CN106432485B - 全人源化抗狂犬病毒的中和性抗体 - Google Patents
全人源化抗狂犬病毒的中和性抗体 Download PDFInfo
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Abstract
本发明公开了一种全人源化抗狂犬病毒的中和型抗体,编码该抗体及其结合片段的DNA序列,以及包含所述DNA的宿主细胞和表达载体。本发明还公开了具有高亲和力的全人源抗狂犬病病毒抗体的制备方法,以及所述抗体在治疗和/或预防狂犬病的药物中的用途。
Description
技术领域
本发明本发明属于细胞免疫学、基因工程领域,涉及一种全人源化抗狂犬病毒的中和性抗体。
背景技术
狂犬病被动免疫制剂能够中和伤口局部的狂犬病毒,阻止病毒扩散并侵入神经系统,其半衰期长达14~21天,能够有效发挥治疗或紧急预防狂犬病的作用。
最早应用狂犬病被动免疫制剂治愈患者的例子可以追溯到1891年,但是在几十年后人类才生产出第一个商品化的人用抗狂犬病免疫血清。这种人用抗狂犬病免疫血清来源于马,由于为异源性血清,使用后不良反应可高达40%以上。目前市场上主要产品还是马源抗狂犬病血清球蛋白(ERIG),虽然为降低ERIG不良反应改进了生产工艺,但是其仍存在明显的副反应,且对某些疫苗的抗体反应有抑制作用。针对这一问题,美国免疫实践咨询委员会(ACIP)建议选用人源抗狂犬病血清球蛋白(HRIG)。由于HRIG来源于人而受到极大的限制,且存在潜在的病原污染风险,临床应用也受到了极大地限制。事实上在狂犬病毒流行地区尤其是狂犬病高发的发展中国家,无论是HRIG还是ERIG均远远无法满足市场需求,因此寻找可靠的RIG替代品是目前研究的首要目标。
应用单克隆抗体技术开发抗狂犬病病毒特异性的单克隆抗体(monoclonalantibodies,mAbs)防治狂犬病可以克服多抗血清的诸多弊端,应用前景广阔。1978年首次通过B淋巴细胞杂交瘤单克隆抗体技术获得了能够保护动物抵抗致死性狂犬病毒攻击的抗狂犬病毒mAbs,此后多种抗狂犬病毒mAbs相继问世。2007年,Muhamuda等获得了中和滴度及蛋白活性高于市场上的ERIG2000倍的鼠源性抗狂犬病毒mAbs,有望进一步研究以应用于人群暴露后预防。目前有部分抗狂犬病病毒特异性的mAbs进入临床研究阶段的报道,但至今仍没有药物被批准上市。
随着抗体技术的发展,制备抗体的方法已由多克隆抗体技术和单克隆抗体技术,发展为基因工程抗体技术。基因工程抗体以其对人体毒副作用小、人源化和高度特异性,越来越显示其优势。针对狂犬病毒糖蛋白的单克隆抗体Mab57是目前研究最为深入的人源杂交瘤单克隆抗体。但早期的基因工程抗体也存在不可忽视的缺点,如基因取自杂交瘤细胞,生产抗体的流程复杂,嵌合抗体和人源化抗体不能完全解决HAMA反应,难以对抗体进行改造获得高亲和力抗体等。Mab57因而一直未能完成临床前研究。随着人源抗体的技术的开发,如噬菌体展示、核糖体展示、酵母展示或是使用转基因技术产生的具有人免疫球蛋白基因的小鼠均能获得高亲和力人源抗体。
近些年,越来越多的研究者在基因工程抗体的研究中通过从人B细胞中筛选抗体的方法,获得全人源的单克隆抗体。由于这些抗体是在人体内自然产生的,在安全性、有效性和与人类疾病的相关性都体现了巨大的优势。噬菌体抗体库、B细胞永生化和单细胞克隆表达是3种目前常用的从B细胞中获得全人源单克隆抗体的方法。随着研究的继续和深入,各种新型抗狂犬被动免疫制剂的商品化生产将成为可能,人源或全人源单克隆抗体有望很快用于临床治疗。
发明内容
为了弥补现有技术的不足,本发明的目的之一,提供一种全人源的抗狂犬病毒的单克隆抗体。
本发明的目的之二,提供一种治疗狂犬病的药物组合物和手段。
