CN103221063A - 针对人类巨细胞病毒(cmv)gb蛋白质的高亲和力人类抗体 - Google Patents
针对人类巨细胞病毒(cmv)gb蛋白质的高亲和力人类抗体 Download PDFInfo
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Abstract
已从人类B细胞分离出针对人类巨细胞病毒CMV gB蛋白质的抗体。这些抗体的亲和力高于先前报道的最佳抗体。由于高亲和力对于阻止病毒跨胎盘转移至关重要,故本发明抗体可用作阻止或改善妊娠期间CMV感染对胎儿的影响的治疗性和预防性药剂。
Description
相关申请案的交叉参考
本申请案主张2010年6月16日提出申请的美国临时专利申请案第61/355,499号的优先权,所述案件的全部内容以引用的方式并入本文中。
经由EFS-WEB提交的序列表参考
如MPEP§1730II.B.2(a)(C)中所审定和阐述,经由USPTO EFS-WEB服务器电子提交的以下序列表的全部内容出于所有目的全文以引用的方式并入本文中。在电子申请的文本文件上序列表鉴别如下:
文件名称 | 创建日期 | 大小(字节) |
388512012740seqlist.txt | 2011年6月16日 | 70,987个字节 |
技术领域
本发明涉及对抗CMV的gB蛋白质的人类单克隆抗体(mAb),其治疗性和预防性地用于阻止或改善妊娠期间CMV感染对胎儿的影响,和治疗免疫减弱患者(包括移植患者)的CMV感染。
背景技术
CMV在移植患者、其它免疫减弱的患者和新生儿中是重要的致病原。在美国,每年约40,000名婴儿出生时脱落CMV。这些婴儿中,8,000名婴儿出生时具有症状和/或严重残障,且多达8,000多名婴儿会在以后发生进行性听力损失。约一半的妊娠母亲天然地具有充足的免疫。因此,已知在人类血液中存在有效的mAb。这也可由静脉内投与的γ球蛋白(IVIG)实现成功免疫被动转移显示出来,所述静脉内投与的γ球蛋白已显示非常高的保护胎儿的功效。这与在移植环境中观察到的IVIG的有限功效形成对比,在所述移植环境中细胞免疫明显比体液免疫更重要。
大部分对CMV的天然反应是针对gB蛋白质(帕克J.W.(Park,J.W.)等人,韩国医学科学期刊(J.Korean Med.Sci.)(2000)15:133-138)。托尼疫苗(Towne vaccine)是在活体外广泛传代的减毒活病毒疫苗,其诱导中和成纤维细胞感染但不中和内皮细胞感染的抗体。已知这种疫苗是安全的且已研究了20年(阿黛乐S.P.(Adler,S.P.)等人,儿科感染性疾病期刊(Pediatr.Infect.Dis.J.)(1998)17:200-206)。可用于如下文所述分离gB抗体的血液供体包括先前暴露于CMV的血清阳性个体以及接种托尼疫苗之前和之后的血清阴性个体。
已制备CMV gB蛋白质的抗体(野泽N.(Nozawa N.)等人,临床病毒学期刊(J.Clin.Virol.)(2009)[印刷出版前的电子版(Epub ahead of print)];中岛N.(Nakajima,N.)等人,(US2009/0004198A1);兰扎韦基亚A(Lanzavecchia,A)等人,(US2009/0004198A1);奥林M.(Ohlin,M.)等人,(病毒学期刊(J Virol)(1993)67:703-710)。已报道gB的AD-2结构域的中和抗体ITC88(兰托(Lantto),病毒学(Virology)(2003)305:201-209)。然而,克隆人类抗CMV抗体的先前努力虽然是成功的,但范围有限,且未曾阐述高亲和力(亚纳摩尔)抗体。高亲和力是一个重要的参数,因为弱亲和力的CMV抗体实际上促进传染穿过人类胎盘(野泽,见上文),此病理学方面在啮齿类动物中未曾观察到。人类CMV具有大约236kb的双链DNA基因组,且是β-疱疹病毒家族的典型成员。基因组的高复杂性意味着存在许多种潜在目标抗原。表征中和抗体和其有关表位的努力产生基于糖蛋白B(gB)的亚单位疫苗,所述疫苗引发有效的中和反应,但在血清阴性妇女队列中测试时,仅具有50%功效。这似乎是任何CMV疫苗的最高功效。由于疫苗通常诱导具有一定范围亲和力的抗体,因此,迄今为止测试疫苗的令人失望的功效可归于对于高亲和力抗体的需要,此支持直接提供高亲和力mAb作为预防性策略。
还已假定不能使免疫反应集中在特定的中和表位是较差功效的原因(马歇尔B.C.(Marshall,B.C.)等人,病毒免疫学(Viral Immunol.)(2003)16:491-500)。研发抗CMV疫苗中的另一可疑的技术问题是,仅对其产生中和成纤维细胞感染的抗体的能力进行评估,但其它细胞类型的感染日益成为理解病毒病理学的焦点。此偏向反映关于病毒在活体外生长的技术障碍。认为在成纤维细胞中重复病毒传代已造成许多实验室株对于内皮和上皮细胞失去向性。在最近几年期间,已将此缺陷与病毒表面上gH/gL/UL131-UL128糖蛋白复合物的一种或一种以上组份的失去联系起来。
显然,当前需要更有效的抗CMV预防策略。
发明内容
已制备与CMV gB蛋白质特异性地免疫反应、与先前抗体(人类或鼠类)相比具有提高的亲和力且具有中和能力的人类抗体。体液免疫系统能够产生数百万种具有成千上万种充分区别的结合能力的抗体结构,而保护性抗体仅是这些抗体中的极小亚群。本发明者使用细胞斑点(CellSpot)TM技术(哈里曼W.D.(Harriman,W.D.)等人,免疫学方法期刊(J.Immunol.Methods)(2009)34:135-145;克拉瑞尼E.J.(Collarini,E.J.)等人,免疫学期刊(J.Immunol.)(2009)183:6338-6345;和美国专利第7,413,868号,所有文献均以引用的方式并入本文中),以便从供体血液产生一组经验证对CMV具有高效价的mAb。
因此,一方面,本发明涉及结合gB蛋白质上的表位的人类单克隆抗体或其免疫反应性片段,其中优选实施例是结合其中的保守序列。这些抗体在针对CMV中和的标准噬斑形成分析中呈现中和能力,且在所述分析中展示小于500ng/ml、优选小于200ng/ml、更优选小于100ng/ml的EC50。本发明抗体还对CMV株AD169的gB蛋白质具有小于10nM或小于5nM或小于1nM的亲和力。
对于在本发明治疗CMV感染或增强对CMV的抗性的方法中的使用,本发明的单克隆抗体或片段可与多种CMV株免疫反应,且单一单克隆抗体可足够具有期望效应。或者,可向欲治疗或使得具有抗性的个体投与多于一种单克隆抗体,所述抗体结合相同或不同的CMV蛋白质。
本发明还包括可用于预防或治疗的医药组合物,所述医药组合物含有本发明的单一抗体或免疫反应性片段或不多于两种本发明抗体或片段作为活性药剂。
本发明的其它方面包括使用所述抗体治疗人类个体的CMV或在人类个体中诱导对感染的抗性的方法。
本发明的单克隆抗体可重组产生,且因此,本发明还包括用于所述产生的重组材料以及用于产生这些抗体的细胞系或永生细胞和非人类多细胞生物体或其细胞、或微生物细胞。在一个实施例中,从人类个体获得的细胞是以“永生”形式产生,其中其已经修饰以容许在充足的时间段内分泌抗体,以便抗体可经表征且相关编码序列经克隆。
附图说明
图1A和1B显示4A2和19B10与gB蛋白质和其保守区的结合。
图2A和B显示由HUVEC和HFF细胞的mAb4A2、310、313、338和345中和VR1814。
图3显示由HUVEC中的mAb4A2、310、313、338和345中和VR1814。
图4显示由HFF细胞中的mAb4A2、310、313、338和345中和VR1814。
具体实施方式
本文所用的术语“治疗”是指降低已受CMV感染的个体中的病毒负荷或改善所述个体的疾病症状。所述症状包括视网膜炎和肝炎。
