CN113735970A - 一种抗新冠病毒全人源广谱中和抗体及应用 - Google Patents
一种抗新冠病毒全人源广谱中和抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种抗SARS‑CoV‑2的全人源单克隆抗体ZWC6，所述抗体通过流式分选‑单细胞PCR技术筛选获得，具有独特的CDR分区，其抗原识别表位位于S1蛋白的RBD区。所述抗体对新冠病毒野生型的EC50是0.266μg/mL，中和Beta株的EC50是0.115μg/mL，中和Delta株的EC50是0.115μg/mL，显示本发明公开的单克隆抗体对目前的主要变异株具有广谱高效中和活性。本发明公开的单克隆抗体还具有高表达、全人源、稳定性好的特点，适合产业化生产，对于应对目前的变异株及将来可能出现的变异株导致的爆发流行具有重大临床应用价值。
Description
技术领域
本发明公开了一种抗体，属于微生物学和免疫学领域。
背景技术
新冠肺炎的病原体是新型冠状病毒-2(SARS-CoV-2)，SARS-CoV-2属于冠状病毒科的β-冠状病毒属，是一类有囊膜的单股正链RNA病毒，其基因组长度约为30kb。基因组的前2/3是非结构基因ORF1a/b，主要编码与病毒复制相关的酶(RNA依赖的RNA聚合酶，RdRp)，后1/3依次编码四种结构蛋白：刺突蛋白(S)，包膜蛋白(E)，膜蛋白(M)和核衣壳蛋白(N)。其中，S蛋白含有病毒受体结合区，可与人细胞表面的血管紧张素转换酶2(ACE2)受体结合，介导病毒吸附和进入细胞，是病毒入侵宿主易感细胞的关键蛋白。
病毒在传播过程中会不断地随机产生突变，其中有些突变会增强S蛋白与ACE2受体的结合能力，加快病毒在人群中的传播，如N501Y，E484K，P681R等。因而一些携带关键位点突变的病毒变异株表现出更强的感染能力或更强的免疫逃逸能力，使现有的公共卫生干预措施或疫苗的有效性降低，如被世界卫生组织(WHO)列为“关注变异株”(variants ofconcern,VOC)的Alpha株(B.1.1.7)、Beta株(B.1.351)、Gamma株(P.1)和Delta株(B.1.617.2)等，这些变异株的出现对疫情防控提出了新的严峻挑战。
靶向病毒表面刺突蛋白(S蛋白)的单克隆中和抗体成为潜在的有效治疗新冠肺炎的手段，它通过与新冠病毒结合，抑制病毒的活性，保护细胞免受侵害。相比小分子药物和血浆疗法，单抗药物机理清晰，对靶点的选择性高、特异性强、副作用小。根据chineseantibody网站信息，已有25项靶向S蛋白的单抗进入临床研究，这些单抗对于应对野生型新型冠状病毒都是有效的。目前已有三种靶向S蛋白的单抗获得了FDA的紧急使用授权，包括再生元的单抗REGN10933和REGN10987联用，礼来的LY-COV555和LY-COV016，Vir公司的VIR-7831。然而，随着SARS-CoV-2的不断进化和变异，研发中的大部分单抗对一种或多种变异株失去中和活性，其中，礼来研发的LY-CoV555和LY-CoV016、再生元研发的REGN10933对Beta株病毒失去中和活性。为应对病毒免疫逃逸，研发具有保守中和表位的广谱单抗，包括研发广谱的中和单抗或两种高效中和单抗的组合物，具有重大临床应用价值。
当前，中和单抗可以通过杂交瘤技术，人源化转基因小鼠，噬菌体文库筛选以及单细胞PCR技术制备。单细胞PCR技术具有全人源，天然稳定性好等优点，被广泛用于新冠中和抗体的研发。单细胞PCR技术的原理是新型冠状病毒感染恢复者或新冠疫苗接种者体内存在对抗病毒的保护性单克隆抗体，编码抗体的基因位于人体外周血单个淋巴细胞内，通过流式细胞仪分选和单细胞PCR技术可以“钓取”此基因。然后通过基因工程手段，可实现体外规模化制备此分子。
本发明拟采用流式分选-单细胞PCR技术从重组新型冠状病毒疫苗接种者的外周血中获得具有优异广谱中和活性的单抗，目的是提供针对COVID-19具有良好保护效果的全人源单克隆治疗性抗体，以应对当前流行的变异株以及将来可能出现的变异株。
发明内容
基于上述发明目的，本发明通过流式分选-单细胞PCR技术筛选到了一种抗SARS-CoV-2的单克隆抗体，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-111位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-32、50-52、89-97位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“ZWC6”。
在一个优选的实施方案中，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:5所示。
在一个更为优选的实施方案中，所述单克隆抗体的重链恒定区的氨基酸序列如SEQ ID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7所示。
第二，本发明还提供了一种编码上述单克隆抗体重链和轻链的多核苷酸，编码所述单克隆抗体的重链可变区的多核苷酸序列由SEQ ID NO:2所示，编码所单克隆述抗体的轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