本发明的目的之三,提供一种检测狂犬病毒的工具和手段。
本发明目的之四,提供一种诊断狂犬病的方法。
为了实现上述目的,本发明采用如下技术方案:
本发明提供了一种抗狂犬病毒的单克隆抗体TRN079,其特征在于,所述单克隆抗体包括轻链CDR1-3和重链CDR1-3氨基酸序列;
所述轻链CDR1的氨基酸序列如SEQ ID NO:2所示;
所述轻链CDR2的氨基酸序列如SEQ ID NO:3所示;
所述轻链CDR3的氨基酸序列如SEQ ID NO:4所示;
所述重链CDR1的氨基酸序列如SEQ ID NO:6所示;
所述重链CDR2的氨基酸序列如SEQ ID NO:7所示;
所述重链CDR3的氨基酸序列如SEQ ID NO:8所示。
进一步,所述抗体轻链的氨基酸序列如SEQ ID NO:1所示。
进一步,所述抗体重链的氨基酸序列如SEQ ID NO:5所示。
进一步,所述抗体分子选自具有全长重链和轻链的完整抗体分子或其片段。
本发明提供了一种上述单克隆抗体的制备方法,包括以下步骤:
(1)获得具有编码上述抗体的DNA分子;
(2)将步骤(1)所述的DNA分子与表达载体相连,构建重组表达载体;
(3)将步骤(2)的重组表达载体,共转染转入宿主细胞后,表达,纯化,即得。
本发明中抗体分子可仅包含重链或轻链多肽,在这种情况下,仅需将重链或轻链多肽编码序列用于转染宿主细胞。对于产生包含重链和轻链的产物,可使用两种载体(编码轻链多肽的第一载体以及编码重链多肽的第二载体)转染细胞系。或者,可使用单一载体,载体包括编码重链和轻链多肽的序列。
本发明提供了一种分离的DNA序列,所述DNA序列编码上述所述的抗体的重链和/或轻链。
本发明提供了一种克隆或表达载体,所述克隆或表达载体包含一种或多种DNA序列,所述DNA序列编码上述所述的抗体的重链和/或轻链。
本发明提供了上述单克隆抗体在制备用于检测狂犬病毒的工具中的应用。
进一步,所述工具包括试剂盒、试纸、芯片等。其中,所述芯片包括蛋白质芯片;所述蛋白质芯片包括固相载体以及固定在固相载体的上述单克隆抗体;所述蛋白免疫检测试剂盒;所述蛋白免疫检测试剂盒包括上述单克隆抗体。
本发明提供了一种检测狂犬病毒的工具,所述工具包含上面所述的单克隆抗体。
本发明提供了上面所述的单克隆抗体在制备治疗和/或预防狂犬病的药物组合物中的用途。
本发明提供了一种制备上述抗体的方法,所述方法包括如下步骤:
(1)分离狂犬病毒阳性患者血清的静脉血中的单个核细胞,之后利用流式细胞术进行细胞分选,分选出单个记忆性B细胞;
(2)利用单细胞RT-PCR扩增步骤(1)获得的单个B细胞中的抗体轻链和重链可变区的核苷酸片段;
(3)将步骤(2)获得的抗体轻链和重链可变区的核苷酸片段融合到含有人类抗体恒定区的表达载体中构建重组表达载体;
(4)将步骤(3)的重组表达载体共转染入宿主细胞后表达、纯化,获得具有结合活性和中和活性的本发明的全人源抗狂犬病毒的单克隆中和抗体。
本发明还提供了一种检测狂犬病毒的方法,所述方法包括以下步骤:
(1)获取样品;
(2)将步骤(1)获得的样品处理后与权利要求1-4任一项所述的抗体反应;
(3)检测样品与抗体的中和效应。
本发明中“单克隆抗体”指从一群基本上同质的抗体获得的抗体,即构成群体的各个抗体相同和/或结合相同表位,除了生产单克隆抗体的过程中可能产生的可能变体外,此类变体一般以极小量存在。此类单克隆抗体典型的包括包含结合靶物的多肽序列的抗体,其中靶物结合多肽序列是通过包括从众多多肽序列中选择单一靶物结合多肽序列在内的过程得到的。