术语“赋予抗性”是指预防性效应,其中在攻击后RSV病毒感染至少严重程度有所降低。
“永生细胞”是指与未经修饰之原代分离细胞相比可存活显著更多传代的细胞。如在本发明上下文中所使用,“永生”不一定意味着细胞在非常长的时间段内持续分泌抗体,只是意味着与原代细胞培养物相比其可存活较长时间。分泌抗体的时间仅需足以鉴定抗体和回收编码核苷酸序列。
人类抗体(例如,本文中从人类细胞分离的那些)不引发强的免疫反应。已知,人类抗体在5-10%的治疗人类中引发反应,甚至对于从人类分离的抗体也是如此,这是由于在所引发的免疫反应中存在一定水平的背景“噪声”。免疫反应可为体液免疫反应或细胞免疫反应或二者。具体来说,可在此百分比的个体中发现提高水平的细胞因子。
CMV的gB蛋白质是以130kDa的前体蛋白质合成,其裂解成116kDa(N端)和58kDa(C端)的仍然共价连接的片段;观测到的分子量可依赖于糖基化状态而变化。AD-2抗原决定簇是指gp116的残基67到82。相当大部分的天然CMV免疫是由对AD-2的结合引起(即,可由覆盖这个区域的肽阻断),奥林(见上文)。
如实例1中所述,本发明抗体已使用专利的细胞斑点TM方法从暴露于CMV的人类供体回收,所述方法阐述于美国专利第7,413,868号、PCT公开案WO2005/045396及WO2008/008858中,所述案件以引用的方式并入本文中。
本发明人类或人源化抗体的产生是通过常规的重组技术达成,例如在中国仓鼠卵巢细胞或其它真核细胞系(例如昆虫细胞)中产生。或者,还已知在植物中和在转基因动物中(例如在牛乳中)或在得自微生物或植物或昆虫的单细胞系统中或在所述细胞的不含细胞的提取物中产生重组材料(包括抗体)的技术。
另外,由于可以获得编码抗体的核苷酸序列,因此可重组(或通过蛋白质本身的蛋白水解处理)产生结合相同表位的相关片段(例如,Fab、F(ab′)2或Fv片段),且可产生呈单链形式的抗体。业内已知多种用于操作重组抗体产生的技术。
嵌合、人源化和人类抗体全部在本发明的范围内,因为其是基于其它蛋白质支架(例如纤维连接蛋白、转铁蛋白或脂质运载蛋白)的抗体模拟物。同样,现存在多种制造纳入来自两种单独抗体的抗原特异性结构域的单一抗体样分子(双特异性抗体)的技术。适宜技术已由宏观基因(Macrogenics)(罗克维尔(Rockville),马里兰州(MD))、微麦特(Micromet)(贝塞斯达(Bethesda),马里兰州)和梅里麦克(Merrimac)(剑桥市(Cambridge),马萨诸塞州(MA))阐述。(参见,例如,奥卡特KD(Orcutt KD),阿克曼ME(Ackerman ME),西埃里维兹M(Cieslewicz M),奎罗兹E(Quiroz E),史拉萨斯基AL(Slusarczyk AL),弗兰焦尼JV(Frangioni JV),维特如普KD(Wittrup KD)。模块IgG-scFv双特异性抗体拓扑学(A modular IgG-scFv bispecific antibody topology).蛋白质工程设计与选择(ProteinEng Des Sel.)(2010)23:221-228;菲茨杰拉德J(Fitzgerald J),卢戈夫斯基A(LugovskoyA).靶向多种致癌基因途径的抗体治疗剂的合理改造(Rational engineering of antibodytherapeutics targeting multiple oncogene pathways).单克隆抗体(MAbs).(2011)1;3(3);贝耶乐PA(Baeuerle PA),莱因霍尔德C(Reinhardt C).用于癌症疗法的双特异性T细胞参与抗体(Bispecific T-cell engaging antibodies for cancer therapy).癌症研究(Cancer Res.)(2009)69:4941-4944。)
因此,可使用对不同CMV表位具有反应性的个别抗体的Fab结构域来构建具有非常宽广的株反应性的单一抗体,以使例如双特异性抗体具有对抗gB和gH复合物二者的活性,或另一选择为,可与来自相同或不同株的gB蛋白质反应。高亲和力gH抗体已由以下阐述:例如,马卡诺A(Macagno A),贝那斯科尼NL(Bernasconi NL),万泽他F(Vanzetta F),丹德E(Dander E),萨拉西尼A(Sarasini A),芮武罗MG(Revello MG),戛纳G(Gerna G),萨鲁斯托F(Sallusto F),兰扎韦基亚A(Lanzavecchia A).通过靶向gH/gL/UL128-131A复合物上的不同表位有效中和人类巨细胞病毒感染的人类单克隆抗体的分离(Isolation of human monoclonal antibodies that potently neutralize humancytomegalovirus infection by targeting different epitopes on the gH/gL/UL128-131Acomplex).病毒学期刊,(2010)84:1005-1013。还可产生和使用针对来自其它株的gH蛋白质的高亲和力抗体。
对于用于疗法中,使用适宜的赋形剂将重组产生的抗体或片段调配成医药组合物,并根据标准方案投与。医药组合物可具有本发明的单克隆抗体或片段、尤其与所有CMV株的gB蛋白质交叉反应的单克隆抗体或片段作为其唯一的活性成份。或者,两种单克隆抗体可为唯一的活性成份,其中一种抗体更强烈地与一种病毒株的gB蛋白质反应,而另一种抗体更强烈地与另一病毒株的gB蛋白质反应。在所有这些情况下,均可存在额外治疗剂,包括结合其它CMV蛋白质的那些。而且,化合物可包括营养物质,例如维生素,或除抗体以外的任何其它有益化合物。
在一个实施例中,当使用供投与的调配物来增加对感染的抗性时,使用包括含补体的Fc区的完整抗体。通常,抗体是以0.01-20mg/kg人类个体的剂量水平或以介于0.01-5mg/kg范围内的量或这些范围内的中间量投与。在一个实施例中,使用介于0.1-1.0mg/kg范围内的量。间隔若干天或若干周或若干个月重复投与可为有利的。
在另一实施例中,对于降低病毒载量的治疗效果,也使用含有含补体的Fc区的完整抗体。所述方案中所投与的量大约为.001-50mg/kg,或使用这个范围内的中间值,例如0.01、1或10mg/kg。还可使用重复投与。在感染诊断后尽可能快地投与治疗性治疗,但在数天内投与也在本发明的范围内。还可使用重复投与。为减轻肺中的炎症反应,仅需要使用抗体的免疫特异性片段。剂量水平与全抗体的剂量水平类似。投与免疫特异性片段和完整抗体的混合物也包括在本发明的范围内。
本发明的抗体组合物通常通过注射、通常静脉内注射来投与。因此,非经肠投与是优选的。然而,包括任何可操作的投与模式,包括基因疗法(在活体内产生重组抗体)。
以业内众所周知的用于投与抗体组合物的方式来制备调配物。适宜调配物可参见标准配方书,例如雷明顿医药科学(Remington’s Pharmaceutical Sciences),最新版,迈克出版公司(Mack Publishing Co.),伊斯顿(Easton),马萨诸塞州(PA),其以引用的方式并入本文中。调配物通常是适用于非经肠投与的那些,包括等渗溶液(其包括缓冲剂、抗氧化剂等)以及乳液(其包括递送媒剂,例如脂质体、胶束和纳米颗粒)。
期望方案和调配物取决于主治从业人员的判断以及个体的具体病况。如果需要,剂量水平将取决于个体的年龄、总体健康状况和感染的严重程度。
提供以下实例以举例说明而不是限制本发明。
实例1
分泌CMV gB抗体的人类B细胞的分离
针对产生抗病毒抗体的人类B细胞对来自50名经证实具有抗CMV效价的成人的外周血单核细胞进行检测。使用具有期望的对抗CMV gB蛋白质的抗体的个体来克隆特定mAb。检测的结果是约10%的个体具有大于1/50,000的期望细胞频率。
