990),144:209)进行。
任何种类的聚乙二醇对本发明都是适用的,条件是PEG-多肽-二聚体仍然能够阻断依赖于sIL-6R的IL-反应,该反应可以根据本领域已知的方法进行测定。
优选地,本发明的多肽-二聚体的聚乙二醇是PEG 1000,2000,3000,5000,10000,15000,20000或40000,其中PEG 20000或40000是特别优选的。
为了形成所述二聚体,将两个可溶的gp130分子通过简单的共价键、柔性的肽接头或优选通过一个或多个二硫键彼此连接。肽接头可以完全是人工的(例如,包含2-20个独立地选自由下列各项组成的组的氨基酸残基:甘氨酸、丝氨酸、天冬酰胺、苏氨酸和丙氨酸)或采用天然存在的蛋白。二硫键的形成可以,例如,通过重组表达实现,其中编码sgp130Fc单体的核酸序列优选地在Fc结构域的铰链区,包含一个或多个编码半胱氨酸的密码子。
本发明的二聚体优选地通过利用编码该二聚体的单体的多核苷酸和载体优选是含有所述多核苷酸的表达载体重组产生。对于本发明的二聚体的生产,所述多核苷酸获自现有的克隆,即,优选地编码天然存在的多肽或其部分(对于人gp130/IL6ST:GenBank序列NM_002184和支持克隆;对于人IgG1/IGHG1的恒定区:例如,GenBank序列AK057754)。由在高严 格或中等严格条件下(关于定义,参见Sambrook,分子克隆A实验室手册(Molecular Cloning A Laboratory Manual),冷泉港实验室(Cold Spring Harbor Laboratory)(1989)纽约)与天然DNA或RNA的补体杂交的任何多核苷酸编码的多肽,只要所述多肽保持天然序列的生物学活性,对于产生本发明的二聚体也是有用的。
重组载体可以根据本领域技术人员公知的方法构建;参见,例如,Sambrook,分子克隆A实验室手册(Molecular Cloning A Laboratory Manual),冷泉港实验室(Cold Spring Harbor Laboratory)(1989)纽约。许多表达载体/宿主***可以用来包含并且表达编码本发明二聚体的序列。这些包括,但不限于,微生物如转化了重组噬菌体、质粒或黏端质粒DNA表达载体的细菌;转化了酵母表达载体的酵母;感染了病毒表达载体(例如,杆状病毒)的昆虫细胞***;转化了病毒表达载体(例如,花椰菜花叶病毒,CaMV;烟草花叶病毒,TMV)或细菌表达载体(例如,Ti或pBR322质粒)的植物细胞***;或动物细胞***。
在细菌***中,取决于本发明的多肽二聚体的目的应用,可以选择许多表达载体。适用于本发明的载体包括,但不限于,用于在细菌中表达的pSKK表达载体。
在野生型或修饰的(例如,糖改造的)酵母物种,诸如酿酒酵母(Saccharomyces cerevisiae)、粟酒裂殖酵母(Schizosaccharomyces pombe)或巴斯德毕赤酵母(Pichia pastoris)中,可以使用包含组成型或诱导型启动子或启动子***如α因子、醇氧化酶、PGH、四环素葡萄糖等的许多载体;对于综述,参见Grant等.(1987)酶学方法(Methods Enzymol.)153:516-544;Siam等.(2004)方法(Methods)33:189-198;Macauley-Patrick等.(2005)酵母(Yeast)22:249-270,Gellissen等.(2005)FEMS酵母研究(FEMS Yeast Res.)5:1079-1096;Wildt和Gerngross(2005)自然微生物学综述(Nat.Rev.Microbiol.)3:119-128。