克隆抗体在本发明中明确包括“嵌合”抗体,其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗体类别或亚类的抗体中的相应序列相同或同源,而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源,以及此类抗体的片段,只要它们展现出期望的生物学活性。
“完整抗体”在本发明中指包含两个抗原结合区及Fc区的抗体。优选的是,完整抗体具有功能性Fc区。
“抗体片段”包含完整抗体的一部分,优选包含其抗原结合区。抗体片段的例子包括Fab、Fab′、F(ab′)2和Fv片段;双抗体;线性抗体;单链抗体分子;及由抗体片段形成的多特异性抗体。
本发明还包括将上述抗体的氨基酸序列通过对氨基酸残基的添加、删除、修改形成所得到的包括人源与非人源抗体,并具有与TRN079抗体相同功能或改造及优化的一切抗体。
本发明的CDR可包括变体,例如当将本文中所公开的CDR回复突变为不同的框架区时。通常,个体变体CDR与本文所述序列的氨基酸同一性为至少70%或80%,更典型地具有优选至少75%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%和几乎100%的渐增的同一性。
通常,一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰,其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。
可利用取代、缺失、插入或其任意组合来实现最终的衍生物或变体。通常,这些变化在几个氨基酸上进行以使分子的改变最小化,特别是抗原结合蛋白的免疫原性和特异性。然而,在某些情况下可以容忍更大的变化。氨基酸取代通常是单个碱基的;插入通常将为大约一个至大约二十个氨基酸残基的数量级,虽然可能容忍显著更大的插入。缺失的范围为大约一个至大约二十个氨基酸残基,虽然在一些情况下,缺失可以大得多。
通常,一个蛋白质中一个或多个氨基酸的修饰不会影响蛋白质的功能。本领域技术人员会认可改变单个氨基酸或小百分比的氨基酸或对氨基酸序列的个别添加、缺失、插入、替换是保守修饰,其中蛋白质的改变产生具有相似功能的蛋白质。提供功能相似的氨基酸的保守替换表是本领域公知的。
在本发明中,“框架”指除去被定义为CDR的那些区域之外的抗体可变结构域的区域。每个抗体可变结构域框架可被进一步细分为由CDR分隔的连续区域(FRl、FR2、FR3和FR4)。
本发明的抗体包括在单克隆抗体TRN079或其CDR移植的抗体片段上利用单点突变或多点组合突变对抗体恒定区/CDR区部分氨基酸/进行改造及优化后的抗体片段或scFv抗体。
本发明提供了一种全新的全人源的抗狂犬病毒单克隆抗体,该抗体特异性强、中和效果好、无免疫原性。
附图说明
图1显示淋巴细胞分离单个的B细胞的分选结果;
图2显示利用SDS-PAGE鉴定表达纯化后的TRN079抗体;
图3显示利用ELISA检测TRN079抗体的结合活性。
具体的实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring HarborLaboratoryPress,1989)中所述的条件,或按照制造厂商所建议的条件。
实施例1淋巴细胞分离与单个记忆性B细胞分选
一、材料:
1.试剂:狂犬病疫苗(诺华,批号:1928)。Ficoll淋巴细胞分离液为Cedarlane公司产品。CD3-APC、CD14-APC、CD16-PE、CD20-APC、CD27-PE、IgD-FITC等均为Invirogen公司产品等。
二、方法结果
1.狂犬病疫苗的志愿者免疫
选择2例接种狂犬病疫苗的25岁健康志愿者,第7、21d时分别加强接种一次,28dIgG定量检测为阳性结果;抽取其外周血100mL,抗凝。