在一个优选的实施方案中，编码所述单克隆抗体的重链恒定区的多核苷酸序列由SEQ ID NO:4所示，编码所述单克隆抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8所示。
第三，本发明还提供了一种表达上述编码单克隆抗体重链和轻链的多核苷酸的功能元件，这种功能元件可以是传统的表达载体。
在一个优选的实施方案中，所述功能元件为线性表达框。
在另一个优选的实施方案中，所述功能元件为哺乳动物表达载体。
第四，本发明还提供了一种含有上述线性表达框的宿主细胞。
在一个优选的实施方案中，所述细胞为Expi 293F细胞。
在另一个优选的实施方案中，所述细胞为CHO-K1或CHO-S细胞，本发明可以使用CHO-K1或CHO-S细胞构建稳转工程细胞株，实现产业化生产。
最后，本发明还提供了上述单克隆抗体在制备COVID-19治疗药物中的应用。
本发明提供的单克隆抗体通过流式分选-单细胞PCR技术筛选获得，具有独特的CDR分区，其抗原识别表位位于S1蛋白的RBD区。所述抗体与SARS-CoV-2野生型S-ECD的亲和力为1.492nM,与Alpha株、Beta株、Gamma株、Delta株S-ECD的亲和力分别为0.938nM、0.658nM、1.204nM、4.211nM。在假病毒中和实验中，对新冠病毒野生型的EC50是6.4ng/mL，中和Alpha株的EC50是3.803ng/mL，中和Beta株的EC50是3.649ng/mL，中和Gamma株的EC50是3.529ng/mL，中和Delta株的EC50是4.446ng/mL。在真病毒中和实验中，对新冠病毒野生型的EC50是0.266μg/mL，中和Beta株的EC50是0.115μg/mL，中和Delta株的EC50是0.115μg/mL。显示ZWC6对目前的主要变异株具有广谱高效中和活性。本发明公开的单克隆抗体具有高表达、全人源、稳定性好的特点，适合产业化生产，对于应对目前的变异株及将来可能出现的变异株导致的爆发流行具有重大临床应用价值。
附图说明
图1.流式细胞仪单细胞分选图；
图2.H、κ、λ三种链基因的巢式PCR扩增后毛细管电泳的鉴定图谱；
图3.单克隆抗体重链和轻链线性表达框的核酸电泳鉴定图谱；
图4.单克隆抗体可变区序列的检索结果输出图；
图5.抗体表达上清与SARS-CoV-2的结合活性图；
图6.亲和层析纯化后的单抗SDS-PAGE检测图谱；
图7.ELISA检测单抗ZWC6与S蛋白、S1蛋白、RBD蛋白、NTD蛋白、S2蛋白的结合活性随浓度变化的曲线图；
图8.ELISA检测ZWC6的交叉结合活性；
图9.ZWC6对新冠假病毒的广谱中和活性；
图10.ZWC6在细胞模型上对真病毒的EC50测定曲线图；
图11.ZWC6与WT S-ECD蛋白的结合动力学曲线图；
图12.ZWC6与Alpha株S-ECD蛋白的结合动力学曲线图；
图13.ZWC6与Beta株S-ECD蛋白及的结合动力学曲线图；
图14.ZWC6与Gamma株S-ECD蛋白的结合动力学曲线图；
图15.ZWC6与Delta株S-ECD蛋白的结合动力学曲线图；
图16.ZWC6与WT RBD蛋白的结合动力学曲线图。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1人源抗SARS-CoV-2单克隆抗体的筛选和制备
1.血液样品的采集
在获得知情同意书后，采集重组新型冠状病毒疫苗免疫接种者第二次免疫14天后血液样品20mL，用于后续实验。
2.流式分选记忆B细胞
将采集的血样利用Ficoll密度梯度离心法分离PBMC，过程如下：
1)取新鲜抗凝全血，EDTA抗凝。
2)在离心管中加入与血液样本等体积的分离液，将血样平铺到分离液液面上方，保持两液面界面清晰。
3)配平，室温，水平转子800g，加减速度3，离心30min。
4)离心结束后，管底是红细胞，中间层是分离液，最上层是血浆/组织匀浆层，血浆层与分离液层之间是一层薄且较致密的白膜，即：单个核细胞(包括淋巴细胞和单核细胞)层。
5)将白膜层小心吸取到新的50mL离心管中，用PBS稀释3倍，颠倒混匀。室温，水平转子600g，离心10min，弃上清。重复洗涤2次。
6)用PBS将淋巴细胞重悬备用。
7)将用来分选的细胞计数，按照下表推荐用量，先加入除Anti-His tag和Anti-FLAG tag抗体以外的所有抗体以及抗原，4℃孵育1h，随后PBS+2％FBS清洗两次，再加入Anti-His tag和Anti-FLAG tag抗体，用PBS+2％FBS补足反应体系，4℃孵育1h。
表1.流式分选荧光抗体/抗原
8)使用含2％FBS的PBS重复洗涤2-3次，1mL FPBS重悬，用40μm细胞筛去除细胞团，4℃避光保存供分选。
9)使用细胞分选仪(Beckman MofloXDP)分选SARS-CoV-2 S-ECD特异的单个记忆B细胞。分选策略为：CD3-/CD19+/IgG+/CD27+/SARS-CoV-2 S-ECD+，如图1，图1-A中圈出淋巴细胞，图1-B中圈出去除粘连的细胞，图1-C中圈出CD3-/CD19+的B细胞，图1-D中圈出IgG+/CD27+的记忆B细胞，图1-E中圈出SARS-CoV-2 S-ECD+的记忆B细胞。直接将单个记忆B细胞分选至96孔板中，96孔板中每孔预先加入20μL去RNA酶水和20U RNA酶抑制剂，-80℃保存。
