CN111732655A - 靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体及应用 - Google Patents
靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种靶向RBD的高中和活性抗SARS‑CoV‑2全人源单克隆抗体，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序列，本发明还公开了所述抗体在制备2019‑冠状病毒病治疗药物中的应用。本发明公开的单克隆抗体具有高效、特异的抗SARS‑COV‑2病毒活性，还具有高表达、全人源、稳定性好的特点，适合产业化生产。
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
目前，已知引起人致病的冠状病毒包括7种，其中HCoV-229E、HCoV-OC43、HCoV-NL63和HCoV-HKU1致病性较低，一般引起轻微的呼吸道症状。另外三种引起严重的疾病。SARS-CoV引起严重呼吸窘迫综合征(SARS)，MERS冠状病毒引起中东呼吸综合征，SARS-CoV-2(新型冠状病毒)引起COVID-19(新冠肺炎)。
SARS-CoV-2属冠状病毒科(Coro-naviridae)冠状病毒属(Coronavirus)，是一类有包膜的单股正链RNA病毒。基因组全长为30kb，依次由5＇-端非编码区、非结构蛋白开放阅读框(ORF)1a/b编码区、刺突糖蛋白S编码区、包膜蛋白E编码区、膜蛋白M蛋白编码区、核衣壳蛋白N蛋白编码区及3＇-端非编码区组成。其中，非结构蛋白ORF1a/b区编码的聚蛋白可被切割，形成RNA依赖的RNA聚合酶(RdRp)等，指导病毒基因组复制、转录和翻译。结构蛋白S可特异性地与宿主细胞的受体结合，是病毒入侵宿主易感细胞的关键蛋白。不同冠状病毒可利用不同的细胞受体来完成入侵，如SARS-CoV的受体是血管紧张素转化酶2(ACE2)，而MERS-CoV的受体是氨基肽酶4(DPP4，也称CD26)。研究证明，氨肽酶N不是SARS-CoV-2的受体，而ACE2可作为其受体。
基因测序结果显示，SARS-CoV-2与SARS-CoV的同源性约79％,与MERS-CoV的同源性约50％。文献报道，新型冠状病毒S蛋白ACE2亲和力较SARS-COV强，提示具有更强的传染性。到目前为止，还没有专门用于预防的疫苗和治疗冠状病毒的特效药物。一般采用非特异性治疗，预防严重的并发症，降低重症发病率和病死率，提高治愈率。研发新型冠状病毒病的疫苗和特异治疗药物成为全球应急科研攻关的重要任务。
新冠肺炎治疗方法中，康复者血浆疗法在治疗重症患者中显示了良好的效果，提示抗体在治疗新冠肺炎上的巨大潜力。单克隆抗体成分单一、易规模化制备、抗病毒机制明确，是新冠肺炎治疗药物研究的重要方向。本发明拟采用单细胞PCR技术从SARS-CoV-2感染者恢复后的外周血中获得具有高亲和力和优异中和活性的单克隆抗体，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。
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27-35、52-54、91-100位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“4B7”。
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，所述抗体的重链恒定区的氨基酸序列如SEQ IDNO:3所示，所述轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9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，编码所述抗体的重链恒定区的多核苷酸序列由SEQ IDNO:4所示，编码所述抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8或者SEQ ID NO:10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/或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细胞，本发明可以使用CHO-K1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显示出对SARS-COV-2感染细胞良好的中和保护效果。本发明的结果显示，所述抗体在制备COVID-19治疗药物中具有广泛应用的前景。本发明公开的单克隆抗体还具有以下的技术优势:(1)全人源，在临床应用上，不需要进行人源化改造以降低人抗鼠抗体反应(HAMA反应)，即低免疫原性。(2)高亲和活性和高中和活性，单抗与RBD抗原的亲和常数KD为0.39nM，在SARS-COV-2感染的细胞模型上，半数有效浓度EC50为2.5nM。(3)作用机制明确：4B7与S、S1和RBD均高度特异结合，显示单抗是靶向受体结合区域的，通过特异性阻断病毒与受体的结合发挥抗病毒效应(4)稳定性好：因为单抗基因来自人体的同一个细胞，是天然配对的，已知人体内IgG1抗体的半衰期是21～28天，理论上公开的单抗具有一致的人体内半衰期。
附图说明
图1.流式细胞仪单细胞分选图；