2.淋巴细胞分离与单个记忆性B细胞分选
将100mL血样用Ficoll分离,吸取单个核细胞(PBMC)层悬液,用PBS洗涤3次,吸弃上清。设置单染管:加AqVd-AmCyan、CD3-PE-Cy5、IgD-PE、CD14-FITC、CD20-APC、CD4-APC-H7,进行补偿调节,样本管中加入AqVd-AmCyan、CD3-PE-Cy5、CD16-PE-Cy5、CD235a-PE-Cy5、CD14-FITC、IgD-PE、CD20-APC、CD27-APC-H7,室温避光静置30min,用PBS洗涤2次后加入500μL PBS吹打混匀。样本置流式细胞仪上,依次选取P1(淋巴细胞门SSC-A/FSC-A)→P2(去粘连SSC-W/SSC-H)→P3(去粘连FSC-W/FSC-H)→P4(活细胞AqVd-AmCyan-)→P5(CD3-/CD14-/CD16-/CD235a-)→P6(IgD-/CD20+)→P7(IgD-/CD27ALL)。选择P7门的细胞,将96孔PCR板(每孔20uL单细胞裂解液)置于收集仓内,设置分选single cell模式,每个孔分选单个的B细胞于PCR板孔中,-80℃冰箱保存备用。
3.结果
结果如图1所示,分选出目标所需的单个的B细胞。
实施例2单B细胞RT-PCR分离抗体可变区基因
一、材料
1.试剂:引物(Invitrogen合成),Superscript III反转录酶、HotStarTaq Plus酶(Invitrogen,Carlsbad,CA)等。
二、方法结果
1.单B细胞RT-PCR(合成cDNA第一条链)
将含有单个B细胞的96孔板加入0.5uM的各亚型重链与轻链的恒定区引物与Superscript III反转录酶,37℃孵育1小时;按以下条件进行PCR扩增:95℃15min;95℃1min,55℃1min,72℃1min,30个循环;72℃10min;4℃5min。产物cDNA-20℃保存。
2.Nest-PCR(分离抗体基因)
每个单细胞分别扩增重链、轻链序列。50uL体系中含有5uL的RT反应产物,HotStarTaq Plus酶,dNTPs,及0.5uM的各亚型重链与轻链抗体的特异性引物,反应条件:预变性95℃5min,然后进行35个PCR循环:95℃×30s,55℃×60s,72℃×90s,最后用72℃延伸7min。PCR产物用1.2%的琼脂糖凝胶电泳进行鉴定。
实施例3PCR产物克隆鉴定和抗体表达
一、材料
1.试剂:琼脂糖、Tris,LB、氨苄青霉素,PolyFect(Qiagen,Valencia,CA),FCS、DMEM、PBS等。
2.载体:pcDNA3.3载体
3.菌株:大肠杆菌DH5α
二、方法结果
1.PCR产物纯化克隆鉴定
取2μL扩增产物经1.2%琼脂糖凝胶电泳检测,目的片段约400bp。将凝胶电泳鉴定为阳性,且重链与轻链可匹配成对的抗体可变区基因PCR产物利用TA克隆的方法连接到pcDNA3.3载体上(已经改造并含有抗体leader与恒定区),将连接产物转化DH5α感受态细菌中,在含有氨苄青霉素的平板上37℃培养过夜,随即挑取10个单菌落用特异性引物进行PCR,反应条件:94℃预变性3min,94℃变性30s,55℃退火30s,72℃延伸1min40s,28个循环,最后72℃再延伸5min。取5μLPCR产物在1%琼脂糖凝胶上进行电泳检测,在阳性转化子中鉴定出了含有抗体重链或轻链基因的转化子。
2.抗体表达
将表达阳性抗体重链和轻链基因的质粒在大肠杆菌DH5α中大量扩增,进行重组质粒快速提取。在T75细胞培养瓶中用转染试剂PolyFect共转染293T细胞,具体操作参见说明书。转染后6-8小时换含有2%FCS新鲜培养基,并在37℃5%CO2培养箱中培养72小时,收集细胞上清进行检测。