结果：分选获得456个SARS-CoV-2 S-ECD+的记忆B细胞。
3.利用单细胞-PCR技术扩增全人源单抗可变区基因
1)反转录PCR
参考说明书(QIAGEN，210212)，程序简单介绍如下：
通过流式细胞仪分选了456个单细胞。向每个反应体系中同时加入以下全部的针对重链(heavy chain，H)、Kappa轻链(kappa chain，κ)、Lamda轻链(Lamda chain，λ)各亚型的特异引物(引物序列见表2)。引物：
H：5′L-VH 1、5′L-VH 3、5′L-VH 4/6，5′L-VH 5、HuIgG-const-anti、3′Cm CH1
κ：5′L Vκ1/2、5′L Vκ3、5′L Vκ4、3′Cκ543–566
λ：5′L Vλ1、5′L Vλ2、5′L Vλ3、5′L Vλ4/5、5′L Vλ6、5′L Vλ7、5′L Vλ8、3′Cλ
表2反转录PCR引物
PCR反应体系中包含：5×缓冲液6μL、dNTP 1.2μL、反转录酶(Qiagen，210212)1.2μL、引物如上、模板为单细胞，水补齐至30μL。
PCR反应条件为：50℃反转录30min，95℃预变性15min，接着95℃40s，55℃30s，72℃1min，40个循环，最后72℃延伸10min。
2)巢式PCR
取反转录产物1μL为模板，进行巢式PCR反应扩增H、κ、λ的可变区，扩增重链可变区、κ轻链可变区和λ轻链可变区的引物如下表3所示。
表3.巢式PCR引物
PCR反应体系中包含：10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶(全式金生物技术有限公司，AP141)0.5μL、引物如上、模板为反转录产物1μL、水补齐至50μL。PCR反应条件为：95℃预变性10min，接着，95℃30s，57℃30s，72℃45s，40个循环，最后72℃延伸10min。
3)毛细管电泳
对巢式PCR扩增产物用QIAGEN DNA Fast Analysis Cartridge(Qiagen，929008)进行毛细管电泳，一个单细胞中重链和轻链基因均扩增成功的克隆，被认为是配对成功的克隆。图2是对H、κ、λ三种链基因的巢式PCR扩增后毛细管电泳的鉴定图谱。
4)序列分析
经PCR鉴定阳性的克隆进行DNA序列测定和分析，登录IMGT网站(http://www.imgt.org/IMGT_vquest/analysis)进行可变区检索，为典型的抗体序列，符合预期。检索结果如图4所示，图4-A显示重链可变区的检索结果，V区同源性最高为90.97％，J区同源性最高为81.25％，D区使用读框2。图4-B显示轻链的检索结果，V区同源性最高为94.62％，J区同源性最高为91.43％。
4.线性表达框表达抗体
相比传统的表达载体构建方法，构建线性表达框更为快速。设计的线性表达框含有单抗在哺乳细胞内表达的所有元件，线性表达框从5’端依次含有CMV启动子序列(Genbank登记号：X03922.1)、抗体前导肽的编码序列、抗体可变区(从单细胞中扩增获得)、抗体恒定区(生工生物合成，重链恒定区序列由SEQ ID NO:3所示，DNA编码序列由SEQ IDNO:4所示，Kappa型轻链恒定区序列由SEQ ID NO:7所示，DNA编码序列由SEQ ID NO:8所示，Lamda型轻链恒定区序列由SEQ ID NO:9所示，DNA编码序列由SEQ ID NO:10所示，多聚A尾(Genbank登记号：X03896.1)连接起来，将该线性形式的DNA转染入细胞中进行抗体表达。
具体过程是通过体外重叠延伸PCR技术将各个PCR片段连接构建：
1)扩增启动子-前导序列
以pMD-CMVH和pMD-CMVL为模板，分别扩增重链和轻链的启动子-前导序列片段。扩增重链启动子-前导序列片段的PCR反应体系中包括：模板质粒pMD-CMVH 10ng，10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶0.5μL、引物5'-CMV-UP(与CMV启动子上游序列匹配)(5'-GATATACGCGTTGACATTGATTATTGAC-3')、引物3'-leader-H(HR)(5'-ACACTGAACACCTTTTAAAATTAG-3',用于重链的融合,信号肽序列的核苷酸序列：
5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTAAAA GGTGT C-3')，编码的氨基酸序列为MNFGLSLIFLVLILKGV。扩增轻链启动子-前导序列片段的PCR反应体系中包括：模板质粒pMD-CMVL 10ng，10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶0.5μL、引物5'-CMV-UP(5'-GATATACGCGTTGACATTGATTATTGAC-3')、引物3'-leader-L(HR)(5'-CCCACAGGTACCAGATACCCATAG-3')，用于轻链的融合，全长信号肽序列核苷酸序列为：