图2.单克隆抗体可变区基因扩增自动核酸电泳仪检测图谱；
图3.单克隆抗体可变区序列检索结果输出图；
图4.亲和层析纯化后的单抗SDS-PAGE检测图谱；
图5.ELISA检测纯化单抗与S蛋白、S1蛋白、S2蛋白、RBD蛋白的结合活性随浓度变化的曲线图；
图6.BIA core T200测定与S蛋白、S1蛋白、RBD蛋白的亲和力结果输出图；
图7.单抗在假病毒细胞模型上的EC50测定曲线图；
图8.单抗在真病毒细胞模型上的EC50测定曲线图。
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
1.1准备96孔板：每孔20μl去RNA酶水+20U RNA酶抑制剂，盖好封板膜，放4℃待用
1.2准备样品：
(1)复苏细胞：将冻存的分离自新冠肺炎恢复者外周血的PBMC细胞从-80℃取出，迅速置于37℃的温水中，待细胞融化后，800rpm离心5min，倒去上清；用2-3ml FPBS重悬到流式管中，配平后800rpm离心5min，倒去上清；再用2-3ml FPBS重悬，离心，倒上清。最后加入100μl FPBS重悬，吸取2μl稀释10倍进行细胞计数。
(2)单染管：5管(包括FITC标记抗原，PE标记IgG，Alexa Fluor 700标记CD19，PerCP标记CD3，APC标记Anti-His抗体)每管5×105个细胞，按照说明书推荐的抗体浓度将染料分别加入到装有细胞的流式管中，用FPBS将各反应体积补足50μl。
(3)裸细胞对照：1管，5×105个细胞，用FPBS补足50μl
(4)用来分选的细胞：1管，先确定细胞的数量，终浓度为100μl体系(1×106cells)，按照推荐的抗体浓度将荧光染料加入，其中把两个细胞样品分别分为两个，各用S-FITC和Anti-His tag抗体染色。用FPBS补足体系。
(5)将样品于4℃避光孵育1h。
(6)每管加入3ml FPBS，于4℃，800g离心5min，倒去上清，重复清洗两次。
(7)用400μl FPBS重悬后，用40μm细胞筛去除细胞团，4℃避光保存供分选。
1.3流式分选：选择CD3-/CD19+/IgG+/S+的细胞进行分选。合计分选到416个细胞。
结果：流式分选的结果见图1，图1-A显示圈定人血单个核细胞群；图1-B显示从图1-A圈定的细胞中选择CD3-CD19+的细胞；图1-C显示从图1-B中圈定的细胞中进一步选择IgG+S+的细胞。
1.4利用单细胞-PCR技术扩增全人源单抗可变区基因
1.4.1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94个细胞。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1)。
引物：
H：5′L-VH 1、L-VH 3、L-VH 4/6，5′L-VH 5、Hu IgG-const-anti、3′CμCH1
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1.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(全式金生物技术有限公司，AT311)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；接着，95℃预变性15min，95℃40s，55℃30s，72℃1min，40个循环，最后72℃延伸10min。
1.4.2巢式PCR
取反转录产物1ul为模板，进行PCR反应扩增H、κ、λ的可变区：扩增重链可变区、kappa轻链可变区和λ轻链可变区的引物如下表2所示。
表2.巢式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2.5μL、10mM dNTP 0.5μL、DNA聚合酶(全式金生物技术有限公司，AP141)0.25μL、引物如上、模板为反转录产物1μL、水补齐至25μL。
PCR反应条件为：94℃预变性4min，接着，94℃30s，57℃30s，72℃45min，40个循环，最后72℃延伸10min。
1.4.3自动核酸电泳仪进行检测
自动核酸电泳检测的部分结果见图2，其中图2-A显示96孔PCR反应板中A和B行的重链可变区检测结果，阳性克隆分别为A06、B03、B07、B12共4个细胞检测阳性。其中图2-B显示96孔PCR反应板中A和B行的轻链可变区检测结果，阳性克隆分别为B03、B05、B07共3个细胞检测阳性，VH和VL检测都阳性的细胞是B03和B07，定义一个单细胞中重链和轻链基因均扩增成功的克隆是配对成功的克隆。本次实验合计检测到11对配对的克隆，图2显示2个配对细胞克隆，其余结果未显示。
1.4.4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3所示，图3-A显示重链可变区的检索结果，V区同源性最高为98.61％，J区同源性最高为91.84％，D区使用读框2。图3-B显示轻链的检索结果，V区同源性最高为98.96％，J区同源性最高为100％。
1.4.5对单抗4B7序列的分析结果如下：
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27-35、52-54、91-100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。
1.5线性表达框的构建