实施例4表达抗体的筛选检测
一、材料
1.疫苗:狂犬疫苗(诺华公司,瑞士)
2.试剂:IgG抗体,BSA,PBST,硫酸钠,丙酮等。
3.病毒:TCID50狂犬病毒(CVS株)。
二、方法
1.表达产物的筛选(ELISA)
以狂犬疫苗为抗原,并用包被液将抗原10倍稀释后包被96孔ELISA板,每孔100μL4℃过夜包被,PBST洗涤3次,每孔加150μL5%BSA常温封闭2个小时,再次用PBST洗涤。加入100μL的瞬时转染上清作为一抗常温孵育2个小时,用HRP标记的羊抗人IgG(1:2000稀释)作为二抗常温孵育1个小时,加入底物显色液100μL/孔,常温避光放置5min后,用2M硫酸钠中止反应,用450nm/630nm波长进行比色。OD450>0.1且实验孔OD450>2倍阴性对照孔OD450即判定阳性。
2.快速荧光灶抑制(RFFIT)检测
将ELISA筛选到的有结合活性的抗体,用WHO推荐的快速荧光灶抑制实验法(RFFIT)检测抗体与狂犬病毒的中和活性,阳性对照为商业化抗狂犬病毒血清标准品(240IU/mL)。将系列稀释的抗体及抗病毒血清标准品与100TCID50狂犬病毒(CVS株)于96孔培养板中混均,37℃孵育1小时,然后加入BSR细胞,放37℃培养24小时,于48小时进行染色观察。PBS洗两次,加入80%丙酮,4℃孵育15min后,PBS洗一次晾干后,每孔加入100μLFITC标记的抗体。37℃孵育1h,PBS洗2次,荧光显微镜蓝色激光激发观察。根据Reed&Muench法计算抗体保护效价(单位为IU/ml)。
三、结果
抗体TRN079(3.8mg/ml)的保护效价约为2400IU/ml,表明表达的重组抗体TRN079具有高纯度的狂犬病毒中和活性。
实施例5抗体大量表达与纯化
一、材料
1.试剂:PolyFect(Qiagen),FCS、DMEM、PBS,Protein-A亲和层析柱(Amersham),SDS-PAGE电泳试剂盒(Invitrogen),低分子量蛋白标准,溴酚蓝,考马斯亮蓝R250。
二、方法结果
1.抗体大量表达与纯化
将TRN079抗体重链与轻链的表达载体用PolyFect(Qiagen)转染试剂盒共转染生长于175cm2细胞培养瓶的293T细胞,转染后6-8小时换含有2%FCS新鲜培养基,并在37℃5%CO2培养箱中培养72小时。收集转染上清,4000rpm离心1小时,采用Amersham公司的Protein-A亲和层析柱直接纯化表达上清。
2.SDS-PAGE检验
利用SDS-PAGE检验抗体TRN079的表达及纯化情况。
三、结果
SDS-PAGE结果如图2所示,解链后的抗体轻、重链清晰可见。
实施例6TRN079抗体的活性检验
一、材料
1.试剂:IgG抗体,BSA,Biacore偶联试剂盒等。
2.动物:昆明小鼠(10-12g)
3.载体:载体pcDN A3.1
二、方法结果
1.ELISA测定结合活性
用前文提到的相同的ELISA方法对表达纯化的抗体的结合活性进行检测:以狂犬疫苗为抗原,并用包被液将抗原10倍稀释后包被ELISA 96孔板,每孔100uL 4℃过夜包被,并用封闭液常温封闭2个小时。将起始浓度为100,ug/mlTRN079抗体上清稀释10倍后作为一抗进行系列稀释,加一抗常温孵育2个小时,同时用未转染的细胞上清作为阴性抗体对照,用HRP标记的羊抗人IgG(1:2000稀释)作为二抗常温孵育1个小时,加入底物显色液100μl/孔,常温避光放置5min后,用2M硫酸钠中止反应,用450nm/630nm波长进行比色。
结果如图3所示,表明把表达纯化的抗体TRN079进行大于50,000倍稀释后(抗体浓度约为:0.0024μg/ml),TRN079抗体依然可以与抗原有结合。