5-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATC TGGTACCTGTGGG，氨基酸序列为MDSQAQVLMLLLLWVSGTCG，信号肽序列来源鼠源单抗可变区)、水补齐至50μL。
PCR反应条件：95℃预变性10min，接着95℃30s，60℃30s，72℃1min，30个循环，最后72℃延伸10min。
2)扩增抗体恒定区-多聚A尾片段
H链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKH 10ng、10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶0.5μL、引物5'-CH(5'-ACCAAGGGCCCATCGGTCTTCCCC-3')、引物3'-TK-POLY(A)(5'-AAGTGTAGCGGTCACGCTGCGCGTAACC-3')、水补齐至50μL。
κ链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKκ10ng、10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶0.5μL、引物5'-Cκ(5'-ACTGTGGCTGCACCATCTGTCTTC-3')、引物3'-TK-POLY(A)(5'-AAGTGTAGCGGTCACGCTGCGCGTAACC-3')、水补齐至50μL。
λ链恒定区-多聚A尾片段PCR体系中包含：模板质粒pMD-TKλ10ng、10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶0.5μL、引物5'-Cλ(CTACGTCAGCCCAAGGCTGCCCCC)、引物3'-TK-POLY(A)(5'-AAGTGTAGCGGTCACGCTGCGCGTAACC-3')、水补齐至50μL。
PCR反应条件为：95℃预变性10min，接着95℃30s，60℃30s，72℃2min，30个循环，最后72℃延伸10min。
3)扩增抗体可变区
取巢式PCR产物1μL为模板，使用TransStart Taq DNA polymerase并按照产品说明书，分别使用对应的混合引物对抗体的H链、κ链、λ链进行扩增，对应引物如下表4所示。
表4.PCR引物
※单独划线部分用于与上游片段融合，划线黑体部分用于与下游片段的融合。
PCR反应体系中包含：10×缓冲液5μL、2.5mM dNTP 4μL、DNA聚合酶(全式金生物技术有限公司，AP141)0.5μL、引物如上、模板为巢式PCR产物1μL、水补齐至50μL。
PCR反应条件为：95℃预变性4min，接着，95℃30s，57℃30s，72℃45s，40个循环，最后72℃延伸10min。
4)分别扩增重链和轻链的线性表达框
PCR反应体系中包括：
模板：纯化后的启动子-前导序列片段10ng、重链/轻链可变区片段10ng、重链/轻链恒定区-多聚A尾片段10ng，10×缓冲液5μL、12.5mM dNTP 4μL、DNA聚合酶(全式金生物技术有限公司，AP151-13)0.5μL、引物5'-CMV-UP(5'-GATATACGCGTTGACATTGATTATTGAC-3')和3'-TK-POLY(A)(5'-AAGTGTAGCGGTCACGCTGCGCGTAACC-3'、水补齐至50μL。
PCR反应条件为：95℃预变性10min，接着95℃30s，60℃30s，72℃3min，30个循环，最后72℃延伸10min。
5)PCR产物回收纯化和定量
PCR反应产物直接用OMEGA公司回收试剂盒回收。DNA定量：用Nano(GEHealthcare)对PCR回收产物进行定量。
6)细胞接种：将293T细胞以2×105/mL接种于96孔细胞培养板中，在含有5％CO2的细胞温箱中，37℃培养过夜。
7)细胞共转染：次日，96孔板每孔加入20μL无血清的Opti-MEM培养基中，构建成功的重链和轻链线性表达框PCR产物各0.1μg，混匀后加入0.4μL转染试剂Turbofect(ThermoScientific,R0531)，共同孵育15-20min后逐滴加至过夜培养的293T细胞培养孔中。在含有5％CO2的细胞温箱中，37℃培养48h后收细胞培养上清备用。
线性表达框的PCR融合扩增核酸电泳检测结果见图3，图3中泳道1，2分别是重链、轻链的启动子-前导序列，扩增片段750bp左右，符合预期；泳道3，4，5分别是H链、κ链、λ链的恒定区-多聚A尾片段，扩增片段分别为2000bp、1350bp、1350bp左右，符合预期；泳道6，7分别是构建成功的ZWD12的重链、轻链表达框，扩增片段分别为3100bp、2450bp左右，符合预期；泳道8，9分别是构建成功的ZWC6的重链、轻链表达框，扩增片段分别为3100bp、2450bp左右，符合预期；泳道M是Trans5K DNA Marker，分子量从大到小分别为5000、3000、2000、1500、1000、750、500、250、100bp。