在使用植物表达***的现有技术的情形中(关于综述,参见,例如,Stoger等.(2005)现代生物技术观点(Curr.Opin.Biotechnol.)16:167-173;Gomord等.(2005)生物技术趋势(Trends Biotechnol.)23:559-565),编码本发明的二聚体(或其单体)的序列的表达可以由许多启动子驱动。例如, 病毒启动子如CaMV的35S和19S启动子可以单独或与TMV的ω前导序列组合使用(Takamatsu(1987)EMBO J.6:307-311)。备选地,可以使用植物启动子如RUBISCO小亚基或热激启动子(Coruzzi等.(1984)EMBO J.3:1671-1680;Broglie等.(1984)科学(Science)224:838-843;和Winter等.(1991)细胞分化中的结果和问题(Results Probl.Cell Differ.)17:85-105)。通过直接的DNA转化或病原体-介导的转染,可以将这些构建体引入到植物细胞中。这样的技术在许多通常可获得的综述中描述(参见,例如,Hobbs和Murry,在科学和技术McGraw Hill年鉴(McGraw Hill Yearbook of Science and Technology)中(1992)McGraw Hill,纽约,N.Y.;第191-196页)。
昆虫***也可以用来表达本发明的二聚体(或其单体)。例如,在一种这样的***中,苜蓿银纹夜蛾多核型多角体病毒(Autographa californica nuclear polyhedrosis virus)(AcNPV)被用作在Spodoptera frugiperda细胞中或在粉夜蛾属(Trichoplusia)幼虫中表达外源基因的载体。可以将所述序列克隆到病毒的非必需区域中,诸如多角体蛋白基因中,并且置于多角体蛋白启动子的控制下。成功***编码sgp130Fc单体或其片段或变体的DNA序列将使该多角体蛋白基因失活,并且产生缺少外壳蛋白的重组病毒。然后,将重组病毒用于感染,例如,S.frugiperda细胞或粉夜蛾属幼虫,在它们中可以表达本发明的sgp130Fc(Engelhard等.(1994)美国国家科学院学报(Proc.Nat.Acad.Sci.)91:3224-3227)。
在哺乳动物宿主细胞中,可以使用基于如细胞的基于脂质的转染或病毒转导的许多表达***。在将腺病毒用作表达载体的情形中,编码本发明的多肽的序列可以连接到由晚期启动子和三联前导序列组成的腺病毒转录/翻译复合物中。***到病毒基因组的非必需的E1或E3区可以用来获得可存活的病毒,其能够在被感染的宿主细胞中表达本发明的多肽(Logan,J.和Shenk,T.(1984)美国国家科学院学报(Proc.Natl.Acad.Sci.) 81:3655-3659)。另外,转录增强子,诸如劳斯肉瘤病毒(RSV)增强子,可以用来增加在哺乳动物宿主细胞中的表达。
在引入重组载体后,将宿主细胞生长在选择培养基上,其选择含有载体的细胞的生长。可以使用任何数目的选择***来回收转化的细胞系。这 些包括,但不限于,单纯疱疹病毒胸苷激酶(Wigler,M.等(1977)细胞(Cell)11:223-32)和腺嘌呤磷酸核糖转移酶(Lowy,I.等.(1980)细胞(Cell)22:817-23)基因,它们分别可以用在tk.sup.-或aprt.sup.-细胞中。此外,抗代谢物、抗生素或除草剂抗性可以用作选择的基础;例如,赋予针对甲氨蝶呤抗性的dhfr(Wigler,M.等.(1980)美国国家科学院学报(Proc.Natl.Acad.Sci.)77:3567-70);赋予针对氨基糖苷类新霉素和G-418抗性的npt(Colbere-Garapin,F.等(1981)分子生物学杂志(J.Mol.Biol.)150:1-14),以及als或pat,其分别赋予针对chlorsulfuron和膦丝菌素乙酰转移酶(phosphinotricin acetyltransferase)的抗性。已经描述了其它选择基因,例如,trpB,其允许细胞使用吲哚代替色氨酸,或hisD,其允许细胞使用histinol代替组氨酸(Hartman,S.C.和R.C.Mulligan(1988)美国国家科学院学报(Proc.Natl.Acad.Sci.)85:8047-51)。可见标记的使用已经获得了普及性,其中这样的标记,如花青苷,β-葡糖醛酸糖苷酶和其底物GUS,和萤光素酶及其底物萤光素,不但广泛地用于鉴定转化体,还用来定量归因于特异性载体***的瞬时或稳定的蛋白表达的量(Rhodes,C.A.等(1995)分子生物学方法(Methods Mol.Biol.)55:121-131)。
