CN114989292A - 一种抗SARS-CoV-2全人源化单克隆抗体及应用 - Google Patents
一种抗SARS-CoV-2全人源化单克隆抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种抗SARS‑CoV‑2全人源化单克隆抗体及应用。所述抗体6G2通过流式分选‑单细胞PCR技术筛选获得，具有独特的CDR分区，并且能够结合SARS‑CoV‑2的Spike糖蛋白的RBD区域，阻断Spike蛋白与宿主膜蛋白ACE2的结合，从而中和新型冠状病毒。本发明公开的单克隆抗体6G2不仅具有高效、特异的抗SARS‑COV‑2中和活性，还具有高表达、全人源、稳定性好的特点，适合产业化生产。本发明公开的抗体具有广泛的应用前景，例如可用于制备新冠病毒检测产品，或用于制备预防和治疗新冠病毒疾病的药物，具有重要的生物学和医学意义。
Description
技术领域
本发明涉及免疫学、病毒学及生物医药领域，具体涉及一种抗SARS-CoV-2全人源化单克隆抗体及应用。
背景技术
SARS-CoV-2(也称为2019-nCov)属于正链RNA病毒的一种，属于冠状病毒家族的β属，其编码四种结构蛋白：spike(S)，envelope(E)，membrane(M)，和nucleocapsid(N)、16种非结构蛋白，及5-8种辅助蛋白质。SARS-CoV-2利用病毒表面的S蛋白与宿主细胞受体-血管紧张素转换酶II(ACE2)进入细胞。S蛋白根据蛋白结构功能又被分为两个功能单位，即S1和S2蛋白亚基。S1可分为NTD(N-terminal domain)和RBD(Receptor binding site)，RBD区域长约240个氨基酸，主要与宿主细胞受体结合，S2在病毒和细胞膜融合起作用。根据已有的报导，中和抗体主要作用于RBD区域，抗体结合于RBD，阻碍RBD与ACE2的结合，从而阻止病毒感染细胞。
到目前为止，虽有针对新型冠状病毒的各类疫苗上市，但随着病毒的不断变异，疫苗的保护效果也受到严重挑战。另外，目前还缺乏治疗冠状病毒的特效药物，临床上一般采用非特异性治疗，预防严重的并发症，降低重症发病率和死亡率，提高治愈率。因此，研发具有高保护效率的通用型新型冠状病毒疫苗和特异性治疗药物成为全球应急科研攻关的首要任务。当病毒感染人体时，人体免疫系统接受病毒抗原刺激就会激发机体的体液免疫和细胞免疫反应，从而逐渐控制感染、清除病毒。现有临床资料表明，新冠肺炎康复者血清及其制品可以有效控制新冠肺炎患者疫情发展，其中起关键作用的就是康复者血液中的中和抗体。中和抗体在病毒感染康复中一直扮演着重要的角色，新冠病毒感染人体也可诱发较强的体液免疫反应，产生特异性针对新冠病毒的中和抗体。因此新冠病毒中和抗体被普遍认为可以作为一种有效而特异的抗新冠病毒的治疗手段。但多抗血浆不仅来源有限，同时其临床应用也受到诸如难以质控、供受体血型差异、潜在的传染性因子等条件的限制。从新冠肺炎康复者体内分离可中和SARS-CoV-2的全人源单克隆抗体，可有效克服上述问题，该种抗体不仅能通过阻断SARS-CoV-2与受体细胞的结合，从而避免病毒的侵入，达到保护的作用，而且相对人源化或人鼠嵌合抗体具有副作用更小的优点，将为COVID-19的特异性预防和治疗提供新的手段。
目前世界上有多家机构正在开展针对新冠肺炎抗体研究，但尚处于实验研发阶段。由于SARS-CoV-2是RNA病毒，在传播流行过程中病毒的基因组序列容易产生突变。当这些抗体所识别的非保守区域位点发生突变，产生新的流行毒株时，会导致抗体失去对突变病毒的保护效果。因此，本领域技术人员仍然希望能够开发出新的对于包括 SARS-CoV-2在内的冠状病毒具有结合能力和中和能力的抗体。
发明内容
本发明所要解决的技术问题是为了克服现有技术中预防、治疗或检测新型冠状病毒 SARS-CoV-2药物的不足，提供了一种靶向SARS-CoV-2的单克隆抗体及其应用。
为了解决上述技术问题，本发明采用如下技术方案：
第一方面，提供一种靶向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 IDNO:1第26-33，51-57，96-113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27-33，51-53，89-98位氨基酸序列所示。所述单克隆抗体在本申请中被命名为“6G2”。
在一个优选的实施方案中，所述全人源单克隆抗体重链的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所示。
第二方面，提供一种编码第一方面所述的靶向RBD的高中和活性抗SARS-CoV-2 全人源单克隆抗体的多核苷酸，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所示。
第三方面，提供一种表达第一方面所述的靶向RBD的高中和活性抗SARS-CoV-2 全人源单克隆抗体的表达载体，所述载体含有第二方面所述编码所述抗体重链的可变区的多核苷酸以及编码所述抗体轻链的可变区的多核苷酸。
第四方面，提供一种表达第一方面所述的靶向RBD的高中和活性抗SARS-CoV-2 全人源单克隆抗体的宿主细胞，所述宿主细胞含有第三方面所述的表达载体。
在一个优选的实施方案中，所述宿主细胞为HEK293T细胞。
第五方面，提供一种用于治疗或预防新冠病毒感染的药物组合物，所述药物组合物包括第一方面所述的靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体。
第六方面，提供第一方面所述靶向RBD的高中和活性抗SARS-CoV-2全人源单克隆抗体在制备检测新冠病毒产品或制备防治新冠病毒感染药物中的应用。
本发明的有益效果：
