CN103864923A - 糖基化抗体 - Google Patents
糖基化抗体 Download PDFInfo
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- CN103864923A CN103864923A CN201410108197.0A CN201410108197A CN103864923A CN 103864923 A CN103864923 A CN 103864923A CN 201410108197 A CN201410108197 A CN 201410108197A CN 103864923 A CN103864923 A CN 103864923A
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Abstract
本发明提供了人IgG1或IgG3型抗体及其用途,所述抗体在Asn297处被糖链糖基化,其特征在于所述糖链内岩藻糖的量至少为99%,此外,NGNA的量为1%或以下,和/或N-末端α-1,3-半乳糖的量为1%或以下。
Description
本申请为2007年4月10日提交的、发明名称为“糖基化抗体”的PCT申请PCT/EP2007/003164的分案申请,所述PCT申请进入中国国家阶段的日期为2008年10月8日,申请号为200780012678.X。
发明领域
本发明涉及其Fc区得以表达且被糖基化的重组抗体,由此与抗体Fc区相连的主要核心糖结构被充分岩藻糖基化(fucosylated)。本发明还涉及CHO(中国仓鼠卵巢)宿主细胞,选择此类CHO宿主细胞的方法,以及此类重组抗体的用途。
发明背景
天然形式的免疫球蛋白或抗体通常是由两条轻链和两条重链组成的四聚体糖蛋白。抗体含有恒定结构域,据此将抗体归属为不同的类,如IgA、IgD、IgE、IgG和IgM,以及若干亚类,如IgG1、IgG2、IgG3和IgG4。IgG1和IgG3类的人抗体通常介导ADCC(抗体依赖性细胞介导的细胞毒作用)。
也存在已知的其他抗体样分子,含有例如异源蛋白质如受体、配体或酶的结合结构域,和抗体的Fc区。例如,这样的Fc融合蛋白由Stabila,P.等,Nature Biotech16(1998)1357-1360和US5,610,297描述。
单克隆抗体引发四种效应功能:ADCC,吞噬作用,补体依赖性细胞毒作用(CDC),影响半衰期/清除率。ADCC和吞噬作用通过细胞结合的抗体与FcγR(Fcγ受体)之间的相互作用介导;CDC则通过细胞结合的抗体与构成补体系统的系列蛋白之间的相互作用。CDC与C1q结合C3激活和/或Fc受体与Fc部分的结合相关。如果需要降低C1q结合C3激活和/或Fc受体与抗体恒定部分的结合,通常使用IgG4抗体,它不激活补体系统,不结合C1q,不激活C3。可选地,使用含有γ1重链恒定区的Fc部分,其中所述恒定区具有某些突变,如L234A和L235A或D265A和N297A(WO99/51642)。
本领域众所周知如何修饰抗体的恒定结构域以改善效应功能。例如,这样的方法描述在WO99/54342中。
Routier,F.H.等,Glycoconjugate J.14(1997)201-207报道了在CHO-DUKX细胞中表达的人源化IgG1抗体的糖基化模式。此抗体显示出Fuc∶Man为0.8∶3.0的摩尔比,述及80%的岩藻糖基化比率。Niwa,R.等,J.Immunol.Methods306(2005)151-160报道了在CHO DG44中重组生产的岩藻糖基化约为90%的抗CD20IgG1和IgG3抗体。Mimura,Y等,J.Immunol.Methods247(2001)205-216报道了丁酸盐增加CHO-K1细胞中人嵌合IgG的产量,同时维持其功能和糖型分布。寡糖分布显示相当可观量的无岩藻糖基化的(afucosylated)聚糖结构。Raju,T.S.,BioProcessInternational1(2003)44-53报道了表达系统糖基化差异对治疗性免疫球蛋白生物活性的影响以及命名法。Ma,S.,Anal.Chem.71(1999)5185-5192报道了利妥昔单抗(rituximab)的糖分析。利妥昔单抗显示出9-10%的岩藻糖基化(Niwa,R.等,J.Immunol.Methods306(2005)151-160)。Fujii,S.,J.Biol.Chem.265(1990)6009-6018报道牛IgG包括大约11%无岩藻糖基化的IgG。Mizouchi,T.,J.Immunol.129(1982)2016-2020报道人IgG大约14%是无岩藻糖基化的。Bergwerff,A.A.,Glycoconjugate J.12(1995)318-330报道了在小鼠SP2/0中生产的抗体含有大量N-羟乙酰神经氨酸(N-glycolylneuraminic acid,NGNA)寡糖。