5.ELISA筛选具有结合活性的抗体
1)包被：实验前一天96孔酶联板，取重组的SARS-CoV-2 S-ECD抗原及羊抗人IgG(H&L)抗体(Abcam，ab97221)用包被液稀释至浓度2μg/mL，包被酶标板，每孔100μL，4℃包被过夜。
2)封闭：实验当天用洗板机(BIO-TEK，405_LS)洗3次，每孔加入100μL封闭液，37℃孵育1小时。
3)样品孵育：洗板3次，加入50μL的转染细胞培养上清和50μL稀释液，37℃孵育1小时。
4)二抗孵育：洗板3次，将HPR标记的羊抗人IgG二抗(Abcam，ab97225)以1:10000用稀释液进行稀释，每孔100μL加入到ELISA板对应孔中，37℃孵育1小时。
5)显色：洗板3次，每孔加入100μL的TMB单组份显色液，显色6min，室温避光，之后每孔加入50μL终止液终止反应。
6)用酶标仪上检测450-630nm处的OD值，以未加待测样品的孔为阴性对照，OD450-630＞阴性对照2.1倍以上的孔为阳性。。
结果：将42株单抗进行表达，并对SARS-CoV-2 S-ECD的结合活性进行鉴定，有21株单抗与SARS-CoV-2 S-ECD能够特异性结合(参见图5)。
6.表达载体的构建和酶切鉴定
对ZWC6构建轻、重链重组表达质粒，进行单抗的表达制备。
1)pCDNA3.4-ZWC6-H表达质粒构建：
以线性表达框为模板，扩增重链，切胶回收1.4kb大小的重链片段，表达载体pCDNA3.4(ThermoFisher Scientific，A14697)使用EcoR I/BamH I酶切后回收，将重链和载体片段通过同源重组(NEBuilder HiFi DNA Assembly Master Mix,E2621L)方法进行连接，转化TOP10挑取克隆进行测序鉴定，构建成功重链的表达载体pCDNA3.4-ZWC6-H。
2)pCDNA3.4-ZWC6-κ表达质粒构建：
以轻链表达框为模板，扩增轻链，胶回收约0.7kb的轻链片段，将轻链和载体片段通过同源重组方法连接，转化TOP10挑取克隆进行测序鉴定，构建成功轻链的表达载体pCDNA3.4-ZWC6-κ。
3)单抗的瞬时表达和亲和层析纯化
使用Expi293表达系统，取15μg重链和15μg轻链混合后转染Expi 293F细胞，按照说明书进行操作(ThermoFisher Scientific，A14635)，5-6天后收获培养液，离心后上清约30mL，使用体积为5mL的预装Protein A亲和层析柱，上样前使用20mM PBS平衡，待电导显示到基线后进样，上样结束后，使用20mM PBS洗涤色谱柱至基线平稳，使用0.1M pH 3.0的甘氨酸缓冲液洗脱目的蛋白，待OD280近基线后，停止收集，使用至少3个柱体积的20mM的PBS洗涤色谱柱，至基线平稳后，用20％的乙醇洗涤色谱柱。亲和层析纯化后的单抗SDS-PAGE检测结果见图6：泳道1为单抗ZWC6的还原电泳结果，泳道2为单抗ZWD12的还原电泳结果，重链和轻链的理论分子量分别为50kDa和25kDa，符合预期，泳道M为分子量标记(分子量从大到小依次为：250，130，100，70，55，55，35，25，15，10kDa)。
实施例2.抗体ZWC6识别表位分析
1)包被：实验前一天96孔酶联板，取重组的SARS-CoV-2 S-ECD抗原、S1抗原、RBD抗原和S2抗原用包被液稀释至浓度2μg/mL，包被酶标板，每孔100μL，4℃包被过夜。
2)封闭：实验当天用洗板机(BIO-TEK，405_LS)洗3次，每孔加入100μL封闭液，37℃孵育1小时。
3)样品孵育：洗板3次，除首孔外，每孔加入100μL稀释液，将抗体稀释至首孔1μg/mL，4倍梯度稀释，100μL/孔，每个抗体设置三个复孔，在37℃孵育1h。
4)二抗孵育：洗板3次，将HPR标记的羊抗人IgG二抗(Abcam，ab97225)以1:10000用稀释液进行稀释，每孔100μL加入到ELISA板对应孔中，37℃孵育1小时。
5)显色：洗板3次，每孔加入100μL的TMB单组份显色液，显色6min，室温避光，之后每孔加入50μL终止液终止反应。
6)用酶标仪上检测450-630nm处的OD值，以未加待测样品的孔为阴性对照，OD450-630＞阴性对照2.1倍以上的孔为阳性。
结果：检测ZWC6与不同抗原表位的结合活性，具体见图7，ZWC6与SARS-CoV-2WT的S-ECD、S1和RBD蛋白均特异结合，呈现剂量反应关系,而与NTD蛋白和S2蛋白均不结合。结果表明，单抗ZWC6识别的表位位于S1蛋白的RBD区。
单抗ZWC6的序列分析结果如下：
重链可变区的氨基酸序列如SEQ ID NO:1所示，重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-111位氨基酸序列所示，编码重链可变区的多核苷酸序列由SEQ ID NO:2所示；轻链可变区的氨基酸序列如SEQ ID NO:5所示，轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-32、50-52、89-97位氨基酸序列所示，编码轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