为实现稀少有利细胞的检测和回收,使用先前所述的细胞斑点TM技术(U.S.7,413,868,其以引用的方式并入本文中)。细胞斑点TM分析方法通过捕获从单一细胞所分泌的IgG作为所述细胞附近的印迹有效地将酶联免疫吸附分析(ELISA)等效分析缩小到几乎单一细胞尺寸的虚拟孔,由此可容易地分析数百万个细胞。此外,通过使用显微多重试剂(组合显色的荧光胶乳微球体,参照US6,642,062,其以引用的方式并入本文中),各克隆体的分泌抗体印迹可使用多重生物化学探针针对特异性和/或亲和力详细地进行表征。定量分析的保真度足以能够实现从检测群体拯救极其稀少的有利细胞,其中克隆的表达细胞显示与最初识别分析一致的表型。
筛选准则是结合经纯化的gB蛋白质以及病毒裂解物。gB蛋白质是从感染CMV的AD169株的293细胞纯化得到。克隆的亲和力等级排序是通过将珠粒上的抗原用血清白蛋白稀释来实现。此降低多齿结合分泌的IgG印迹(“亲合力(avidity)”效应)的机会,由此针对较高内在亲和力进行选择。
使用标准磁分离方法从人类供体血浆中的PBMC去除非B细胞。将细胞以1e6/ml再悬浮于IMDM/20%HI-FCS中;并用EBV永生化(由受感染B95-8细胞的上清液直接集结成球)。EBV按1∶100稀释来添加,且将细胞在37℃下培育2hr。洗掉过量的EBV,且将细胞:
(1)以2e6/ml在IMDM、20%HI-FCS、20%巨细胞肿瘤条件培养基、2μg/ml CpG(ODN2006)和10ng/ml IL-10中培养(仅用于检测),或
(2)使用磁性阳性选择针对表面IgG进一步选择。
将细胞以200-300个细胞/孔在受照射的人类肺细胞(MRC-5,5,000个细胞/孔)上在IMDM、20%HI-FCS、20%巨细胞肿瘤条件培养基、2μg/ml CpG(ODN2006)和10ng/mlIL-10中培养。每2-3天补充培养基。在第6天在细胞斑点TM中分析各孔的一半内容物。随后将检测分析呈阳性的少数孔中的剩余细胞利用相同的喂养细胞和培养条件稀释到10个、5个、1个和0.5个细胞/孔。4-5天之后,再次通过ELISA或细胞斑点TM分析这些限制性稀释板。
使细胞斑点TM纳米颗粒与病毒裂解物或经纯化的gB蛋白质偶联以筛选期望抗体。由受CMV AD169病毒感染的细胞产生的裂解物购自威若西(Virusys)(目录号为CV046)。(所述裂解物是在正常人类真皮成纤维细胞(NHDF)细胞系中产生。)重组CMV gB抗原在293细胞中以带有His标签的融合蛋白形式产生,且使用镍螯合柱进行纯化。将经纯化的gB蛋白质用于ELISA和细胞斑点分析。使AD169裂解物和经纯化的gB蛋白质的制剂分别如先前所述与纳米颗粒偶联,参见哈里曼等人(见上文)和克拉瑞尼等人(见上文)。
随后使用逆转录酶-PCR处理限制性稀释的阳性孔的内容物,以便回收抗体重链和轻链的编码mRNA。从使PBMC解冻到经由RT-PCR回收编码mRNA序列的总时间是10-12天。
实例2
将人类抗体克隆到CMV gB
使用半巢式聚合酶链反应(PCR)来实现来自阳性酶联免疫吸附分析孔的重排Ig重链和Ig轻链基因的扩增。对于先前未知的V-基因重排的扩增,构建家族特异性V-基因引物的集合,所述引物识别人类Ig基因座中的几乎所有V-基因区段。使用5’引物与对Cγ、Cκ和Cλ基因区段具有特异性的引物混合物。限制稀释CMV-gB特异性B细胞的克隆体形成能力(clonality)由来自不同后代细胞的V-基因扩增产物的序列比较明确地测定,并将扩增的全长V-基因重排克隆到IgG表达载体中。
详细来说,使用市售的RNA纯化试剂盒(RNeasyTM;凯杰(Qiagen)(德国))从分离的人类B细胞提取总mRNA。通过使用总RNA制备品和低聚核苷酸作为引物实施逆转录-PCR。各试样运行三次PCR反应:一次针对卡帕轻链(κ),一次针对兰布达轻链(λ),且一次针对伽马重链(γ)。使用凯杰一步骤(OneStep)RT-PCR试剂盒扩增(凯杰目录号为210212)。在偶合的RT-PCR反应中,利用RT酶(欧姆尼柯瑞普(Omniscript)TM和森西柯瑞普(Sensiscript)TM)的独特掺合物使用对应于C-κ、C-λ或对应于Cγ基因的CH1区的共有序列的反义序列特异性引物合成cDNA,在50℃下实施RT达1小时,随后由HotStarTaq DNA聚合酶针对高特异性和灵敏性实施cDNA的PCR扩增。各PCR反应使用5’正义引物的混合物。引物序列是基于VH、VK和VL的前导序列。PCR反应在95℃下运行15分钟,初始热启动,随后为95℃下30秒(变性)、60℃下45秒(复性)及72℃下1分钟(延伸),进行20个循环。
用于检测可变Ig片段并将其克隆到表达载体中的巢式PCR。在第二轮中,施加第一扩增反应的5μl分液。所用引物携载5’BglII和3’XbaI限制位点。实施30个PCR循环。第一和第二轮扩增使用相同的条件。在1%琼脂糖凝胶上加载5μl各反应,并分离,且随后用溴乙啶染色。预计V-C PCR产物分别将VH和VL的重排片段扩增到500bp和450bp。分子大小大约为500bp的PCR带指示阳性结果。纯化PCR产物(凯杰凝胶纯化试剂盒,目录号为28704),并使用特定的恒定区引物直接测序所提取的PCR产物。通过对制备用于重组产生的质粒测序来证实克隆片段的序列。
将上文所述的PCR片段消化并克隆到携载人类γ1或人类κ或λ的恒定区的个别表达载体中,以便在哺乳动物细胞中在活体外产生抗体。将编码重链和轻链的表达载体共转染到293(人类肾)细胞系(英杰(Invitrogen))中。经由使用基于阳离子脂质的转染试剂(293fectinTM;英杰)引入表达质粒。对于各转染反应,将20μg经纯化质粒和40μL293fectinTM与1mL(英杰)混合,并在室温下培育5min,随后合并,并容许在室温下形成复合物达20min。将DNA-293fectin复合物添加到接种在90mm陪替氏培养皿(petri plate)中并在37℃、8%CO2下培育的3×106个细胞中。在最后的程序中,在转染后72hr通过离心(3,000g,15min,在4℃下)收获上清液,以便回收分泌的抗体。
从约200万个淋巴细胞分离45种结合AD169裂解物的克隆体。这些克隆体中,大多数还结合重组gB蛋白质。
结合AD169和gB二者的mAb中的两者(4A2和19B10)具有中和能力。这些mAb中的一者(4A2)结合AD-2肽,所述AD-2肽是gB蛋白质上的保守位点。另一mAb(5C5)结合AD-2,但不中和病毒。
4A2和19B10的重链和轻链的氨基酸序列显示于下文中,包括可变区、D和J连接区、框架区(FR)和互补决定区(CDR)。重链上的分泌信号序列用斜体表示,且CDR1-3加下划线。
4A2HC,VH3-30核酸(SEQ ID NO:1)和氨基酸(SEQ ID NO:2)atggaattggggctgagctgggttttcgtcgttgctcttttaagaggtgtccagtgtcaa gtgttgttggaggagtctgggggaggcgtggtccagcctgggaggtctctgagactctccV L L E E S G G G V V Q P G R S L R L StgtgcaggctctggattcaccttcaataggcatggaattcactgggtccgccaggctccaC A G S G F T F NRHG I H W V R Q A PggcaaggggctggagtgggtgactgttatatcatctgatggagcaaatcaacagtatgcaG K G L E W V T V I S S D G A N Q Q Y AgagtccgtgaagggccgattcatcatctccagagacaattccaagaacacggtatatctaE S V K G R F I I S R D N S K N T V Y LgaaatgaatagcctgaggaatgacgacacgggtgtgtatttctgcgcgagagacggtcgtE M N S L R N D D T G V Y F C A R D G RtgtgaaggcgagaggtgctactccggtgtcacggacttctggggccagggaacactggtcC E G E R C Y S G V T D F W G Q G T L V