Nahrgang,S.等在Animal CellTechnology:Products from Cells,Cells as Products,Bernard,A.等(编辑),Kluwer Academic Publishers,Dordrecht,NL,1999,第259-261页中报道了IgG1的CHO表达,在瞬时转染之后发现总体糖基化弱。Lund,J.等,Mol.Immunol.30(1993)741-748报道了在小鼠转染瘤细胞中重组生产小鼠-人嵌合抗体。13%量的IgG1抗体是无岩藻糖基化的。Patel,T.P.等,Biochem.J.285(1992)839-845报道了来自杂交瘤细胞和小鼠腹水的抗体的糖基化。Niwa,R.等,J.Immunol.Methods306(2005)151-160报道了在CHO DG44中重组生产CD20IgG1抗体之后91%的岩藻糖基化,而Mori,K.等,Biotech.Bioeng.88(2004)901-908报道了94%的岩藻糖基化。Davies,J.等,Biotechnol.Bioeng.74(2001)288-294报道表达具有改变的糖型的抗体导致ADCC增加。Sheeley,D.M.等,Anal.Biochem.247(1997)102-110比较了不同表达系统中的抗体糖基化。Shields,R.L.等,J.Biol.Chem.277(2002)26733-26740报道人IgG1Fc上缺乏岩藻糖提高FcγRIII结合和ADCC。Zhu,L.等,Nature Biotechnol.23(2005)1159-1169报道了在鸡蛋中生产人抗体。WO2004/087756和WO2005/005635公开了抗IGF-1R的改良的抗体。
发明概述
本发明包括人IgG1或IgG3型抗体,所述抗体在Asn297处被糖链糖基化,其特征在于在所述糖链内岩藻糖的量至少为99%,此外,NGNA的量为1%或以下,和/或N-末端α-1,3-半乳糖的量为1%或以下。
根据本发明,“量”是指在Asn297处糖链内所述糖的量,以G0、G1、G2(不含甘露糖(4和5))之和作为100%,如实施例3所述进行计算。
根据本发明,有可能提供岩藻糖基化甚至为99.4%或以上、99.5%或以上、或99.9%或以上的抗体和/或CHO宿主细胞。
优选NGNA的量为0.5%或以下,更优选0.1%或以下,甚至LCMS(液相色谱/质谱)不可检测。
优选N-末端α-1,3-半乳糖的量为0.5%或以下,更优选0.1%或以下,甚至LCMS不可检测。
糖链优选表现出与CHO细胞中重组表达抗体的Asn297处相连的N-连接聚糖的特征。
优选抗体是单克隆抗体。优选抗体是嵌合、人源化或人抗体。
本发明还包括能够重组表达人IgG1或IgG3型抗体的CHO细胞,所述抗体在Asn297处被糖链糖基化,其特征在于在所述糖链内岩藻糖的量至少为99%,此外,NGNA的量为1%或以下,和/或N-末端α-1,3-半乳糖的量为1%或以下。
这样的细胞系为细胞系hu MAb<IGF-1R>B1-4E10_9-16),根据国际承认用于专利程序的微生物保存布达佩斯条约,于2006年6月21日保藏在德国的德意志微生物保藏中心(Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH,DSMZ),保藏号DSM ACC2795。
优选的糖的量如上文所述。
优选CHO细胞是这样的CHO细胞:包括两个DHFR等位基因缺失(如DG44)或功能性失活,或者一个DHFR等位基因缺失,第二个DHFR等位基因功能性失活(如DXB11)。
本发明还包括根据本发明用于人类医疗的组合物。
根据本发明组合物的抗体优选是嵌合抗体、人抗体、人源化抗体、非人抗体、包含IgG1或IgG3重链恒定部分的单链抗体、或者IgG1或IgG3重链恒定部分。
本发明还包括根据本发明的抗体用于制备药物的用途。优选药物用于免疫抑制、用以治疗T细胞介导的紊乱、自身免疫紊乱、传染病、癌病。
本发明还包括药物组合物,其包含根据本发明的抗体。
本发明的另一目的是选择用于重组生产人IgG1或IgG3型单克隆抗体的CHO细胞的方法,所述抗体在Asn297处被糖链糖基化,其特征在于在所述糖链内岩藻糖的量至少为99%,此外,NGNA的量为1%或以下,和/或N-末端α-1,3-半乳糖的量为1%或以下,所述方法包括在DHFR和MTX选择压力下培养用IgG1或IgG3抗体和DHFR基因转染的CHO细胞,挑取单个的克隆,扩增所述克隆,选择产生本发明糖基化模式抗体的克隆。优选培养进行至少两周、优选至少三周。
本发明的另一目的是根据本发明的CHO细胞用于重组生产单克隆抗体的用途。
本发明的另一目的是在根据本发明的CHO细胞中重组生产单克隆抗体的方法。