本发明提供的单克隆中和抗体表现出对SARS-CoV-2良好的中和保护效果。本发明的结果显示，所述抗体在制备检测新冠病毒产品与制备防治新冠病毒感染药物中具有广泛应用的前景。本发明公开的单克隆中和抗体还具有如下的技术优势:(1)本单克隆抗体属于全人源化抗体，具有低免疫原性，在临床应用中，不需要进行人源化改造以降低人抗鼠抗体反应。(2)单抗与RBD抗原具有较高的结合力，在SARS-CoV-2野生型假病毒感染的细胞模型上的半数有效浓度EC50为224.7nM，且单抗对多种SARS-CoV-2 突变假病毒具有较高的中和活性，另外单抗在SARS-CoV-2真病毒感染的细胞模型上的半数有效浓度EC50为113.4nM。(3)作用机制明确：本发明提供的单克隆抗体6G2 与RBD具有高度特异结合，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。
附图说明
图1.流式细胞仪单细胞分选图；
图2.单克隆抗体6G2可变区基因扩增核酸电泳仪检测图谱；
图3.亲和层析纯化后的单克隆抗体6G2考马斯亮蓝染色检测图谱；
图4.ELISA检测纯化单克隆抗体6G2蛋白和RBD蛋白的结合活性随浓度变化的曲线图；
图5.单克隆抗体6G2在假病毒细胞模型中的EC50测定曲线图；
图6.单克隆抗体6G2在真病毒细胞模型中的EC50测定曲线图；
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook 等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1人源抗SARS-CoV-2单克隆抗体的筛选和制备
1.1溶液配制：按每孔20μl无RNA酶水+20μl RNA酶抑制剂的量配制混合溶液，然后加入每孔40μl溶液，用封板膜封住96孔板，放4℃冰箱待用。
1.2样品准备：
(1)复苏细胞：将装有分离自新冠肺炎恢复者外周血PBMC细胞的细胞冻存管从液氮中取出，迅速置于37℃的温水中轻轻摇晃，待冻存液融化后，将细胞转移到新的离心管中，800rpm离心5min，弃上清；使用3ml含2％FBS的PBS(FPBS)重悬细胞，再次800rpm离心5min，弃上清；重复重悬细胞一次，收集细胞沉淀；最后加入100μl FPBS 重悬细胞，吸取2μl稀释10倍进行细胞计数。
(2)单染管：5管(包括FITC标记CD38，PE标记CD19，BB700标记IgD，BV510 标记CD3，APC标记抗原S)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将各反应体积补齐到50μl。
(3)阴性对照组细胞：1管，细胞5×105，用FPBS补齐至50μl。
(4)实验组细胞：1管，先进行细胞计数，要求总共1×106个细胞，体积为100μl。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FITC 和APC标签抗体染色，用FPBS补齐体积。
(5)将样品置于4℃避光环境下孵育1h。
(6)每管加入3ml FPBS，于4℃，800rpm离心5min，弃上清，重复清洗两次。
(7)用500μl FPBS重悬细胞后，用40μm细胞筛去除细胞团，4℃避光保存待上机分选。
1.3流式分选：选择CD3-/CD19+/IgG+/S+的细胞进行分选，合计分选到215个细胞。实验结果如下：流式分选的结果见图1，图1-A显示圈定的人血单个核细胞群；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出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54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：5×缓冲液6μL、dNTP 2μL、反转录酶2μL、引物如上、模板为单细胞抽提的RNA，最后用ddH2O补齐至30μL。
PCR反应条件为：50℃反转录30min；接着，95℃预变性15min，95℃变性40s， 55℃退火30s，72℃延伸1min，进行40个循环，最后72℃延伸10min。
1.4.2巢式PCR
取上述实验得到的反转录产物1μL作为模板，进行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2所示。
表2.巢式PCR引物
PCR反应体系：10×buffer 2.5μL、10mM dNTP 0.5μL、DNA聚合酶0.25μL、引物如上、模板为反转录产物1μL、ddH2O补齐至25μL。
PCR反应条件：94℃预变性4min，接着94℃变性30s，57℃退火30s，72℃延伸45min，进行40个循环，最后72℃延伸10min。
1.4.3核酸电泳仪检测
自动核酸电泳检测的部分结果见图2，其中图2-A显示PCR反应中A和B行的重链可变区检测结果，阳性克隆共6个细胞检测阳性。其中图2-B显示PCR反应板中A 和B行的轻链可变区检测结果，阳性克隆共7个细胞检测阳性，VH和VL检测都阳性的细胞有6个，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。图2显示6个配对细胞克隆。
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。