实施例3：抗体ZWC6的交叉结合活性鉴定
对ZWC6与SARS-CoV-2的受关注变异株(Variants of concern)S蛋白的交叉结合活性进行鉴定，方法同上，结果如图8所示。ZWC6与Alpha株、Beta株、Gamma株、Delta株的S-ECD蛋白均特异结合，且呈现剂量反应关系。结果表明，单抗ZWC6能够交叉结合Alpha株、Beta株、Gamma株、Delta株的S-ECD蛋白。
实施例4.抗体ZWC6的假病毒中和活性鉴定
1)将纯化单抗用培养基DMEM+10％FBS自初始浓度(ZWC6单抗初始浓度3.7μg/ml),3倍系列稀释，加入96孔培养板，设置3个复孔，体积50μL/孔；随即每孔加入50μL新冠病毒野生型或突变株的假病毒悬液(用DMEM+10％FBS稀释病毒至合适滴度)，充分混匀，另设置存活对照(不加病毒和抗体)和死亡对照(只加病毒)，置37℃5％CO2细胞培养箱孵育1h。
2)将HEK293T细胞用0.25％的胰酶消化后，用培养基(DMEM+10％FBS)稀释至2.5×105cells/mL浓度，接种到96孔细胞培养板中，接种体积100μL/孔，置37℃5％CO2细胞培养箱培养过夜。
3)48h后弃100μL细胞培养上清，加入100μL显色底物，避光孵育2min。
4)吸取150μL转移到96孔白色微孔板，利用Tecan Spark多功能微孔板检测仪读取Luciferase信号值；用(Luc样本孔-Luc死亡对照)/(Luc存活对照-Luc死亡对照)计算细胞活率，用GraphPad Prism 8拟合曲线，计算抗体EC50值。
结果见图9，本发明公开的单抗ZWC6对新冠病毒野生型假病毒的EC50是6.4ng/mL，中和Alpha株假病毒的EC50是3.803ng/mL，中和Beta株假病毒的EC50是3.649ng/mL，中和Gamma株假病毒的EC50是3.529ng/mL，中和Delta株假病毒的EC50是4.446ng/mL。结果显示ZWC6对目前的主要变异株的假病毒具有广谱高效中和活性。
实施例5.抗体ZWC6的真病毒中和活性鉴定
1)将Vero E6细胞用0.25％的胰酶消化后，用培养基(DMEM+10％FBS)稀释至3×105cells/mL浓度，接种到96孔细胞培养板中，接种体积100μL/孔，置37℃5％CO2细胞培养箱培养过夜。
2)实验当天，将纯化单抗用培养基DMEM+2％FBS自初始浓度(ZWC6单抗初始浓度100μg/ml,3倍系列稀释，加入96孔培养板，体积120μL/孔；随即每孔加入120μL COVID-19病毒悬液(用DMEM+2％FBS稀释病毒，加入100TCID50/孔)，充分混匀，置细胞培养箱孵育1h。
3)弃去96孔板中细胞培养上清，每孔加入200μL共孵育后的病毒-抗体混合悬液；另设置存活对照(不加病毒和抗体)和死亡对照(只加病毒)，置37℃5％CO2细胞培养箱培养72h。
4)72h后弃去细胞培养上清，加入50μL结晶紫染色液室温染色30min，弃去染液，加入200μL/孔纯水，重复洗涤6次。
5)弃尽洗液，用吸水纸拍干板孔中水分，加入100μL脱色液充分溶解，以OD620为参考，用酶标仪测OD570值；用(OD样本孔-OD死亡对照)/(OD存活对照-OD死亡对照)计算细胞活率，用GraphPad Prism 8拟合曲线，计算抗体EC50值。
结果见图10，本发明公开的单抗ZWC6的对新冠病毒野生型的EC50是0.266μg/mL，中和Beta株的EC50是0.115μg/mL，中和Delta株的EC50是0.115μg/mL。说明ZWC6对SARS-CoV-2的野生型、Beta和Delta变异株的真病毒均具有高效中和活性。
实施例6.表面等离子共振法(SPR)测定单抗与S抗原的亲和力
1)配置缓冲液：量取50mL 10×HBS-EP+缓冲液、450mL去离子水，混匀后放入500mL缓冲液瓶。
2)蛋白换液：使用脱盐柱将抗体和抗原蛋白换液至HBS-EP+缓冲液中，将脱盐柱置于空的收集管中，拧松脱盐柱盖子，1500g离心1min去除柱中原有液体，加入300μL HBS-EP+缓冲液，1500g离心1min，重复4次，将脱盐柱置于新的收集管，将100μL蛋白溶液加入柱中，1500g离心2min，收集滤下液体，使用NanoVue测定蛋白浓度。
3)打开Biocore T200机器，将进液管A插入HBS-EP+缓冲液瓶中，放入ProteinA芯片，运行Prime程序。
4)样品准备：用HBS-EP+缓冲液将配体(抗体)稀释至0.5μg/mL，将分析物(抗原)稀释至100nM、50nM、25nM、12.5nM、6.25nM、3.125nM、1.5625nM、0.78125nM。