4A2LCL6,IgKV3-11核酸(SEQ ID NO:3)和氨基酸(SEQ ID NO:4)atggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccaccggaM E A P A Q L L F L L L L W L P D T T GgaaattgtattgacacagtctccagccaccctgtctttgtctccaggggagagagccaccE I V L T Q S P A T L S L S P G E R A TctctcctgcagggccagtcagaatattggcggctacttggcctggttccaacaaaaagctL S C R A S Q N I G G Y L A W F Q Q K AggccaggctcccaggctcctcatctatgatgcatccatcagggccactggcatcccagccG Q A P R L L I Y D A S I R A T G I P AaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctR F S G S G S G T D F T L T I S S L E PgaagattttgcagtttattactgtcagcagcgtaacagttggcctccactcactttcggcE D F A V Y Y C Q Q R N S W P P L T F G
19B10HC VH4-31,D2,J6核酸(SEQ ID NO:5)和氨基酸(SEQ ID NO:6)atgaaacatctgtggttct tcctcctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagcagtcgggcccaggactggtgaagccttcacagaccctgtccctcaccV Q L Q Q S G P G L V K P S Q T L S L TtgcactgtctctggtggctccatcagtagcggtgatttttgctggaattggatccgccagC T V S G G S I S S G D F C W N W I R QcccccagggaagggcctggagtggattgggtacatctgttacaccggggacacctactacP P G K G L E W I G Y I C Y T G D T Y YaacccgccccttaacagtcgagttaccatatcagtcgacaggtccaggaaccaaatctccN P P L N S R V T I S V D R S R N Q I SctgaggctgagttctgtgactgccgcagacacggccgtgtattattgtgccagagaggatL R L S S V T A A D T A V Y Y C A R E DaggagacaactacactctcgcccctacttctactacggtttggacgtctggggccgagggR R Q L H S R P Y F Y Y G L D V W G R GaccaaggtcaccgtctcctcagcttccaccaagggcccatcggtcttccccctggtacccT K V T V S S A S T K G P S V F P L V PtctagcS S
19B10LC,A3,IgKV2核酸(SEQ ID NO:7)和氨基酸(SEQ ID NO:8)atgaggctccctgctcagcttctggggctgctaatgctctgggtctctggatccagtgggM R L P A Q L L G L L M L W V S G S S GgagattgtgatgactcagtctccgctctccctgcccgtcacccctggagagacggcctccE I V M T Q S P L S L P V T P G E T A SatctcctgcaggtctagtcagagcctcctgcatagtaatggacacaactatttggattggI S C R S S Q S L L H S N G H N Y L D WtatctgcagaagccagggcagtctccacacctcctgatctatttgggttctattcgggccY L Q K P G Q S P H L L I Y L G S I R AtccggggtccctgacaggttcagtggcagtggaacaggcacagattttacactgaaaatcS G V P D R F S G S G T G T D F T L K IagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcctS R V E A E D V G V Y Y C M Q A L Q T PaacacttttggccaggggaccaagctggagatcagacgaactgtggctgcaccatctgtcN T F G Q G T K L E I R R T V A A P S V
通过在荧光珠粒上制备以高或低密度经全长gB蛋白质或Ad-2肽涂布的细胞斑点探针来产生高亲和力抗体。不受限于理论,由于低密度降低多齿亲合力效应,所以本研究偏向于具有高内在亲和力的抗体。分离多种高亲和力抗体并测序。测定与CMV反应的其它单克隆抗体的序列,并显示为SEQ ID NO:9-36和38-66。人类IgG1重链恒定区的核苷酸序列显示于SEQ ID NO:37中。
在本发明抗体中,重链可具有GFTFNRHG(SEQ ID NO:67)或GSISSEDFC(SEQ IDNO:68)的CDR1;和/或SSDGANQ(SEQ ID NO:69)或ICYTGD(SEQ ID NO:70)的CDR2区;和/或ARDGRCEGERCYSGVTDF(SEQ ID NO:71)或AREDRRQLHSRPYFYYGLDV(SEQ ID NO:72)的CDR3区。在其它实施例中,轻链具有QNIGGY(SEQ ID NO:73)或QSLLHSNGHNY(SEQ ID NO:74)的CDR1区;和/或DAS(SEQ ID NO:75)或LG(SEQ IDNO:76)的CDR2区;和/或QQRNSWPPLT(SEQ ID NO:77)或QALQTPNT(SEQ ID NO:78)的CDR3区。
实例3
亲和力测定
通过福特生物(门洛帕克(Menlo Park),加利福尼亚州(CA))生物传感器分析测定本发明抗体的亲和力。在这个方法中,用EDC/NHS激活胺反应性生物传感器上的羧酸基团。将稀释在MES缓冲液(pH5)中的抗体附接到探针的经激活表面,并用乙醇胺阻断剩余的活性羧基化基团。将gB蛋白质与涂布Ab的探针一起培育,并通过福特生物仪器测定与涂布Ab的探针缔合的速率和从涂布Ab的探针解离的速率。
在一个实验中,测得4A2具有168pM的亲和力,且19B10具有697pM的亲和力,如表1中的对应IC50(μg/ml)所显示。这些亲和力常数实质上好于已公布的单克隆gB抗体。
表1
mAb的中和效能的比较.