重组多肽的纯化通过用于这一目的已知的任何一种方法进行,即,任何常规方法,包括提取、沉淀、层析、电泳等。可以使用的进一步纯化步骤是亲和层析,其使用例如蛋白质A、蛋白质G或单克隆抗体,其结合靶多肽,并且其被产生并固定在包含在柱内的凝胶基质上。使含有所述重组多肽的不纯的制备物流过柱。多肽将通过与亲和凝胶基质的特殊相互作用而与柱结合,而杂质将流过。洗涤后,通过改变pH或离子强度,多肽从凝胶上洗脱下来,然后,如果它是以单体产生的,则进行二聚体化,并且如果需要,进行PEG化。
因此,本发明还涉及产生本发明的多肽二聚体的方法,所述方法包括培养转化了编码所述多肽的单体的DNA序列的宿主细胞,并且从所述宿主细胞或培养物回收多肽-单体或二聚体。
本发明的多肽二聚体有效用于所有病理的治疗和/或预防,其中激动复合物IL-6/sIL6R的活性应该被抑制。
因此,本发明还涉及包含有效量的本发明的多肽二聚体、优选与载体组合的药物组合物。“药用”意欲包括任何载体,其不干扰活性成分的生物活性的功效,并且其对于施用其的宿主是没有毒性的。适当的药学载体的实例在本领域中是公知的,并且包括磷酸缓冲盐溶液、水、乳液、诸如油/水乳液、各种类型的湿润剂、无菌溶液等。这样的载体可以通过常规方法配制,并且可以以有效剂量施用给受试者。
“有效量”是指足以影响疾病的病程和严重性、引起这样的病理的减少或减缓的活性成分的量。
有效用于治疗和/或预防这些疾病或病症的“有效剂量”可以使用本领域技术人员已知的方法确定(参见,例如,Fingl等,治疗剂的药学基础(The Pharmocological Basis of Therapeutics),Goodman和Gilman,编.Macmillan出版公司,纽约,第1-46页((1975))。
组合物的施用可以通过不同方式实施,例如,通过静脉内、腹膜内、皮下、肌内、局部或皮内施用。剂量方案将由主治医师和其它临床因素确定。如在医学领域中公知的,对于任一名患者的剂量取决于许多因素,包括患者的大小、体表面积、年龄、性别、要施用的具体化合物、施用的时间和途径、治疗的类型、综合健康和同时施用的其它药物。
本发明还涉及上述定义的多肽二聚体用于制备治疗和/或预防疾病或病症的药物组合物的应用,其中阻断激动复合物IL-6/sIL-6R具有有利作用。本发明的多肽-二聚体的优选医学用途是治疗/预防骨吸收、高钙血症、恶病质、肿瘤或其它类型的癌症(例如,结肠癌、多发性骨髓瘤、淋巴瘤、白血病或霍奇金病)、自体免疫病(例如,多发性硬化(MS)或1型糖尿病)、炎性或特应性疾病(例如,局限性回肠炎、溃疡性结肠炎、类风湿性关节炎、青少年类风湿性关节炎、哮喘、银屑病、结节病、红斑狼疮或葡萄膜炎)、感染(例如,细菌、病毒、真菌、或其它病原体感染)、以及内分泌紊乱和代谢病或分解代谢疾病(例如,2型糖尿病、肥胖症、高血糖症或高胆固醇血症)。
下述实施例更详细地解释本发明。
实施例1
构建和产生sgp130Fc突变蛋白CR5/18
(A)材料
Gateway克隆***成分(AccuPrime Pfx DNA聚合酶,供体载体pDONR221,CMV启动子-控制的表达载体pcDNA-DEST40,用于***转移的BP和LR重组酶和感受态大肠杆菌(E.coil)细胞)购自Invitrogen(Karlsruhe,德国)。QuikChange II位点定向诱变试剂盒获自Stratagene(阿姆斯特丹,荷兰)。PAGE纯化的诱变引物来自Microsynth(Balgach,瑞士)。CHO-K1细胞购自德国微生物和细胞培养物保藏中心(German Collection of Microorganisms and Cell Cultures)(Braunschweig,德国)。