5)使用多循环检测方法进行样品检测：打开Run/Wizard中的Kinetics/Affinity，Flow path选择2-1或4-3，Chip type选择Protein A，选择Ligand capture，点击下一步；在Setup界面中，在Startup下的Solution中填写HBS-EP+，Number of cycles选择3，点击下一步；在Injection Parameters界面：Ligand名称填写抗体名称，Contact time为60s，Flowrate为10μL/min，Stabilization period为0s；Sample的Contact time为120s，Flow rate为30μL/min，Dissociation time为900s；Regeneration中Solution为Glycine pH 1.5，Contact time为30s，Flow rate为30μL/min，Stabilization period为30s；点击下一步；在Sample界面中填写分析物的信息：S-ECD分子量为134kDa，RBD分子量为26.5kDa，浓度为0nM、0.78125nM、1.5625nM、3.125nM、6.25nM、12.5nM、25nM、50nM、100nM、0.78125nM，选择0.78125nM作为重复，点击下一步；在System Preparations界面中设置分析温度和样品舱温度为25℃，点击下一步；在Rack Position界面中，选择Sample and Reagent Rack1，设置样品位置，按照样品位置和用量进行样品准备和放置，点击下一步；确认运行缓冲液体积达到实验所需，点击Start，对实验方法和结果进行保存，程序开始自动运行，运行时间5小时。
6)结果分析：打开数据分析软件Biacore T200 Evaluation Software，打开运行的结果文件：点击Kinetics/Affinity，选择Surface bound；在Select Curves界面中选择0nM和至少5个合适浓度，点击下一步；在Select Data界面中点击Kinetics；在FitKinetics界面中，Method选择1:1Binding，点击fit进行数据拟合；记录结合动力学数据ka、kd、KD等。表5依据Report显示的分析数据，记录单抗ZWC6与不同抗原的结合动力学数据ka、kd、KD。图11-16分别是ZWC6与WT、Alpha、Beta、Gamma、Delta株的S-ECD和WT RBD的亲和力常数测定图，其KD依次为1.492nM、0.938nM、0.658nM、1.204nM、4.211nM及0.683nM。结果显示该中和抗体与SARS-CoV-2的野生型及目前主要变异株的S抗原均具有很好的亲和力，使其发展成新冠肺炎特效药成为可能。
表5单抗ZWC6与不同抗原的结合动力学数据
| 抗原 | K<sub>on</sub>(1/Ms) | K<sub>off</sub>(1/s) | K<sub>D</sub>(M) |
| WT S-ECD | 1.708*10^5 | 2.548*10^-4 | 1.492*10^-9 |
| Alpha S-ECD | 1.749*10^5 | 1.641*10^-4 | 9.384*10^-10 |
| Beta S-ECD | 1.835*10^5 | 1.208*10^-4 | 6.583*10^-10 |
| Gamma S-ECD | 1.512*10^5 | 1.820*10^-4 | 1.204*10^-9 |
| Delta S-ECD | 7.239*10^4 | 3.048*10^-4 | 4.211*10^-9 |
| WT RBD | 5.346*10^5 | 3.653*10^-4 | 6.832*10^-10 |
序列表
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<120> 一种抗新冠病毒全人源广谱中和抗体及应用
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ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
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aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacaca 960
cagaagagcc tctccctgtc tccgggtaaa 990
<210> 5
<211> 108
<212> PRT
<213> Homo sapiens
<400> 5
Ala Ile Arg Met Thr Gln Ser Pro Pro Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Asn Tyr
20 25 30
Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Thr Phe Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg
100 105
<210> 6
<211> 324
<212> DNA
<213> Homo sapiens
<400> 6
gccatccgga tgacccagtc tccacccacc ctgtctttgt ctccagggga aagagccacc 60