CHO细胞是用于重组表达异源多肽的宿主细胞。
附图的简短说明
图1的柱形图显示了本发明抗体以及对照和参比抗体的ADCC活性或该活性的不足。
发明详述
抗体在重链恒定区的保守位置上含有糖结构,其中每个同种型拥有独特排列的N-连接糖结构,它们可变地影响着蛋白质的装配、分泌或功能活性(Wright,A.和Morrison,S.L.,Trends Biotechnol.15(1997)26-32)。视加工程度不同,所连的N-连接糖的结构相当地变化多样,可以包括高甘露糖、多分支以及二天线复杂型寡糖(Wright,A.和Morrison,S.L.,TrendsBiotechnol.15(1997)26-32)。
IgG1和IgG3型抗体是在各CH2结构域的Asn297处具有保守N-连接糖基化位点的糖蛋白。与Asn297相连的两个复杂型二天线寡糖被遮蔽在CH2结构域之间,与多肽主链形成广泛接触,且其存在对于抗体介导效应功能如抗体依赖性细胞细胞毒作用(ADCC)是必不可少的(Lifely,M.R.等,Glycobiology5(1995)813-822;Jefferis,R.等,Immunol Rev.163(1998)59-76;Wright,A.和Morrison,S.L.,Trends Biotechnol.15(1997)26-32)。
如本文所用,术语“人IgG型Fc区”优选还包括天然存在的免疫球蛋白(抗体)Fc区的等位基因变体,以及具有取代、添加或缺失改变但不影响Ans297糖基化作用的变体。例如,免疫球蛋白Fc区的N-末端或C-末端可以缺失一个或多个氨基酸,而不会导致生物功能的实质损失。这样的变体可以根据本领域公知的一般规则进行选择,从而对活性具有最小的影响(参见例如Bowie,J.U.等,Science247(1990)1306-1310)。
术语“抗体”涵盖多种形式的抗体,包括但不限于全抗体、抗体片段、人抗体、人源化抗体和遗传改造的抗体,只要保留根据本发明的特征性特性即可。因此,根据本发明的抗体至少含有IgG1或IgG3型功能活性(结合FcR的)Fc部分,包含糖基化的Asn297。
如本文所用的术语“单克隆抗体”或“单克隆抗体组合物”是指具有完全相同氨基酸组成的抗体分子制品。相应地,术语“人单克隆抗体”是指具有来源于人生殖系免疫球蛋白序列的可变和恒定区、呈现出单一结合特异性的抗体。
术语“嵌合抗体”是指包含来自一种来源或物种的可变区即结合区,以及至少一部分来源于不同来源或物种的恒定区的单克隆抗体,通常通过重组DNA技术制备。特别优选包含鼠可变区和人恒定区的嵌合抗体。此类鼠/人嵌合抗体为免疫球蛋白基因的表达产物,所述免疫球蛋白基因包含编码鼠免疫球蛋白可变区的DNA区段以及编码人免疫球蛋白恒定区的DNA区段。生产嵌合抗体的方法包括目前本领域众所周知的常规重组DNA技术和基因转染技术(参见例如Morrison,S.L.等,Proc.Natl.Acad Sci.USA81(1984)6851-6855;美国专利No.5,202,238和5,204,244)。
术语“人源化抗体”是指这样的抗体,其中框架或“互补决定区”(CDR)已经被修饰,以包含与亲本免疫球蛋白相比具有不同特异性的免疫球蛋白CDR。在优选的实施方案中,将鼠CDR移植至人抗体的框架区,以制备“人源化抗体”(参见例如Riechmann,L.等,Nature332(1988)323-327;以及Neuberger,M.S.等,Nature314(1985)268-270)。特别优选的CDR对应于提供这样序列的CDR,所述序列识别上述嵌合及双功能抗体的抗原。
如本文所用,术语“人抗体”旨在包括具有来源于人生殖系免疫球蛋白序列的可变和恒定区的抗体。这样的区域由例如Jobnson,G.和Wu,T.T.,Nucleic Acids Res.28(2000)214-218和文中引述的数据库描述,只要保留根据本发明的特性就是可用的。人抗体在本领域是众所周知的(van Dijk,M.A.和van de Winkel,J.G.,Curr.Opin.Chem.Biol.5(2001)368-374)。人抗体也可以在转基因动物(如小鼠)中生产,所述转基因动物经免疫后能够生产全部或选择的人抗体,而无内源免疫球蛋白生产。将人生殖系免疫球蛋白基因排列转移到这样的生殖系突变小鼠中导致在抗原刺激时生产人抗体(参见例如Jakobovits,A.等,Proc.Natl.Acad.Sci.USA90(1993)2551-2555;Jakobovits,A.等,Nature362(1993)255-258;Bruggemann,M.等,Year Immunol.7(1993)33-40)。人抗体也可以在噬菌体展示文库中生产(Hoogenboom,H.R.和Winter,G.,J.Mol.Biol.227(1992)381-388;Marks,J.D.等,J.Mol.Biol.222(1991)581-597)。还可以利用Cole等及Boerner等的技术制备人单克隆抗体(Cole等,Monoclonal Antibodies and CancerTherapy,Alan R.Liss,第77页(1985);和Boerner,P.等,J.Immunol.147(1991)86-95)。人抗体涵盖多种形式的抗体结构,优选单克隆抗体,包括但不限于全抗体、抗体片段和遗传改造的抗体(变体或突变抗体),只要保留根据本发明的特征性特性即可。尤其优选重组人抗体。