培养基成分购买如下:Ham’s F12培养基,低IgG FBS和PBS(PAA实验室; 德国),FBS(Biochrom;柏林,德国),胰蛋白酶/EDTA溶液(Invitrogen)和G418溶液(西格玛-奥德里奇(Sigma-Aldrich);Taufkirchen,德国)。转染试剂脂转染胺试剂(Lipofectamine 2000)购自Invitrogen。Santa Cruz(海德尔堡,德国)提供蛋白质A/G加琼脂糖用于免疫沉淀。对于免疫沉淀和蛋白质印迹中的一级检测,使用小鼠抗-人IgG(Fc)单克隆抗体(CBL102;Chemicon;Hofheim,德国)。蛋白质印迹二级检测使用抗-小鼠IgG HRP-连接的抗体、ECL-加蛋白质印迹底物和Hyperfilm ECL(全购自GE卫生保健(GE Healthcare);慕尼黑,德国)进行。摇瓶(2.1L,2,5X表面)购自Greiner Bio-One(Frickenhausen,德国)。用于真空滤器装置的醋酸纤维素滤器(0.45μm)购自Sartorius( 德国)。用于亲和和大小排阻层析(SEC)的材料全获自GE卫生保健(GE Healthcare)(慕尼黑,德国):在XK16/20柱中的MabSelect材料(产品编号17-5199-01),PD-10脱盐柱和用于SEC的HiLoad 26/60 Superdex 200pg柱。Amicon Ultra-15 50kDa Ultracel-PL膜浓缩装置购自密理博(Millipore)(Eschborn,德国)。已经制备好的用于PAGE的丙烯酰胺-二溶液(19∶1,30%)由Bio-Rad(慕尼黑,德国)提供。
(B)CR5/18的构建
将包含完整的gp130细胞外结构域和野生型人IgG1 Fc的全长sgp130Fc的cDNA(来源:关于人gp130/IL6ST:GenBank序列NM_002184和关于人IgG1/IGHG1的恒定区的支持克隆,例如,GenBank序列AK057754)关于在CHO-K1细胞中表达进行密码子-最优化,并且使用Gateway引物、AccuPrime Pfx DNA聚合酶和BP重组酶以标准Gateway克隆方法亚克隆到pDONR221中。使用重叠的正向和反向测序引物每250-300bp对亚克隆的***片段进行完全测序验证。在使用QuikChange II试剂盒的位点定向诱变中,IgG1-Fc的下游铰链区(依据EU标号,氨基酸234,235和237)由野生型序列“LLGG”突变为“AEGA”。突变的克隆通过上述完全测序验证。随后,通过Gateway LR重组,将***物转移到表达载体pcDNA-DEST40中。因为***物在Fc部分之后编码2个终止密码子,在pcDNA-DEST40中编码的标记(V5和6xHis表位)在CR5/18中不存在。阳性克隆通过AlwNI限制性酶切消化进行鉴定,并且再次测序验证。
(C)细胞培养和转染
在37℃和5% CO2在水饱和的氛围下,CHO-K1细胞生长在补充了10% FBS的Ham’s F12培养基上。保持培养物每3-4天***(split),并且只使用到20次传代。将细胞用表达构建体pcDNA-DEST40_CR5/18转染,转染使用Invitrogen提供的脂转染胺试剂2000和用于CHO-K1的标准条件进行。对于第一次瞬时表达检测,在6-孔平板中转染CHO-K1细胞,并且在转染24小时后收集细胞和上清液二者。CR5/18使用蛋白质A/G加琼脂糖和抗-人IgG(Fc)抗体,依据供应商的使用说明,从上清液进行免疫沉淀。提取总细胞蛋白,并且使用抗-人IgG(Fc)抗体与细胞裂解物和免疫沉淀物如在Waetzig等,免疫学杂志(J.Immunol.)168:5342(2002)中所述进行蛋白质印迹。
(D)在CHO-K1细胞中生产CR5/18
在成功的瞬时表达后,转染CHO-K1细胞,并且在10cm平板中使用400μg/ml G418选择阳性克隆。为了确定产物质量和性质,将预选择的多克隆CHO-K1合并物转移到摇瓶中,并且用低IgG FBS培养。汇合的细胞的上清液每周收集2-3次,以3,500xg在4℃离心15分钟离心2次,以去除细胞碎片,并且立即处理或冷冻在-80℃。平行地,使用有限稀释方法,从预选择的合并物中选择稳定的细胞克隆,并且通过如上述的蛋白质 印迹表达分析进行表征。将具有最高和最稳定表达的克隆转移到摇瓶中,并且用于永久生产。