测定mAb310、313、345和4A2对gB的AD-2表位的结合亲和力。gB的结合动力学显示于表2中。mAb310、313和338的效能约为mAb4A2的10倍。mAb323、316和338的效能也高于mAb4A2(数据未显示)。其余mAb的结合亲和力也进行了测试,且同样好于已公布的单克隆gB抗体。
表2
gB结合动力学
实例4
酶联免疫吸附分析结合分析和表位作图
针对与经纯化的gB蛋白质和称为AD-2的保守肽:NETIYNTTLKYGDV(SEQ IDNO:79)的结合来评估mAb4A2和19B10。如图1中所显示,4A2充分结合全长蛋白质和肽AD-2二者,而19B10仅结合蛋白质。
实例5
病毒中和分析
mAb4A2和19B10中和MRC5原代成纤维细胞中的AD169CMV株。将抗体的系列稀释物与等体积的AD169混合(将108/ml原液稀释到每孔2000个受感染细胞),并在室温下培育1h,随后添加到存于96孔微量滴定板中的目标细胞单层中。24h之后,将细胞固定,可渗透化处理,并用偶联到HRP的对抗IE1(即刻早期蛋白1,也称为UL123,复制病毒的标记物)的单克隆抗体染色。在沉积HRP底物之后检测受感染细胞。绘制受感染细胞的数量对抗体浓度的图。
还使用VR1814株评估病毒中和(芮武罗(Revello)等人,基因病毒学期刊(J.Gen.Virol.)(2001)82:1429-1438)。mAb4A2、310、313、338和345中和人类脐静脉内皮细胞(HUVEC)和人类包皮成纤维细胞(HFF)二者中的VR1814株。图2A和2B显示mAb4A2、310、313、338和345中的每一者的IC50和IC90值。图3和4分别显示mAb4A2、310、313、338和345中的每一者对HUVEC和HFF细胞的中和。结果重复测试两次。
还测试中和AD169和VR1814株的其它mAb。
序列表
4A2HC,VH3-30核酸(SEQ IDNO:1)和氨基酸(SEQ ID NO:2)atggaattggggctgagctgggttttcgtcgttgctcttttaagaggtgtccagtgtcaa gtgttgttggaggagtctgggggaggcgtggtccagcctgggaggtctctgagactctccV L L E E S G G G V V Q P G R S L R L StgtgcaggctctggattcaccttcaataggcatggaattcactgggtccgccaggctccaC A G S G F T F N R H GI H W V R Q A PggcaaggggctggagtgggtgactgttatatcatctgatggagcaaatcaacagtatgcaG K G L E W V T V I S S D G A N Q Q Y AgagtccgtgaagggccgattcatcatctccagagacaattccaagaacacggtatatctaE S V K G R F I I S R D N S K N T V Y LgaaatgaatagcctgaggaatgacgacacgggtgtgtatttctgcgcgagagacggtcgtE M N S L R N D D T G V Y F C A R D G RtgtgaaggcgagaggtgctactccggtgtcacggacttctggggccagggaacactggtcC E G E R C Y S G V T D F W G Q G T L V
4A2LC L6,IgKV3-11核酸(SEQ ID NO:3)和氨基酸(SEQ ID NO:4)atggaagccccagcgcagcttctcttcctcctgctactctggctcccagataccaccggaM E A P A Q L L F L L L L W L P D T T GgaaattgtattgacacagtctccagccaccctgtctttgtctccaggggagagagccaccE I V L T Q S P A T L S L S P G E R A TctctcctgcagggccagtcagaatattggcggctacttggcctggttccaacaaaaagctL S C R A S Q N I G G Y L A W F Q Q K AggccaggctcccaggctcctcatctatgatgcatccatcagggccactggcatcccagccG Q A P R L L I Y D A S I R A T G I P AaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctR F S G S G S G T D F T L T I S S L E PgaagattttgcagtttattactgtcagcagcgtaacagttggcctccactcactttcggcE D F A V Y Y C Q Q R N S W P P L T F G
19B10HC VH4-31,D2,J6核酸(SEQ ID NO:5)和氨基酸(SEQ ID NO:6)atgaaacatctgtggttcttcctcctgctggtggcagctcccagatgggtcctgtcccaggtgcagctgcagcagtcgggcccaggactggtgaagccttcacagaccctgtccctcaccV Q L Q Q S G P G L V K P S Q T L S L TtgcactgtctctggtggctccatcagtagcggtgatttttgctggaattggatccgccagC T V S G G S I S S G D F C W N W I R QcccccagggaagggcctggagtggattgggtacatctgttacaccggggacacctactacP P G K G L E W I G Y I C Y T G D T Y YaacccgccccttaacagtcgagttaccatatcagtcgacaggtccaggaaccaaatctccN P P L N S R V T I S V D R S R N Q I SctgaggctgagttctgtgactgccgcagacacggccgtgtattattgtgccagagaggatL R L S S V T A A D T A V Y Y C A R E DaggagacaactacactctcgcccctacttctactacggtttggacgtctggggccgagggR R Q L H S R P Y F Y Y G L D V W G R GaccaaggtcaccgtctcctcagcttccaccaagggcccatcggtcttccccctggtacccT K V T V S S A S T K G P S V F P L V PtctagcS S
19B10LC,A3,IgKV2核酸(SEQ IDNO:7)和氨基酸(SEQ ID NO:8)atgaggctccctgctcagcttctggggctgctaatgctctgggtctctggatccagtgggM R L P A Q L L G L L M L W V S G S S GgagattgtgatgactcagtctccgctctccctgcccgtcacccctggagagacggcctccE I V M T Q S P L S L P V T P G E T A SatctcctgcaggtctagtcagagcctcctgcatagtaatggacacaactatttggattggI S C R S S Q S L L H S N G H N Y L D WtatctgcagaagccagggcagtctccacacctcctgatctatttgggttctattcgggccY L Q K P G Q S P H L L I Y L G S I R AtccggggtccctgacaggttcagtggcagtggaacaggcacagattttacactgaaaatcS G V P D R F S G S G T G T D F T L K IagcagagtggaggctgaggatgttggggtttattactgcatgcaagctctacaaactcctS R V E A E D V G V Y Y C M Q A L Q T PaacacttttggccaggggaccaagctggagatcagacgaactgtggctgcaccatctgtcN T F G Q G T K L E I R R T V A A P S V
人类IgG1HC恒定区的氨基酸序列(SEQ ID NO:9)
ASTKGPSVFPLVPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
MAB297HC可变结构域的氨基酸序列(SEQ ID NO:10)
QVQLVQSGGGVVQPGRSLRLSCSASGFTFSNYNMHWVRQAPGKGPEWVAVISKDGNEKHYAESAKGRFTISRDNSKNTLYMEMHSLTPEDTAMYYCTRDGRTDGTGYSGILDIWGQGTKVIVS
MAB309和318HC可变结构域的氨基酸序列(SEQ ID NO:11)
QVQLVQSGGGVVQPGTSLRLSCAASGFMFNTYNMHWVRQAPGKGLEWVAVISNDGTYKHFADSLKGRFSISRDDSKNTLYLHMNSLRPDDTAIYYCARDGRSVGGFSGILDPWGQGTLVTVSS
MAB310HC可变结构域的氨基酸序列(SEQ ID NO:12)
QVQLVQSGGGVVQPGTSLRLSCAASGFMFNTYNMHWVRQAPGKGLEWVAVISNDGTYKYSADSLKGRFSISRDNSKNTLYLHMNSLRPDDTAVYYCARDGRSVGGFSGILDPWGQGTLVTVSS
MAB313HC可变结构域的氨基酸序列(SEQ ID NO:13)
QVQLVQSGGGVIQPGRSLTLSCAASGFTFSAYSLHWVRQAPGKGLQWVAVISFDGNFKHFADSLRGRFTISRDNSKNRFYLQMNGLRGEDTAVYYCARDGRAVDGFSGILDFWGQGTLVSVSS
MAB314HC可变结构域的氨基酸序列(SEQ ID NO:14)
QVQLQESGGGLVQPGGSLKLSCAVSGFSFGGSAMHWVRQASGKGLEWIGHIRSGANNFETAYAPSLDGRFTISRDDSKNTAYLHMNSLKTDDTAMYFCTTGLIASGDANFDYWGQGTQVTVSS
MAB316HC可变结构域的氨基酸序列(SEQ ID NO:15)
QVQLVQSGGGVVQPGRSLTLSCAASGFTFSGFSLHWVRQAPGKGLQWVAVISFDGNHKHFADSLKGRFTISRDNSKNTLYLQINDLRGEDTAVYYCARDGRAVDGFSGILDFWGQGTLVSVSS
MAB319HC可变结构域的氨基酸序列(SEQ ID NO:16)
QVQLVESGGGVVQPGRSLRLSCSASGFTFSDYNLHWVRQAPGKGLEWVAVISIDGSDKHHADSVKGRFTVSRDNSKNTVSLQMDSLRPEDTAVYYCARDGRSVGGYSGILDPWGQGTLVTVSS
MAB321HC可变结构域的氨基酸序列(SEQ ID NO:17)
EVQLVESGAEVKKPGESLKISCQGSGYRFTNYWIAWVRQMPGKGLEWMGIIYPGDSDTRYHPSFQGQVTISSDKSLNTAYLQWSSLKPSDTAVYYCARHHCLSTNCQTAVAGYNDYWGQGNPGRRLLS
MAB322HC可变结构域的氨基酸序列(SEQ ID NO:18)
QVQLVQSGGGVVQPGRSLRLSCSASGFTFTNYNMHWVRQAPGKGLEWVAVTSKDGNEKHFADSVKGRFTISRDNSKNTLYLEMNTLTAEDTAIYYCTRDGRTDGTGYSGILDIWGQGTKVTVSS