如本文所用,术语“重组人抗体”旨在包括通过重组手段生产制备、表达、创建或分离的所有人抗体,例如,利用转染到根据本发明的宿主细胞中的重组表达载体,从这样的宿主细胞中分离的抗体。
“恒定结构域”不直接参与抗体与抗原的结合,但表现出其他功能如效应功能。对应于IgG1的重链恒定区称为γ1链。对应于IgG3的重链恒定区称为γ3链。人恒定γ重链由Kabat,E.A.等,Sequences of Proteins ofImmunological Interest,第5版,Public Health Service,National Institutesof Health,Bethesda,MD.(1991)以及Brueggemann,M.等,J.Exp.Med.166(1987)1351-1361;Love,T.W.等,Methods Enzymol.178(1989)515-527详细描述。IgG1或IgG3型的恒定结构域在Asn297处糖基化。根据本发明的“Asn297”表示大致位于Fc区第297位上的氨基酸天冬酰胺;根据抗体微小的序列变异,Asn297也可以位于上游或下游的某些氨基酸位置上(通常不超过±3个氨基酸)。例如,在根据本发明的一个抗体中,“Asn297”位于氨基酸位置298处。
人IgG1或IgG3的糖基化发生在Ash297处,为核心岩藻糖基化的二天线复杂型寡糖糖基化,由至多2个Gal残基封端。根据末端Gal残基量的不同,这些结构命名为G0、G1(α1,6或α1,3)或G2聚糖残基(Raju,T.S.,BioProcess International1(2003)44-53)。抗体Fc部分的CHO型糖基化由例如Routier,F.H.,Glycoconjugate J.14(1997)201-207描述。
如本文所用的“可变区”(轻链可变区(VL)、重链可变区(VH))表示直接参与抗体与抗原结合的每一对轻链和重链。
根据本发明,可以选择生产抗体的CHO宿主细胞,其能够经由重组表达提供表现出根据本发明糖基化模式的单克隆抗体的组合物。这样的CHO宿主细胞含有用于重组表达这类抗体的一个或多个表达载体。优选宿主细胞用所述载体稳定转染,抗体编码核酸稳定整合到CHO宿主细胞基因组中。
术语“CHO细胞”涵盖以两个缺失的dhfr等位基因(二氢叶酸还原酶缺陷型(dhfr-))为基础的多种形式的中国仓鼠卵巢(CHO)细胞。这样的dhfr-细胞及其产生方法描述在例如Urlaub,G..等,Cell33(1983)405-412;Urlaub,G.等,Som.Cell Molec.Genet.12(1986)555-566;Kolkekar等,Biochemistry36(1997)10901-10909中。优选细胞是DG44细胞系。可以利用γ射线消除整个的dhfr基因座生产这样的CHO dhfr-细胞。在非突变的野生型细胞中,dhfr是从甘氨酸、嘌呤和胸苷酸从头合成必不可少的酶。这使得质粒上编码的dhfr基因能够在dhfr-缺陷型细胞系中用作蛋白质表达的显性选择标记和基因放大器(amplifier)。细胞中的dhfr-突变稳定且不可逆转。成功地共转染了人IgG1或IgG3型抗体和DHFR基因的表达载体的CHO细胞将拥有dhfr+表型,并且通过在缺乏胸苷和次黄嘌呤、任选含有扩增用甲氨蝶呤(MTX)的培养基中培养细胞集落能容易地进行选择。
DG44细胞在本领域众所周知,例如,可以作为细胞系从例如美国英骏公司(Invitrogen Corp.,USA)商购获得。DG44细胞可以贴壁、在悬液中和/或在无血清培养基中培养。如本文所用,表述“细胞”、“细胞系”和“细胞培养物”互换使用,且所有这样命名的CHO dhfr-细胞系(两个缺失的dhfr等位基因)包括子代。从而,措辞“转化株”和“转化细胞”包括最早的受试细胞和由之获得的培养物,而不考虑转移次数。也应当理解,由于故意或无意的突变,所有的子代在DNA内容方面可能并不精确相同。具有在原始转化的细胞中进行筛选的根据本发明的糖基化特性的变体子代包括在本发明的范围内。