2.小鼠体内中和试验
用WHO推荐的小鼠中和试验(MNT)检测纯化抗体与狂犬病毒的抗感染能力,阳性对照为商业化抗狂犬病毒血清标准品(240IU/mL)。将系列稀释抗体(TRN079)及抗病毒血清标准品与等体积中和毒(100LD50/mL)37℃中和1小时后,颅内注射10-12g昆明幼鼠,0.03mL/只,每个抗体稀释度攻击6只,共注射8个稀释度。逐日观察并于第4天后记录小鼠发病及死亡数至第14天。在攻击后4天内死亡的小鼠为非特异性死亡,应剔除不计。根据Reed&Muench法计算样品的效价。
结果表明抗体TRN079(3.8mg/ml)其保护效价约为2400IU/ml,说明表达的重组抗体TRN079是高纯度的具有狂犬病毒中和活性的全人源单克隆抗体。
3.TRN079抗体亲和力测定
先进行CM5芯片偶联捕获分子,再活化芯片的葡聚糖表面,以进样时间确定偶联量。最后利用CM5芯片捕获分子捕获配体:将制备的全人源抗狂犬病病毒中和抗体作为配体,以计算得到的信号值确定单抗的进样浓度及接触时间。单抗与狂犬病病毒G蛋白(抗原)结合的亲和力和动力学分析:狂犬病病毒G蛋白用HBS-EP缓冲液稀释作为分析物,分析物以逐渐增高的浓度依次流过芯片,分别得到信号曲线。每个浓度作为1个循环,完成1次循环后用10mmol/L的甘氨酸-盐酸再生芯片以回复到原始未结合抗原的状态。用BiaCore X-100System软件进行分析。
结果显示,TRN079抗体的亲和力可达10-8mol数量级。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出,对于本领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以对本发明进行若干改进和修饰,这些改进和修饰也将落入本发明权利要求的保护范围内。
SEQUENCE LISTING
<110> 广州泰诺迪生物科技有限公司 暨南大学
珠海泰诺麦博生物技术有限公司
<120> 全人源化抗狂犬病毒的中和性抗体
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Claims (5)
1.一种全人源抗狂犬病毒的单克隆抗体TRN079,其特征在于,所述单克隆抗体TRN079轻链的氨基酸序列如SEQ ID NO:1所示,重链的氨基酸序列如SEQ ID NO:5所示。
2.一种分离的DNA序列,其特征在于,所述序列编码权利要求1所述的抗体的重链和轻链。
3.一种克隆或表达载体,其特征在于,所述克隆或表达载体包含一种或多种DNA序列,所述DNA序列编码权利要求1所述的抗体的重链和轻链。
4.权利要求1所述的全人源抗狂犬病毒的单克隆抗体TRN079在制备治疗和/或预防狂犬病的药物组合物中的用途。
5.权利要求1所述的全人源抗狂犬病毒的单克隆抗体TRN079在制备狂犬病毒检测工具中的应用。
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Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis.;Paola De Benedictis et al.;《EMBO Mol Med.》;20160401;全文 |
全人源抗狂犬病病毒单克隆抗体的制备与鉴定;张夏玲;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20150315;全文 |
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