MAB323HC可变结构域的氨基酸序列(SEQ ID NO:19)
QVQLVQSGGGVVQPGRSLRLSCAASGFTFSNFAMHWVRQAPGKGLEWVAVISNAGRETHYADSVKGRFTVSRDNSKNMLSLQMNSLRGEDTAVYYCARDGRTDGSGYSGVLDIWAQGTLVTVSS
MAB338HC可变结构域的氨基酸序列(SEQ ID NO:20)
QVQLVESGGGVVQPGRSLRLSCSGSGFTFSDYNLHWVRQAPGKGLEWVAVISIDGTNKHHADSVKGRFTISRDNSKNTVNLEMSRLKAEDTAVYYCVRDGRSIGGYSGIFDPWGQGTLVTVSS
MAB343HC可变结构域的氨基酸序列(SEQ ID NO:21)
QVQLQESGGGVVQPGRSLRLSCAASGFTFNTYNMHWVRQAPGKGLEWVAVISNDGTYKYSADSVKGRFSISRGNSKNTLYLQMNSLRPDDTAVYYCARDGRSVGGFSGILDPWGQGTLATVSS
MAB345HC可变结构域的氨基酸序列(SEQ ID NO:22)
QVQLVESGGGVVQPGRSLRLSCAASGFTFSDYNMHWVRQAPGKGLEWVAVISIDGTYKYSADSVAGRFSLSRDNSKNTLYLQMNSLRPDDTAIYYCARDGRSVGGFSGILDPWGQGTLVTVSS
人类LC恒定κ区的氨基酸序列(SEQ ID NO:23)
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
MAB297和MAB322LC可变结构域的氨基酸序列(SEQ ID NO:24)
EIVMTQSPATLSLSPGERATLSCRASQSVGGYLAWYQQKPDQAPRLLIYDVSNRAAGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNTWPPLTFGGGTKVEIKR
MAB309LC可变结构域的氨基酸序列(SEQ ID NO:25)
EIVLTQSPATLSLSPGDRATLSCRASQTVGRYLAWYQQKPGQAPRLLIYDASDRATGISARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIKR
MAB310LC可变结构域的氨基酸序列(SEQ ID NO:26)
EIVLTQSPATLSLSPGDRATLSCRASQTVGRYLAWYQQKPGQAPRLLIYDASDRATGISARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIKR
MAB313LC可变结构域的氨基酸序列(SEQ ID NO:27)
EIVMTQSPATLSLSPGERATLSCRASQSVGRYLTWFQQKPGQAPRLLIYDASERATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRANWPPLTFGGGTKVEIK
MAB314LC可变结构域的氨基酸序列(SEQ ID NO:28)
EIVMTQSPGTLSLFPGERATLSCRASQTVRNGYLAWYQQKPGQAPRLLIYGASIRATGIPDRFSGSGSETDFTLSITRVEPEDFAVYYCQQYGRLSSTFGQGTKLDLK
MAB316LC可变结构域的氨基酸序列(SEQ ID NO:29)
EIVMTQSPATLSLSPGERATLSCRASQSVGRYLTWFQQKPGQAPRLLIYDASERATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK
MAB318LC可变结构域的氨基酸序列(SEQ ID NO:30)
EVVLTQSPATLSLSPGDRATLSCRASQTVGRYLAWYQQKPGQAPRLLIYDASDRATGISARFSGSGSGTDFTLTIGSLEPEDFAVYYCQQRSSWPPLTFGGGTKVEIK
MAB319LC可变结构域的氨基酸序列(SEQ ID NO:31)
EIVLTQSPATLSLSPGERATLSCRASQSVGSYLAWYQQKPGQAPRLLIYDASERATGIPARFSGSGSGTDFTLTISSLEPEDVAVYYCQQRNNWPPLTFGGGTKVEIK
MAB321LC可变结构域的氨基酸序列(SEQ ID NO:32)
EIVMTQSPDSLAVSLGERATINCKSSQSILFSSKNQNHLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNIPHTFGGGTKVEIK
MAB323LC可变结构域的氨基酸序列(SEQ ID NO:33)
ETVLTQSPATLSLSPGERATLSCRASQSVNRYLAWFQHRPGQPPRLLIYDASKRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK
MAB338LC可变结构域的氨基酸序列(SEQ ID NO:34)
RIVLTQSPATLSLSPGERATLSCRASQSVDRYLAWYQQKPGQAPRLLIYDASQRATGIPARFSGSGSGTDFTLAISSLEPEDVAVYYCQQRSNWPPLTFGGGTKIEIK
MAB343LC可变结构域的氨基酸序列(SEQ ID NO:35)
EIVMTQSPATLSLSPGDRATLSCRASQSVGSYLAWYQQKPGQAPRLLIYDASDRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK
MAB345LC可变结构域的氨基酸序列(SEQ ID NO:36)
EIVMTQSPATLSLSPGDRATLSCRASQSVGSYLAWYQQKPGQAPRLLMYDSSVRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNNWPPLTFGGGTKVEIK
人类IgG1HC恒定区的核苷酸序列(内含子加下划线)(SEQ
ID NO:37)
GCCTCCACCAAGGGCCCATCAGTCTTCCCCCTGGCACCCTCTACCAAGAGCACCTCTGGGGGCACAACGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCT CAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGC AGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGA GAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAA CCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATAT CCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCC TCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGC AGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGC CCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCA GGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCT GTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA
MAB297HC可变结构域的核苷酸序列(SEQ ID NO:38)
CAGGTGCAACTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTTCAGCCTCTGGATTCACCTTCAGCAACTATAATATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCCGGAGTGGGTGGCAGTTATATCAAAAGATGGAAACGAAAAACACTATGCAGAGTCTGCGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATATGGAAATGCACAGCCTGACACCTGAGGACACGGCTATGTATTACTGTACGAGAGATGGGCGAACCGATGGTACTGGGTACTCCGGTATTCTTGATATCTGGGGCCAAGGGACAAAGGTCATCGTCTCT
MAB309和318HC可变结构域的核苷酸序列(SEQ ID NO:39)
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATGTTCAATACCTATAATATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTATAAGCATTTCGCTGACTCCCTGAAGGGCCGATTCAGCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCACATGAACAGCCTGAGACCTGACGACACGGCTATATATTACTGTGCGAGAGATGGCCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
MAB310HC可变结构域的核苷酸序列(SEQ ID NO:40)
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATGTTCAATACCTACAATATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTATAAGTACTCCGCTGACTCCCTGAAGGGCCGATTCAGCATCTCCAGAGACAATTCCAAGAACACGTTGTATCTGCACATGAACAGCCTGAGACCTGACGACACGGCTGTATATTACTGTGCGAGAGATGGCCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
MAB313HC可变结构域的核苷酸序列(SEQ ID NO:41)
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGATCCAGCCTGGGAGGTCCCTGACACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGCCTATTCTCTACACTGGGTCCGCCAGGCTCCAGGCAAAGGGCTACAGTGGGTGGCGGTTATCTCATTTGATGGGAATTTTAAACACTTCGCAGACTCCCTGAGGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACAGATTCTATTTGCAAATGAATGGCCTGAGAGGTGAGGACACGGCTGTATATTACTGTGCGAGAGATGGACGTGCTGTTGACGGGTTTAGTGGGATCCTCGACTTCTGGGGCCAGGGAACCCTAGTCAGCGTCTCCTCAG
MAB314HC可变结构域的核苷酸序列(SEQ ID NO:42)
CAGGTGCAGCTGCAGGAGTCGGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAAACTCTCCTGTGCAGTCTCTGGATTCTCCTTCGGTGGCTCTGCAATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGATTGGCCATATTAGAAGCGGAGCTAATAATTTCGAGACAGCATATGCTCCGTCGCTGGATGGCAGGTTCACCATCTCCAGAGACGATTCAAAGAACACGGCGTATCTGCACATGAACAGCCTGAAAACCGATGACACGGCCATGTATTTCTGCACTACCGGACTTATAGCGTCAGGTGATGCAAATTTTGACTACTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCGG
MAB316HC可变结构域的核苷酸序列(SEQ ID NO:43)
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGACACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGGCTTTTCTCTACACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTACAGTGGGTGGCGGTTATCTCATTTGATGGGAACCATAAACACTTCGCAGACTCCCTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACATTGTATTTGCAAATTAATGACCTGAGAGGTGAGGACACGGCTGTATATTACTGTGCGAGAGATGGACGTGCTGTTGACGGGTTTAGTGGGATTCTCGACTTCTGGGGCCAGGGAACCCTGGTCAGCGTCTCCTCAG
MAB319HC可变结构域的核苷酸序列(SEQ ID NO:44)
CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTTCAGCCTCAGGATTCACCTTCAGTGACTATAATCTACACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTCATCTCAATTGATGGAAGCGATAAACACCACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAACACAGTGAGTCTACAAATGGACAGCCTGAGACCTGAAGACACGGCTGTATATTACTGTGCGAGAGATGGCCGTAGTGTGGGCGGCTACAGTGGGATCCTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
MAB321HC可变结构域的核苷酸序列(SEQ ID NO:45)
GAGGTGCAGCTGGTGGAGTCCGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTCAGGGTTCTGGATACAGGTTTACCAATTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATATCACCCGTCCTTCCAAGGCCAGGTCACCATCTCATCCGACAAATCCCTCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGCCCTCGGACACCGCCGTGTATTACTGTGCGAGACACCACTGCCTTAGTACCAACTGCCAAACCGCAGTGGCTGGATATAATGACTACTGGGGCCAGGGAAACCCTGGTCGCCGTCTCCTCAG
MAB322HC可变结构域的核苷酸序列(SEQ ID NO:46)
CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTTTCCTGTTCAGCCTCTGGATTCACCTTCACCAACTATAACATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACGTCAAAAGATGGAAACGAAAAACACTTTGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGGAAATGAACACCCTGACAGCTGAGGACACGGCGATATATTACTGTACGAGAGATGGGCGAACCGATGGTACTGGGTACTCCGGTATTCTTGATATCTGGGGCCAAGGGACAAAGGTCACCGTCTCCTCA
MAB323HC可变结构域的核苷酸序列(SEQ ID NO:47)
CAGGTGCAGCTGGTGCAGTCTGGGGGAGGGGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTTTGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGCTGGAAGGGAAACACACTACGCAGACTCCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATTCCAAGAATATGTTGTCTCTGCAAATGAACAGCCTGAGAGGTGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGCGAACCGATGGTAGTGGCTATTCCGGTGTTCTTGATATCTGGGCCCAAGGGACACTGGTCACTGTCTCCTCA
MAB328HC可变结构域的核苷酸序列(SEQ ID NO:48)
CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTTCAGGCTCTGGATTCACCTTCAGTGACTATAATCTACACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAATGGGTGGCAGTCATTTCAATTGATGGAACTAATAAACACCACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCAAGAATACAGTGAATCTGGAAATGAGTCGGCTGAAAGCAGAAGACACGGCTGTATATTACTGTGTGAGAGATGGGCGAAGTATTGGCGGCTACAGTGGAATCTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
MAB343HC可变结构域的核苷酸序列(SEQ ID NO:49)
CAGGTGCAGCTGCAGGAGTCAGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATACCTACAATATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCAAATGATGGAACCTATAAATACTCCGCTGACTCCGTGAAGGGCCGATTCAGCATCTCCAGAGGCAATTCCAAGAACACGTTGTATCTGCAGATGAACAGCCTGAGACCTGACGACACGGCTGTATATTACTGTGCGAGAGATGGGCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGCCAGGGAACCCTGGCCACCGTCTCCTCA
MAB345HC可变结构域的核苷酸序列(SEQ ID NO:50)
CAGGTGCAGCTGGTGGAGTCCGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACAATATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTCAATTGATGGAACGTATAAATACTCCGCTGACTCCGTGGCGGGCCGATTCAGTCTCTCCAGAGACAATTCCAAGAACACGTTGTATTTGCAGATGAATAGTCTGAGACCTGACGACACGGCTATATATTATTGCGCGAGAGATGGGCGTAGTGTTGGCGGGTTTAGTGGGATCCTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
人类LC恒定κ区的核苷酸序列(SEQ ID NO:51)
ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
MAB297和MAB322LC可变结构域的核苷酸序列(SEQ ID NO:52)
GAAATTGTAATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCGGCTACTTAGCCTGGTACCAACAGAAACCTGACCAGGCTCCCAGGCTCCTCATCTATGATGTTTCCAATAGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGGAACACCTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA
MAB309LC可变结构域的核苷酸序列(SEQ ID NO:53)
GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGCATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAGCTGGCCGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA
MAB310LC可变结构域的核苷酸序列(SEQ ID NO:54)
GAAATTGTGTTGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGCATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAACTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA
MAB313LC可变结构域的核苷酸序列(SEQ ID NO:55)
GAAATTGTGATGACTCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGATACTTAACTTGGTTCCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGAGAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGCGTGCTAACTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGA
MAB314LC可变结构域的核苷酸序列(SEQ ID NO:56)
GAAATTGTGATGACCCAGTCTCCAGGCACCCTGTCCTTGTTTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTAGGAACGGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCTTCCATCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGAGACAGACTTCACCCTCAGCATCACCAGAGTGGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGTATGGAAGGTTATCGTCCACTTTTGGCCAGGGGACCAAGCTGGACCTCAAACGA
MAB316LC可变结构域的核苷酸序列(SEQ ID NO:57)
GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGATACTTAACTTGGTTCCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGAGAGGGCCACTGGCGTCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAACAGCGTAGTAACTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