优选CHO dhfr-细胞系至少与DHFR一起进行共扩增,后者作为一种选择标记基因。例如,将含有选择标记的哺乳动物表达载体和抗体基因共转染到受体CHO细胞中。可以对产生的集落进行选择,呈现出预期表型的集落能够表达抗体。额外的选择标记是或者可能不是显性的。用于共转染的额外选择标记的实例包括:腺苷脱氨酶(Kaufman,R.J.等,Proc.Natl.Acad.Sci.USA83(1986)3136-3140)、天冬酰胺合成酶(Cartier,M.等,Mol.Cell Biol.7(1987)1623-1628)、大肠杆菌(E.coli)trpB基因和沙门氏菌属(Salmonella)hisD基因(Hartman,S.C.和Mulligan,R.C.,Proc.Natl.Acad.Sci.USA85(1988)8047-8051)、M2小鼠核糖核苷酸还原酶(Thelander,M.和Thelander,L.,EMBO J.8(1989)2475-2479)、人多药耐药基因(Kane,S.E.等,Gene84(1989)439-446)、谷氨酰胺合成酶(Bebbington,C.R.等,DNA Cloning,Vol.III,D.M.Glover(编辑),IRL Press,第163-188页,1987)、黄嘌呤鸟嘌呤磷酸核糖转移酶(gpt)(Mulligan,R.C.和Berg,P.,Science209(1980)1422-1427)、潮霉素B(Santerre,R.F.等,Gene30(1984)147-156)、新霉素基因(Southern,P.J.和Berg,P.,J.Mol.Appl.Genet.1(1982)327-341)。
选择标记还可以提供抗体编码基因得以扩增的基础。在CHO细胞系的共转染过程中,各载体DNA通常整合到细胞染色体相同的基因座上。从而,一般仅仅使用一种选择标记作为扩增基础就引起两种基因拷贝数的平行增加。以这种方式使用的一种特别的选择标记是dhfr,通过使用浓度渐增的MTX,能够获得期望的扩增。又一优选的选择标记是GS,通过添加甲硫氨酸亚砜胺(methionine sulphoximine,MSX)能够进行扩增。
选择标记当然要处于DNA调控元件的控制之下以确保其表达。在使用dhfr作为选择标记的情况下,调控元件优选为病毒来源的,例如来自DNA肿瘤病毒。特别优选使用SV40或腺病毒主要晚期启动子。就此而言,从所述启动子中除去增强子元件从而有效使之“削弱”尤为有利。与否则如果使用强启动子会发生的情况相比,这种修饰能够在各种浓度的甲氨蝶呤选择下增加基因扩增水平。在使用新霉素作为选择标记的情况下,适宜启动子的实例为小鼠金属硫蛋白启动子。
如本文所用,术语“核酸”或“核酸分子”旨在包括DNA分子和RNA分子。核酸分子可以是单链或双链的,但优选为双链DNA。
当核酸与另一核酸序列处于功能关联状态时,其“有效连接”。例如,如果前序列或分泌前导序列的DNA与多肽的DNA的连接表达为参与所述多肽分泌的前蛋白,则他们有效连接;如果启动子或增强子的连接影响编码序列的转录,则他们有效连接;或者如果核糖体结合位点与编码序列的排置有利于翻译,则他们有效连接。通常,“有效连接”表示连接的DNA序列是顺式的,而且在分泌前导序列的情况下是相邻且同读框连接的。不过,增强子不必相邻。连接通过便利的限制性位点处的连接实现。如果不存在这样的位点,则按照常规操作使用合成的寡核苷酸接纳体(adaptor)或接头。
根据本发明的抗体优选通过重组手段生产。此类方法在本领域广为人知,并且包括在原核和真核细胞中表达蛋白质,随后分离抗体多肽,并通常纯化至可药用的纯度。为进行蛋白质表达,通过标准方法将编码轻链和重链或其片段的核酸插入表达载体中。在CHO宿主细胞中进行表达,并从细胞或上清液(优选在裂解后)中回收抗体。
抗体的重组生产在本领域众所周知,并描述在例如Makrides,S.C.,Protein Expr.Purif.17(1999)183-202;Geisse,S.等,Protein Expr.Purif.8(1996)271-282;Kaufman,R.J.,Mol.Biotechnol.16(2000)151-161;Werner,R.G.,Drug Res.48(1998)870-880的综述文章中。
抗体可以存在于完整细胞、上清液、细胞裂解物中,或为部分纯化或基本上纯的形式。通过标准技术,包括本领域众所周知的碱/SDS处理、氯化铯分带(CsCl banding)、柱层析、琼脂糖凝胶电泳等等进行纯化,以除去其他细胞组分或其他杂质,如其他细胞核酸或蛋白质(参见Ausubel,F.等编辑Current Protocols in Molecular Biology,Greene Publishing andWiley Interscience,New York(1987))。
例如,适用于原核生物的控制序列包括启动子,任选的操纵子序列,和核糖体结合位点。真核细胞已知利用启动子、增强子和多聚腺苷酸化信号。