相比传统的表达载体构建方法，构建线性表达框更加快速。设计的线性表达框含有单抗在哺乳细胞内表达的所有原件，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 ID NO: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：
1.5.1以pcDNA3.4(ThermoFisher Scientific，A14697)为模板，扩增启动子-前导序列片段、多聚A尾片段。扩增启动子-前导序列片段的PCR反应体系中包括：模板质粒pcDNA3.4 1ng，10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-CMV-forward(与CMV启动子上游序列匹配)(5'-CGATGTACGGGCCAGATATACGCGTTG-3')、引物3'-leader-sequence(5'-ACACTGGACACCTTTTAAAATTAG-3',用于重链的融合,信号肽序列的核苷酸序列5'-ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTAATTTTA AAAGGTGT C-3')，编码的氨基酸序列为MNFGLSLIFLVLILKGV；用于轻链的融合引物序列为5'-GTCACCAGTGGAACCTGGAACCCA-3'，全长信号肽序列核苷酸序列为5-ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGG，氨基酸序列为MDSQAQVLMLLLLWVSGT CG，信号肽序列来源鼠源单抗可变区)、水补齐至50μL。
扩增多聚A尾片段的PCR反应体系中包括：模板质粒pSecTag2(Invitrogen，V90020)1ng，10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-BGH POLY-(A)(5'-GCCTCGACTGTGCCTTCTAG-TTGC-3')、引物3'-BGH-POLY(A)(5'-TCCCCAGCATGCCTGCTATTGTCT-3')、水补齐至50μL。扩增片段长度215bp。
PCR反应条件：94℃预变性4min，接着94℃30s，60℃30s，72℃1min，30个循环，最后72℃延伸10min。
1.5.2扩增抗体恒定区。
H链恒定区PCR体系中包含：重链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-CH1(5'-ACCAAGGGC CCATCGGTCTTCCCC-3')、引物3'-CH3(5'-GCAACTAGAAGGCACAGT CGAGGCTTTACCCGGAGACAGGGA-3')、水补齐至50μL。
κ链恒定区PCR体系中包含：κ链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'-Cκ(5'-ACTGTGGCTGCACCA TCTGTCTTC-3')、引物3'Cκ(5'-GCAACTAGAAGGCACAGTCGAGGCACACTCTCCCCTGTTGAAGCT-3')、水补齐至50μL。
λ链恒定区PCR体系中包含：λ链恒定区模板10ng、10×缓冲液5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5'Cλ(GAGGAGCTTCAAG CCAA CAAGGCCACA)、引物3'Cλ(GCAACTAGAAGGCACAGTCGAGGCTGAACATTCTGTAGGGGCCAC)、水补齐至50μL。
其中黑体字序列部分GCAACTAGAAGGCACAGTCGAGGC是与polyA互补序列，用于融合扩增。
PCR反应条件为：94℃预变性4min，接着94℃30s，60℃60s，72℃3min，30个循环，最后72℃延伸10min。
1.5.3扩增抗体可变区。
见巢式PCR部分。
1.5.4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/轻链恒定区片段10ng、多聚A尾片段10ng，10×缓冲液2.5μL、10mM dNTP 0.5μL、DNA聚合酶(全式金生物技术有限公司，AP151-13)0.25μL、引物5'-CMV-FORWARD(5'-CGATGTACGGGCCAGATATACGC GTTG-3')和3'-POLY(A)(5'-TCCCCAGCATGCCTGCTATTGTCT-3'、水补齐至25μL。
PCR反应条件为：94℃预变性4min，接着94℃30s，60℃30s，72℃3min，30个循环，最后72℃延伸10min。
1.5.5PCR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。
1.5.6细胞接种：将293T细胞以2×105/mL接种于24孔细胞培养板中，在含有5％CO2的细胞温箱中，37℃培养过夜。
1.5.7细胞共转染：次日，向200μL无血清的MEM培养基中，加入构建成功的重链和轻链线性表达框PCR产物各1μg，混匀后加入4μL转染试剂Turbofect(Thermo Scientific,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。
1.6表达载体的构建和酶切鉴定