(E)通过亲和和大小排阻层析进行纯化
将来自摇瓶培养物的含有CR5/18的上清液在4℃使用P-1蠕动泵和 纯化仪100***(二者均来自GE卫生保健(GE Healthcare);慕尼黑,德国)进行纯化。流程基于供应商关于单克隆抗体纯化的推荐。在离心后,将新鲜或解冻的(在冰上)上清液的pH调整到6.7-7.0。在两轮真空过滤(0.45μm)后,将上清液中的气体去除,并且----如果需要的话,----将pH再调整到6.7-7.0的值。随后,用2-4L上清使用P-1泵以3-10ml/分钟的流速上样到PBS-平衡的亲和层析柱(6-25ml MabSelect,在XK16/20柱中)。在用PBS洗涤后,将柱转移到 纯化仪中,并且再用PBS洗涤,直到在定量去除未结合的蛋白后A280稳定。对于洗脱, ***装配分别处于pH 3.25和5.5的两种50mM柠檬酸钠缓冲液,将它们混合,以产生需要的pH条件。在pH 5.1的一个洗涤步骤后进行在pH 3.7的洗脱。将10ml的级分收集在含有2ml1M Tris-HCl(pH 11)的15ml的管中。合并峰值级分,并且测量pH,并调整到7.5,如果需要的话。通过A280测量合并的蛋白浓度,并且使用Amicon Ultra-15 50kDa Ultracel-PL膜浓缩装置将该合并液小心浓缩到最大1.5mg/ml。使用PBS-平衡的PD-10脱盐柱将柠檬酸缓冲液替换为PBS,然后在280nm进行另一次蛋白浓度测量。
对于大小排阻层析(SEC),推荐在PBS中的最大蛋白浓度为1.2mg/ml。SEC使用 ***在PBS-平衡的HiLoad 26/60 Superdex 200pg柱中以0.8ml/分钟的流速进行。与野生型sgp130Fc相反,CR5/18以单一峰在更高分子量的聚集物的低峰之后洗脱下来(图2)。在第一次运行中,获得所有级分的样品用于PAGE分析。合并峰级分,测量它们的蛋白浓度,并且在PBS中设定为400-500μg/ml,并且将单次使用的等分试样冷冻在-80℃用于长期保存。通过非变性PAGE(7.5%)和随后的银染或考马斯染色分析级分和合并的样品。
如在图2中所示,与在平行实验中纯化的母体化合物sgp130Fc相比,CR5/18的副产物(聚集物)的量显著减少。此外,需要的产物(CR5/18 二聚体)的洗脱清楚地与杂质级分(聚集物)分离,对于野生型sgp130Fc不存在这样的情形。因此,CR5/18制备物的产率(由于更高比例的需要的产物)和质量好于常规sgp130Fc的那些,导致工业生产的更低的成本。这些结果表明CR5/18比母体sgp130Fc分子的明显提高。
实施例2
CR5/18在标准化细胞增殖测定中的生物活性
(A)材料
使用稳定转染的B细胞前体细胞系BAF3/gp130和设计的细胞因子超-IL-6。培养基成分购自下述:DMEM和PBS(PAA实验室; 德国),FB S(B iochrom;柏林,德国)和胰蛋白酶/EDTA溶液(Invitrogen;Karlsruhe,德国)。白细胞介素-6(IL-6)和可溶的白细胞介素-6受体(sIL-6R)分别购自BioSource(Solingen,德国)和R&D***(Wiesbaden,德国),细胞滴定96水相非放射活性细胞增殖测定(Cell Titer 96Aqueous Non-Radioactive Cell Proliferation Assay)(MTS)获自普洛麦格(Promega)(曼海姆,德国)。
(B)sgp130Fc或CR5/18对IL-6/sIL-6R-诱导的BAF3/gp130细胞增殖的阻断