可以通过常规的免疫球蛋白纯化方法如蛋白质A-琼脂糖、羟基磷灰石层析、凝胶电泳、透析或亲和层析从杂交瘤培养基中适当地分离单克隆抗体。编码单克隆抗体的DNA和RNA很容易从杂交瘤中分离,并利用常规方法进行测序。杂交瘤细胞可用作为此类DNA和RNA的来源。DNA一经鉴定和分离,即可插入到表达载体中,随之转染到否则将不生产免疫球蛋白的CHO细胞中,以实现宿主细胞中重组单克隆抗体的合成。
本发明在另一方面提供了药物组合物,包括与可药用载体配制在一起的本发明组合物。优选使用根据WO98/22136的药物组合物。例如,1ml这样的组合物含有2.0mg抗体,15mM磷酸缓冲液pH6.5,30mM氯化钠,25mg甘露醇(mannite),10mg精氨酸,0.1mg20。
如本文所用,“可药用载体”包括生理上相容的任何及所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等等。优选载体适于静脉内、肌内、皮下、肠胃外、脊髓(spinal)或表皮给药(例如通过注射或输注)。
“可药用盐”是指保留抗体期望的生物活性而不带来任何不期望的毒物学效应的盐(参见例如Berge,S.M.等,J.Pharm.Sci.66(1977)1-19)。这样的盐包括在本发明范围内。这样的盐的实例包括酸加成盐和碱加成盐。酸加成盐包括从无毒的无机酸衍生的盐,如盐酸盐。
本发明的组合物可通过本领域公知的多种方法给药。正如技术人员将会理解的那样,给药路径和/或方式将随期望的结果而变动。
为通过某些给药路径给药本发明的化合物,可能需要用防止化合物失活的材料对其进行包衣,或化合物与之共给药。例如,可以向受试者给药处于适宜载体例如脂质体或稀释剂中的化合物。可药用稀释剂包括盐溶液和水性缓冲溶液。
可药用载体包括无菌水性溶液或分散液,以及用于临时制备无菌注射液或分散液的无菌粉剂。此类介质和药剂用于药物活性物质的用途是本领域公知的。
如本文所用的短语“肠胃外给药”和“经肠胃外给药”,表示不同于肠和局部给药的给药方式,通常是通过注射,包括但不限于:静脉内、肌内、动脉内、鞘内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。
这些组合物还可以含有佐剂,如防腐剂、润湿剂、乳化剂和分散剂。通过前述灭菌操作,以及通过纳入多种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸等,均可以确保防止微生物的存在。还可能期望在组合物中纳入等渗剂,如糖、氯化钠等。另外,可注射药物形式的延迟吸收可以通过纳入延迟吸收的药剂如单硬脂酸铝和明胶来实现。
无论所选的施用路径如何,可以以适宜水合物形式使用的本发明的化合物和/或本发明的药物组合物通过本领域公知的常规方法配制为可药用剂型。
根据本发明药物组合物中活性成分的实际剂量水平可有变动,以获得有效量的活性成分,从而对于特定的患者、组合物和给药方式实现期望的治疗反应,而对患者没有毒性。所选的剂量水平将取决于多种药代动力学因素,包括所采用的本发明特定组合物或其酯、盐或氨基化合物(amide)的活性、给药路径、给药时间、所采用特定化合物的排泄速率、与所采用特定组合物组合使用的其他药物、化合物和/或材料、所治疗患者的年龄、性别、体重、病情、总体健康和既往病史、以及医学领域众所周知的类似因素。
组合物必需是无菌和流动的,从而组合物可以通过注射器递送。除了水之外,载体还可以是等渗缓冲盐溶液,乙醇,多元醇(例如甘油,丙二醇,液体聚乙二醇,等等),以及它们适宜的混合物。
例如,可以通过使用包衣如卵磷脂,分散剂的情况下通过维持所需的颗粒大小,通过使用表面活性剂,来维持适当的流动性。在许多情况下,优选在组合物中纳入等渗剂,例如糖,多元醇如甘露醇或山裂糖醇,和氯化钠。可注射组合物的长期吸收可以通过在组合物中纳入延迟吸收的药剂如单硬脂酸铝或明胶来实现。
提供如下实施例和附图以帮助理解本发明,本发明的真正范围阐释在所附权利要求书中。应当理解,可以对所示的操作进行改动而不会偏离本发明的精神。
实施例
细胞系
用来产生重组表达IgG的细胞系的亲本细胞系是中国仓鼠卵巢(CHO)细胞系CHO-DG44(Flintoff,W.F.等,Somat.Cell Genet.2(1976)245-261;Flintoff,W.F.等,Mol.Cell.Biol.2(1982)275-285;Urlaub,G.等,Cell33(1983)405-412;Urlaub.G.等,Somat.Cell Mol.Genet.12(1986)555-566)。CHO-DG44细胞的两个内源二氢叶酸还原酶(DHFR)基因座均已丧失。
CHO-DG44细胞培养在MEM alpha Minus培养基(Gibco No.22561),10%透析的胎牛血清FCS(Gibco No.26400-044)和2mmol/L L-谷氨酸,100μM次黄嘌呤,16μM胸苷(HT增补物)中。
质粒