以线性表达框为模板，扩增重链，切胶回收约1.4kb大小的重链片段，表达载体pCDNA3.4(ThermoFisher Scientific，A14697)使用Eco RI/BamHI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PCR检测、双酶切鉴定和序列测定，构建成功重链的表达载体pCDNA3.4-4B7-H-1。以轻链表达框为模板，扩增轻链，胶回收约0.7kb大小的轻链片段，将轻链和载体片段通过同源重组方法进行连接，TOP10挑取克隆进行PCR检测、双酶切鉴定和序列测定，构建成功轻链的表达载体pCDNA3.4-4B7-L-3。
1.7单抗的瞬时表达和亲和层析纯化
使用Expi293表达系统，取15ug重链和15ug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 pH3.0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4，其中图4的A为非还原SDS-PAGE检测结果，图4的B为还原SDS-PAGE检测结果，泳道1-3为单抗3H3，泳道4为4B7，泳道5为4C12，泳道6为4E1电泳结果。在还原电泳中，预期重链和轻链的分子量分别为50kDa和25kDa，泳道M为分子量标记。在非还原电泳中，预期全分子单抗的分子量为150kDa，符合预期。
实施例2人源抗SARS-CoV-2单克隆抗体4B7与S、S1、S2和RBD的结合活性分析
2.1包被：取重组的S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。
2.2封闭：每孔加入300μL PBST洗液，洗涤3次×3min/次；拍净孔内液体，加入2％BSA，200μL/孔，37℃封闭1h。
2.3样品孵育：每孔加入300μL PBST洗液，洗涤3次×3min/次；拍净孔内液体，加入PBS稀释后的纯化单抗，首孔9ug/ml,3倍系列稀释100μL/孔，37℃孵育1h。
2.4二抗孵育：洗涤，同上；加入HRP羊抗人FC二抗(1:20000稀释)，100μL/孔，37℃孵育1h。
2.5显色：洗涤，同上；每孔加入100μL TMB单组分显色液，37℃显色5min后每孔加50μL终止液终止反应，使用酶标仪检测450nm处的吸光值。使用Graph Pad非线性回归，四参数拟合绘制标准曲线，并根据标准曲线及稀释倍数计算单抗的EC50浓度。
2.6结果：见图5和表3。图5中，圆标曲线表示与S蛋白的检测结果，显示特异结合呈现剂量反应关系；方标曲线表示与S1抗原的检测结果，显示特异结合呈现剂量反应关系；正三角标曲线表示与S2抗原的检测结果，显示不结合；倒三角标曲线显示与RBD的检测结果，显示特异结合呈现剂量反应关系。分析结果证明，单抗4B7识别的表位位于S蛋白，S1区，更具体的是位于RBD区。表3中，单抗4B7与S、S1、RBD均具有高结合活性，说明单抗是RBD特异的。
表3.单抗4B7与S、S1、S2和RBD的结合活性
| 抗原 | S | S1 | S2 | RBD |
| EC<sub>50</sub>:(ug/ml) | 0.002156 | 0.001899 | 0.2758 | 0.001808 |
实施例3 Biacore T200测定单抗与S、S1、RBD抗原的亲和力
测定的基本原理是基于一种表面等离子共振(SPR)的物理光学现象的生物传感分析技术。不必使用荧光标记和同位素标记，从而保持了生物分子的天然活性。当入射光以临界角入射到两种不同透明介质的界面时将产生全反射，且反射光强度在各个角度上都应相同，但若在介质表面镀上一层金属薄膜后，由于入射光可以引起金属中自由电子的共振，从而导致反射光在一定的角度内大大减弱，其中使反射光完全消失的角度称为共振角。共振角会随金属薄膜表面通过的液相的折射率的改变而改变，折射率的变化(RU)又和结合在金属表面的大分子质量成正比。因此，BIA技术可以通过对反应全过程中各种分子反射光的吸收获得初始的数据，并经相关处理获得结果－传感图。
实验步骤：(1)换液：抗体0429-4B7换液到HEPES缓冲液中，nano测定浓度为1.67mg/mL。抗原缓冲液中由于含有Tris，影响包被，需要换液到PBS中，浓度介于0.8-1mg/mL。(2)预富集，确定包被缓冲液pH:S蛋白采用pH4.0的甘氨酸缓冲液，S1和RBD蛋白采用pH5.0的缓冲液。(3)配体固定：使用CM5芯片、氨基偶联试剂盒进行操作，分别将S、S1、RBD蛋白包被在芯片的2、3、4通道，1通道作为参比通道。包被结果：S(437RU)S1(107RU)RBD(41RU)。(4)抗体稀释：最高浓度为100nM，对倍稀释，10个稀释度。最终结果采用25nM、12.5nM、6.25nM、3.125nM、1.5625nM稀释度的曲线进行拟合。(5)结合时间100s，解离时间900s。拟合方法采用1：1model拟合。
分析及结果：通过软件拟合计算得到4B7与S蛋白、S1和RBD的亲和常数，见图6和表4。说明单抗与S、S1和RBD均具有高的亲和力。
表4.单抗4B7与S、S1和RBD抗原的亲和力检测结果
| KD(M) | ka(1/Ms) | kd(1/s) | |
| 4B7和nCoV S | 4.533E-10 | 9.355E+5 | 4.240E-4 |
| 4B7和nCoV S1 | 2.170E-10 | 1.609E+6 | 3.492E-4 |
| 4B7和nCoV RBD | 3.866E-10 | 1.101E+6 | 4.256E-4 |