ctctcctgca gggccagtca gagtgttacc aactacttgg tctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat acattcaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctggagcct 240
gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctcccac ttttggccag 300
gggaccaaag tggatatcaa acgt 324
<210> 7
<211> 106
<212> PRT
<213> Homo sapiens
<400> 7
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
35 40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 8
<211> 318
<212> DNA
<213> Homo sapiens
<400> 8
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120
aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180
aaggacagca cctacagcct cagcagtacc ctgacgctga gcaaagcaga ctacgagaaa 240
cacaaagtct acgcctgcga agtcacccat cagggcctga gttcgcccgt cacaaagagc 300
ttcaacaggg gagagtgt 318
<210> 9
<211> 106
<212> PRT
<213> Homo sapiens
<400> 9
Arg Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 10
<211> 318
<212> DNA
<213> Homo sapiens
<400> 10
cgtcagccca aggctgcccc ctcggtcact ctgttcccac cctcgagtga ggagcttcaa 60
gccaacaagg ccacactggt gtgtctcata agtgacttct acccgggagc cgtgacagtg 120
gcctggaagg cagatagcag ccccgtcaag gcgggagtgg agaccaccac accctccaaa 180
caaagcaaca acaagtacgc ggccagcagc tacctgagcc tgacgcctga gcagtggaag 240
tcccacaaaa gctacagctg ccaggtcacg catgaaggga gcaccgtgga gaagacagtg 300
gcccctacag aatgttca 318
Claims (10)
1.一种抗SARS-CoV-2的全人源单克隆抗体，其特征在于，所述单克隆抗体的重链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:1第26-33、51-58、97-111位氨基酸序列所示；轻链可变区的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO:5第27-32、50-52、89-97位氨基酸序列所示。
2.根据权利要求1所述的单克隆抗体，其特征在于，所述单克隆抗体重链可变区的氨基酸序列如SEQ ID NO:1所示，轻链可变区的氨基酸序列如SEQ ID NO:5所示。
3.根据权利要求2所述的单克隆抗体，其特征在于，所述单克隆抗体重链恒定区的氨基酸序列如SEQ ID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7所示。
4.一种编码权利要求1-3任一所述单克隆抗体重链和轻链的多核苷酸，其特征在于，编码所述单克隆抗体的重链可变区的多核苷酸序列由SEQ ID NO:2所示，编码所述单克隆抗体的轻链可变区的多核苷酸序列由SEQ ID NO:6所示。
5.根据权利要求4所述的多核苷酸，其特征在于，编码所述单克隆抗体的重链恒定区的多核苷酸序列由SEQ ID NO:4所示，编码所述单克隆抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8所示。
6.一种表达权利要求5所述编码单克隆抗体重链和轻链的多核苷酸的功能元件，其特征在于，所述功能元件为线性表达框或哺乳动物表达载体。
7.一种含有权利要求6所述功能元件的宿主细胞。
8.根据权利要求要求7所述的宿主细胞，其特征在于，所述细胞为Expi293F细胞。
9.根据权利要求要求7所述的宿主细胞，其特征在于，所述细胞为CHO-K1或CHO-S细胞。
10.权利要求1-3任一所述的单克隆抗体在制备COVID-19治疗药物中的应用。
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