表达系统包括CMV启动子,并在表1中描述。至于抗体,使用了抗IGF-1R抗体(WO2005005635;AK18或AK22)。
表1
实施例1
转染和选择
表达质粒的转染利用Fugene(罗氏诊断有限公司,Roche DiagnosticsGmbH)进行。在转染1天后,将DG44细胞置于选择压力下:包括MEMalpha Minus培养基、10%透析的胎牛血清FCS、2mmol/L L-谷氨酸和20nM甲氨蝶呤(MTX)。置于选择压力下培养3周后,从板中挑取单个的克隆,并进行扩增。
收集上清液,利用人IgG特异性ELISA分析抗体的存在。亚克隆进一步扩增,并分析特异性抗体的产量。
使克隆适应生长于悬浮培养物和无血清培养基中,即含20nM MTX的HyQ SFM4CHO-Utility(海克隆公司,HyClone#SH30516)。平行测定糖模式分布(glycopattern profile)。选择脱岩藻糖基化(defucosylation)为2.0%或更低(指寡糖总摩尔量)的亚克隆。
实施例2
培养和纯化
细胞在装有30ml培养基的125ml摇瓶(康宁公司,Corning)中于37℃,5%CO2,100rpm条件下培养。通过CASY计数器测量细胞密度,采样上清液,通过蛋白质A亲合层析测定抗体浓度。对大约20ml的各上清液进行纯化,通过蛋白质A亲合层析(用PBS平衡,用25mM柠檬酸钠缓冲液pH5.2洗涤,用洗脱100mM柠檬酸钠缓冲液pH2.8,用10mM NaOH进行CIP原位清洁)进一步进行生化表征。
实施例3
抗体糖结构的分析
通过液相色谱/质谱(LCMS)肽图谱分析对纯化的抗体材料进行分析。对样品进行还原(0.4M TRIS/HCl,8M胍/HCl,pH8.5,DTT(3mg/ml),羧甲基化的(碘乙酸))并用胰蛋白酶切割。肽-糖肽混合物利用RP-HPLC分离,并利用电喷雾质谱进行在线分析。整合了含糖结构的肽的m/z谱,结构示于表2。
表2
糖基化变体的相对量
克隆号 | G0[%] | G1[%] | G2[%] | NonFuc[%] | Man1[%] |
1 | 38,4 | 51,4 | 10,2 | 0,1 | 0,5 |
2 | 44,3 | 47,6 | 8,1 | 0,1 | 0,6 |
3 | 42,8 | 48,7 | 8,5 | 0,2 | 0,8 |
4 | 49,2 | 43,6 | 7,2 | 0,3 | 1,2 |
5 | 62,7 | 33,0 | 4,3 | 0,6 | 1,0 |
6 | 60,4 | 35,5 | 4,2 | 0,5 | 1,2 |
7 | 40,4 | 49,8 | 9,8 | 0,3 | 0,6 |
8 | 46,9 | 45,9 | 7,3 | 0,3 | 1,1 |
Man:高甘露糖结构,分别携带4个和5个甘露糖残基
G0、G1、G2:岩藻糖基化二天线复杂型糖还原重链,具有1、2或3个末端半乳糖残基
nonFuc:二天线复杂型糖还原重链,不含岩藻糖
CHO细胞系克隆5(hu MAb<IGF-1R>B1-4E10_9-16)根据国际承认用于专利程序的微生物保存布达佩斯条约,于2006年6月21日保藏在德国的德意志微生物保藏中心,保藏号DSM ACC2795。
所用的培养基获自海克隆公司(Hyclone)(HyQ SFM4CHO-Utility,用于克隆4-6)或希格玛公司(Sigma)(C-8862,用于克隆1-3和7)。
通过整合所有糖肽所有电荷状态下的特异性离子色谱,进行LCMS肽图谱分析。
以相同的方式测定了平分型GlcNac、NGNA和高甘露糖。
平分型GlcNac和NGNA不可检测,因此NGNA的量为0.5%或更低,优选0.1%或更低。平分型GlcNac的量也为0.5%或更低,优选0.1%或更低。
例示性的糖基化计算(克隆3)示于表3(表3a:克隆3;表3b:克隆5;肽包含asn298,称为H27)。
表3a
加和:G042,4 |
加和:G148,2 |
加知:G28,4 |
总和99,0 |
相对于100%的G0-1-2 |
G042,8 |
G148,7 |
G28,5 |
加和:不含Man99,2 |
加和:G0/1-Fuc0,2 |
不含Fuc的相对量0,2 |
面积:峰面积
H27_G0至H27_G4:岩藻糖基化二天线复杂型糖糖肽H27(含有Asn298),具有x个末端半乳糖(例如,G4具有4个半乳糖单位)
不含Fuc的相对量:Fuc相对于不含甘露糖(4和5个)糖结构(高甘露糖)的所有G0、G1、G2的百分比
H27_G1_1NGNA至H27_G3_2NGNA:岩藻糖基化二天线复杂型糖糖肽H27(含有Asn298),具有x个末端半乳糖单位(例如,G2具有2个半乳糖单位),携带1-2个N-羟乙酰神经氨酸。
表3b
例示性的糖基化计算(克隆5)
1)岩藻糖基化二天线复杂型糖结构,具有x个末端半乳糖(分别为0、1、2、3和4个)