实施例4 SARS-CoV-2假病毒细胞模型上中和活性分析
4.1假病毒包装：将编码全长SARS-CoV-2S蛋白的基因(GenBank ID：QHD43416.1)插入pDC316载体，得到质粒pDC316-SARS-CoV-2-S。将ACE2基因稳定转染入HEK293细胞，构建稳定表达人ACE2的细胞ACE2-293T细胞。将7.0×106ACE2-293T细胞接种在10cm细胞培养皿中，并在37℃，5％CO2下生长过夜。用Lipofectamine 3000转染试剂(Invitrogen)将pDC316-SARS-CoV-2-S和HIV骨架载体pNL4-3.Luc.R-E-共转染到ACE2-293T细胞中。6小时后更换培养基。转染后48小时收集含有HIV假型病毒和S蛋白的上清液，并通过0.45μm过滤器过滤。然后将上清液等分并保存在-80℃。
4.2稀释抗体：4B7(20200525批，原始浓度8.35mg/ml，稀释到1.67mg/mL)将抗体用DMEM培养基稀释到需要的浓度，首孔浓度为6μg/mL(100μL体系)，首孔100μL，吸取50μL加入到下一孔，对倍稀释。每个梯度设置两个复孔。
4.3稀释假病毒：将假病毒与DMEM培养基按照3:7的比例混合，混匀后每孔加入50μL，轻轻混匀。37℃孵育1h。
4.4将ACE2-293T细胞稀释到2×105cells/mL，每孔加入100μL，37℃、5％CO2培养48h。
4.5检测Luciferase读值，使用britelite plus Reporter Gene Assay System(PerkinElmer,6066769)。每孔吸弃100μL培养基，加入100μL britelite plus显色底物，避光孵育2min，用枪头吹吸3次后吸取150μL转移到白色96孔板，使用TECAN SPARK 10M进行读数。
4.6统计分析：没有病毒和抗体的细胞用作空白对照，没有抗体的细胞用作病毒对照。中和百分比计算为(样品信号-空白对照信号)/(病毒对照信号-空白对照信号)×100％。使用GraphPad Prism 7进行统计分析。
结果：见图7(图7横坐标表示对数浓度，纵坐标表示相对阴性对照组的保护率％)。本发明公开的单抗4B7在假病毒模型上EC50是0.052ug/ml(0.35nM)。
实施例5 SARS-CoV-2感染时细胞模型上中和活性分析
5.1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。
5.2实验当天，将纯化单抗用培养基DMEM+2％FBS自初始浓度(4B7单抗初始浓度92.78ug/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100TCLD50/孔)，充分混匀，置细胞培养箱孵育1h。
5.3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细胞培养箱继续培养72h。
5.4 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。
4.5弃尽洗液，用吸水纸拍干板孔中水份，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，细胞活率和抗体浓度用GraphPad Prism 5拟合曲线，计算抗体EC50值。
5.6单抗在细胞模型上的保护效果和IC50的结果，见图8(图8横坐标表示对数浓度，纵坐标表示相对阴性对照组的保护率％)。本发明公开的单抗4B7在病毒感染的细胞模型上EC50是0.38ug/ml(2.5nM)。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体
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Claims (12)
1.一种靶向RBD的高中和活性抗SARS-CoV-2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SEQID NO:5第27-35、52-54、91-100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所述的抗体，其特征在于，所述抗体重链恒定区的氨基酸序列如SEQID NO:3所示，轻链恒定区的氨基酸序列如SEQ ID NO:7或SEQ ID NO:9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，编码所述抗体的轻链恒定区的多核苷酸序列由SEQ ID NO:8或者SEQ ID NO:10所示。
6.一种表达权利要求5所述编码单克隆抗体重链和/或轻链的多核苷酸的功能元件。
7.根据权利要求要求6所述的功能元件，其特征在于，所述功能元件为线性表达框。
8.根据权利要求要求6所述的功能元件，其特征在于，所述功能元件为哺乳动物表达载体。
9.一种含有权利要求7所述线性表达框的宿主细胞。
10.根据权利要求要求9所述的宿主细胞，其特征在于，所述细胞为Expi293F细胞。
11.根据权利要求要求9所述的宿主细胞，其特征在于，所述细胞为CHO-K1细胞。
12.权利要求1-3任一所述抗体在制备COVID-19治疗药物中的应用。
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