MAB318LC可变结构域的核苷酸序列(SEQ ID NO:58)
GAAGTTGTGCTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTGTTGGCAGGTACTTAGCCTGGTACCAACAAAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGCATCTCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCGGCAGCCTGGAGCCTGAAGATTTTGCAGTCTATTACTGTCAGCAGCGGAGCAGCTGGCCGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
MAB319LC可变结构域的核苷酸序列(SEQ ID NO:59)
GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGGGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTATCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCGAGAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATGTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCTCCGCTCACCTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
MAB321LC可变结构域的核苷酸序列(SEQ ID NO:60)
GAAATTGTGATGACCCAGTCTCCAGACTCCCTTGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGTCAGAGTATTTTATTCAGCTCCAAGAATCAGAACCACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTGATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCCGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTCCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAATATTCCTCACACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
MAB323LC可变结构域的核苷酸序列(SEQ ID NO:61)
GAAATTGTGTTGACTCAGTCTCCAGCCACCTTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCCGGGCCAGTCAGAGTGTTAACCGCTACTTAGCCTGGTTCCAACACAGACCTGGCCAGCCTCCCAGGCTCCTCATCTATGATGCGTCCAAGAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAG
MAB338LC可变结构域的核苷酸序列(SEQ ID NO:62)
GAAATTGTGTTGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGACAGGTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGACTCCTCATCTATGATGCATCCCAGAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCCGGGACAGACTTCACTCTCGCCATCAGCAGCCTGGAGCCTGAAGATGTTGCAGTTTATTACTGTCAGCAGCGTAGTAACTGGCCTCCGCTCACCTTCGGCGGAGGGACCAAAATAGAGATCAAA
MAB343LC可变结构域的核苷酸序列(SEQ ID NO:63)
GAAATCGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCTTCCGACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC
MAB345LC可变结构域的核苷酸序列(SEQ ID NO:64)
GAAATTGTGATGACCCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGATAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATGTATGATTCTTCCGTCAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAACAACTGGCCTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
人类LC恒定κ区的核苷酸序列(SEQ ID NO:65)
ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
人类LC恒定λ区的核苷酸序列(SEQ ID NO:66)
GGTCAGCCCAAGGCTGCCCCCTCTGTCACTCTGTTCCCGCCCTCTAGCGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGTCCCTGCAGAATGCTCT
Claims (16)
1.一种分离的单克隆抗体mAb或其免疫反应性片段,其:
(a)以至少10nM的亲和力结合巨细胞病毒CMV株AD169的gB蛋白质;
(b)为人类抗体或片段,且
(c)中和成纤维细胞培养物中的CMV。
2.根据权利要求1所述的mAb,其呈完整抗体的形式。
3.根据权利要求1所述的mAb,其是双特异性抗体。
4.根据权利要求1所述的mAb或片段,其对CMV的所述AD169株的gB具有至少1nM的亲和力。
5.根据权利要求1所述的抗体,其中重链具有GFTFNRHG(SEQ ID NO:67)或GSISSEDFC(SEQ ID NO:68)的CDR1;和/或
其中所述重链具有SSDGANQ(SEQ ID NO:69)或ICYTGD(SEQ ID NO:70)的CDR2区;和/或
其中所述重链具有ARDGRCEGERCYSGVTDF(SEQ ID NO:71)或AREDRRQLHSRPYFYYGLDV(SEQ ID NO:72)的CDR3区。
6.根据权利要求1所述的抗体,其中轻链具有QNIGGY(SEQ ID NO:73)或QSLLHSNGHNY(SEQ ID NO:74)的CDR1区;和/或
其中所述轻链具有DAS(SEQ ID NO:75)或LG(SEQ ID NO:76)的CDR2区;和/或
其中所述轻链具有QQRNSWPPLT(SEQ ID NO:77)或QALQTPNT(SEQ ID NO:78)的CDR3区。
7.根据权利要求6所述的抗体或片段,其是4A2、19B10、313、338或345。
8.一种或一种以上核酸分子,其包含编码根据权利要求1到7中任一权利要求所述的mAb或片段的第一核苷酸序列;或一种或一种以上核酸分子,其包含在其整个长度上与所述第一核苷酸序列互补的第二核苷酸序列。
9.一种重组宿主细胞,其包含产生根据权利要求1到7中任一权利要求所述的mAb或片段的表达系统,其中所述重组宿主细胞是哺乳动物细胞、微生物细胞、昆虫细胞或植物细胞。
10.一种产生mAb或其免疫反应性片段的方法,所述方法包含培养根据权利要求9所述的细胞和回收所述mAb或片段。
11.一种医药组合物,其包含根据权利要求1到7中任一权利要求所述的分离的单克隆抗体或片段以及医药学上可接受的赋形剂。
12.根据权利要求11所述的医药组合物,其进一步含有额外医药剂以及医药学上可接受的赋形剂。
13.一种治疗感染CMV的人类个体的CMV的方法,其包含向需要所述治疗的个体投与有效量的根据权利要求1到7中任一权利要求所述的抗体或片段。
14.一种增强人类个体对CMV感染的抗性的方法,其包含向需要所述增强的个体投与有效量的根据权利要求1到7中任一权利要求所述的抗体或片段。
15.根据权利要求13或14所述的方法,其中所述个体免疫减弱。
16.根据权利要求13或14所述的方法,其中所述个体是孕妇。
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EA201270662A1 (ru) | 2009-12-23 | 2013-01-30 | 4-Антибоди Аг | Связывающие элементы для человеческого цитамегаловируса |
JP6055763B2 (ja) * | 2010-06-16 | 2016-12-27 | トレリス バイオサイエンス リミテッド ライアビリティー カンパニー | ヒトサイトメガロウイルス(CMV)gBタンパク質に対する高親和性ヒト抗体 |
US10611800B2 (en) | 2016-03-11 | 2020-04-07 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
US11440951B2 (en) | 2017-03-13 | 2022-09-13 | The Government Of The United States, As Represented By The Secretary Of The Army | Therapeutic antibodies to Marburg virus |
US11629172B2 (en) | 2018-12-21 | 2023-04-18 | Pfizer Inc. | Human cytomegalovirus gB polypeptide |
CN112898414B (zh) * | 2019-12-04 | 2024-05-10 | 珠海泰诺麦博制药股份有限公司 | 抗人巨细胞病毒抗体及其用途 |
US11857622B2 (en) | 2020-06-21 | 2024-01-02 | Pfizer Inc. | Human cytomegalovirus GB polypeptide |
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US20120020980A1 (en) | 2012-01-26 |
EP2582389A4 (en) | 2014-01-08 |
AU2011268277A2 (en) | 2013-07-25 |
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EP2582389B1 (en) | 2018-02-21 |
WO2011159938A3 (en) | 2012-04-19 |
EP2582389A2 (en) | 2013-04-24 |
JP2013531991A (ja) | 2013-08-15 |
KR20130086036A (ko) | 2013-07-30 |
BR112012032182A2 (pt) | 2016-10-25 |
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WO2011159938A2 (en) | 2011-12-22 |
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