2)岩藻糖基化二天线复杂型糖结构,具有x个末端半乳糖(分别为0、1、2、3和4个),具有额外的N-羟乙酰神经氨酸残基
3)岩藻糖基化二天线复杂型糖结构(主要是本发明方法的人工制品)
4)高甘露糖结构,分别携带4或5个甘露糖残基
5)非岩藻糖基化的糖结构
实施例4
测定抗IGF-IR HuMAb抗体介导的效应功能
为了测定所产生的HuMAb抗体引发免疫效应机制的能力,进行了抗体依赖性细胞细胞毒作用(ADCC)的研究。
为了研究各抗体在ADCC中的效应,表达IGF-IR的DU145前列腺癌细胞(HTB-81;1×106,处于2-4ml RPMI-FM中)在细胞培养箱中用1μl双(乙酰氧基甲基)2,2’:6’,2”-三联吡啶-6,6”-二羧酸(bis(acetoxymethyl)2,2’:6’,2”-terpyridine-6,6”-dicarboxylate,BATDA)溶液于37℃标记25分钟。细胞用10ml RPMI-FM洗涤4次,以200×g制动旋转10分钟。之后,将细胞调整至1×105个细胞/ml的浓度。在圆底板的每个孔中涂布相当于50μl体积的5,000个细胞。添加处于50μl细胞培养基中的HuMAb抗体,终浓度范围为25-0.1ng/ml。随后,以25∶1范围的E∶T比添加50μl效应细胞,即从全血中新鲜分离的PBMC或从白细胞层纯化的效应细胞。板立即以200×g制动离心1分钟,于37℃孵育2小时。孵育后,细胞以200×g10分钟旋沉,将20μl上清液转移到Optiplate96-F微量滴定板中。添加200μl铕溶液(室温),混合物在摇床上孵育15分钟。利用来自珀金埃尔默公司(Perkin Elmer)的EU-TDA方案在时间分辨荧光计中测量所产生的荧光。
ADCC的细胞裂解幅度表达为占去污剂裂解靶细胞中TDA最大释放量[已针对相应靶细胞中的TDA自发释放进行了校准]的百分比。作为显示“无ADCC”抗体参比标准使用的是抗KLH(钥孔戚血蓝蛋白)(单克隆)抗体或从大约35.000个供体分离的IgG混合物(“Redimune”)。利用75%无岩藻糖的抗IGF-IR抗体作为阳性对照。根据本发明的抗体表现出的TDA释放处于标准抗体TDA释放3×SD的范围内(图1)。
与保藏的微生物或其它生物材料有关的声明
(PCT细则第13条之2)
PCT/RO/134表(1998年7月;2004年1月重印)
C.附加声明(附页)
申请人: 弗·哈夫曼-拉罗切有限公司
申请人卷号: 23706WO-SR
国际申请号: PCT/EP/2007/003164
说明书中所述保藏的生物材料的专家方案声明
涉及保藏的生物材料的声明均已包含在说明书中。如下附加声明无需为说明书的一部分,应当视为“其他声明”。这仅涉及专家方案。
如下的附加声明涉及说明书第4页所提及的保藏的生物材料:
huMAb<IGF-1R>B1-4E10_9-16)DSM ACC2795
附加声明如下:
对于CA(加拿大)指定:
对于加拿大指定,依照加拿大专利法专利细则第107和108条的规定,直到本申请被授予加拿大专利、或者直到被驳回之日、或经放弃且不能再恢复、或者被撤回的情况下,仅通过向委员会指定的独立专家发放样品的方式(细则104(4)),提供所保藏的生物材料样品。
对于EP(欧洲专利)指定:
对于EPO指定,依照EPC实施细则28(3)的规定,直到本申请得到欧洲专利授权公开、或者如果本申请被驳回、撤回或视为撤回的情况下,自递交之日起20年,仅通过向请求人指定的专家发放样品的方式(EPC细则28(4)),提供所保藏的生物材料样品。
对于SG(新加坡)指定:
申请人特此声明,依照1995年专利法细则第4方案第3款,仅向专家提供上述培养物样品。
Claims (5)
1.人IgG1或IgG3型单克隆抗体,所述抗体在Asn297处被糖链糖基化,所述抗体的特征在于
a)在所述糖链内岩藻糖的量至少为99%,其中所述量以不含甘露糖4和甘露糖5的G0、G1、G2之和作为100%,并且是通过液相色谱/质谱(LCMS)肽图谱分析所分析的;并且此外,
b)在所述糖链内NGNA的量为1%或以下,其中所述量以不含甘露糖4和甘露糖5的G0、G1、G2之和作为100%,并且是通过液相色谱/质谱(LCMS)肽图谱分析所分析的,
和/或在所述糖链内N末端α-1,3-半乳糖的量为1%或以下,其中所述量以不含甘露糖4和甘露糖5的G0、G1、G2之和作为100%,并且是通过液相色谱/质谱(LCMS)肽图谱分析所分析的。
2.根据权利要求1的抗体,其特征在于NGNA的量为0.5%或以下。
3.根据权利要求1或2的抗体,其特征在于N-末端α-1,3-半乳糖的量为0.5%或以下。
4.根据权利要求1至3的抗体,其特征在于所述抗体是嵌合、人源化或人抗体。
5.药物组合物,包含根据权利要求1至4的抗体。
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