BAF3/gp130细胞的增殖和存活依赖于在培养基中存在IL-6/sIL-6R复合物。对于维持,将BAF3/gp130细胞以低于5x 105细胞/mL的密度培养在具有10%FBS和10ng/mL Hyper-IL-6(由共价连接的IL-6和sIL-6R组成的设计的细胞因子;Fischer等.1997,自然生物技术(Nat.Biotechnol.)15:142-145)的DMEM中。10ng/mL超-IL-6可以由100ng/mL IL-6和50ng/mL sIL-6R代替。细胞每周传代2次。对于测定,将细胞在不含有超-IL-6(或IL-6/sIL-6R)的培养基中洗涤两次,然后以5,000细胞/孔接种在96-孔平板中。CR5/18或母体化合物sgp130Fc以在20μg/mL-78ng/mL范围内变化的浓度(1∶4稀释系列;图3)加入。随后,将细胞在100ng/mL IL-6和50ng/mL sIL-6R的存在下培养3天。对照包括不具有和具有最大浓度的CR5/18或sgp130Fc的未刺激的细胞,以及只用刺激物IL-6和sIL-6R培养的细胞(图3)。
(C)结果
在细胞培养物中的CR5/18或野生型sgp130Fc的生物活性通过在3天后对存活的BAF3/gp130细胞的数量减少(通过MTS底物转化确定)而测量。CR5/18比野生型sgp130Fc更有生物活性,使得其IC50处于约400ng/mL的浓度,其中sgp130Fc(IC50≈800ng/mL)仍然没有表现出显著效果(图3)。这表明CR5/18可以以约为野生型化合物治疗浓度一半的浓度使用。
Claims (17)
1.一种多肽二聚体,其能够抑制激动复合物IL-6/sIL-6R的活性并且包含两个单体,其中所述单体的每一个包含融合到IgG1蛋白Fc结构域的一条链的gp130分子的完整细胞外结构域,并且其中所述Fc结构域的铰链区包含氨基酸序列基序Ala234-Glu235-Gly236-Ala237而不是Leu234-Leu235-Gly236-Gly237。
2.权利要求1的多肽二聚体,其中所述铰链区包含氨基酸序列Asp221-Lys222-Thr223-His224-Thr225-Cys226-Pro227-Pro228-Cys229-Pro230-Ala231-Pro232-Glu233-Ala234-Glu235-Gly236-Ala237-Pro238-Ser239-Val240。
3.权利要求1-2中任一项的多肽二聚体,其中所述gp130分子的完整细胞外结构域直接融合到IgG1蛋白的Fc结构域的铰链区或通过柔性的多肽接头融合。
4.权利要求3的多肽二聚体,其中所述接头是包含2-50个氨基酸残基的接头,所述氨基酸残基独立地选自由下列各项组成的组:甘氨酸、丝氨酸、天冬酰胺、苏氨酸和丙氨酸。
5.权利要求1-2中任一项的多肽二聚体,其中所述单体通过共价键、柔性的肽接头或一个或多个二硫键彼此连接。
6.权利要求1-2中任一项的多肽二聚体,其中所述二聚体的至少一个单体被PEG化,其中所述聚乙二醇是PEG1000,2000,3000,5000,10000,15000,20000或40000。
7.编码权利要求1-5中任一项的多肽二聚体的单体的多核苷酸。
8.表达载体,其含有权利要求7的多核苷酸。
9.宿主细胞,其含有权利要求8的表达载体。
10.生产权利要求1-5中任一项的多肽二聚体的方法,所述方法包括培养权利要求9的宿主细胞,并且从所述宿主细胞或培养物回收所述单体或二聚体。
11.药物组合物,其含有权利要求1-6中任一项定义的多肽二聚体。
12.权利要求1-6中任一项定义的多肽二聚体用于制备治疗和/或预防疾病或病症的药物组合物的应用,在所述疾病或病症中对激动复合物IL-6/sIL-6R的阻断具有有利的作用。
13.权利要求12的应用,其中所述疾病是骨吸收、高钙血症、恶病质、癌症、炎性疾病、或内分泌紊乱。
14.权利要求12的应用,其中所述疾病是自体免疫病。
15.权利要求12的应用,其中所述疾病是代谢病。
16.权利要求12的应用,其中所述疾病是分解代谢疾病。
17.权利要求12的应用,其中所述疾病是特应性疾病。
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