1.4.5对单抗6G2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26-33，51-57，96-113位氨基酸序列所示，编码重链可变区的核苷酸序列由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27-33，51-53，89-98位氨基酸序列所示，编码轻链可变区的核苷酸序列由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)、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×buffer 5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5’-CMV- F(5’-CGATGTACGGGCCAGATATACGCGTTG-3’)、引物3’-L(5’-ACACTGGACACCTT TTAAAATTAG-3’，用于重链的融合，信号肽序列的核苷酸序列5’-ATGAACTTCGGG CTCAGCTTGATTTTCCTTGTCCTAATTTTAAAAGGTGT 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’-GTCACCAGTGGAACCTGGA ACCCA-3’，全长信号肽序列核苷酸序列为5-ATGG ATTCACAGGCCCAGG TTCTTA TG TTACTG CTG CTATGGGTATCTGGTACCTGTGG，氨基酸序列为MDSQAQVLMLLLLWVSGTCG，信号肽序列来源鼠源单抗可变区)、ddH2O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×buff er 5μL、10mMdNTP 1μL、DNA聚合酶0.5μL、引物5’-BGH POLY-(A)(5’-GCCTCGA CTGTGCCTTCTAG-TTGC-3’)、引物3’-BGH-POLY(A)(5’-TCCCCAGCATGCCTGCTA TTGTCT-3’)、ddH2O补齐至50μL。扩增片段长度215bp。
PCR反应条件：94℃预变性4min，接着94℃变性30s，60℃退火30s，72℃延伸1 min，进行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：重链恒定区模板10ng、10×buffer 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’)、ddH 2O补齐至50μL。κ链恒定区PCR体系：κ链恒定区模板10ng、10×buffer 5μL、10mM dNTP 1μL、DNA聚合酶0.5μL、引物5’-Cκ(5’-ACTGTGGCTGCACCATCTGTCTTC-3’、引物3’Cκ(5’-GCAACTAGAAGGCACAGTCGAGGCACACTCTCCCCTGTTGAAGCT- 3’)、ddH2O补齐至50μL。λ链恒定区PCR体系：λ链恒定区模板10ng、10×buffer 5μL、 10mM dNTP 1μL、DNA聚合酶0.5μL、引物5’Cλ(GAGGAGCTTCAAGCCAACAAGGCCACA)、引物3’Cλ(GCAACTAGAAGGCACAGTCGAGGCTGAACATTCTGTAGGGGC CAC)、ddH2O补齐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×buffer 2.5μL、10mM dNTP 0.5μL、DNA 聚合酶0.2 5μL、引物5’-C M V-F(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℃变性30s，60℃退火30s，72℃延伸3min，进行30个循环，最后72℃延伸10min。
1.5.5PCR产物回收纯化和定量：
PCR反应产物电泳后，使用天根公司回收试剂盒回收。DNA浓度测定：用Nanodrop(GE Healthcare)测定PCR回收产物中的DNA含量。
1.5.6细胞接种：将HEK293T细胞以4×105/mL接种于12孔细胞培养板中，在含有5％CO2的细胞培养箱中，37℃培养过夜。
1.5.7细胞共转染：次日，向200μL OPti-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Lipo 2000(Invitrogen,11668019)，共同孵育15min后逐滴加至细胞培养孔中。在含有5％CO2的细胞培养箱中， 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1.5kb大小的重链片段，表达载体pCDNA3.4(ThermoFisher Scientific，A14697)使用EcoRI/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pCDNA3.4-6G2-H-1。以轻链表达框为模板，扩增轻链，胶回收约0.8kb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pCDNA3.4-6G2-L-3。
1.7单抗的瞬时表达和亲和层析纯化
取15μg重链和15μg轻链表达载体混合后转染HEK293T细胞，按照说明书进行操作(Invitrogen，11668019)，4-5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3，在还原SDS-PAGE考马斯亮蓝染色检测结果中，重链和轻链的分子量分别为55kDa和25kDa，符合预期。
实施例2人源抗SARS-CoV-2单克隆抗体6G2与RBD的结合活性分析
2.1包被：取重组的RBD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，每次三分钟；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，每次三分钟；吸净孔内液体，加入PBS稀释后的纯化单抗6G2，首孔9μg/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4和表3。图4中，圆标曲线表示与RBD抗原的检测结果，显示特异结合呈现剂量反应关系。表3中，单抗6G2与RBD具有较高结合活性，说明单抗是 RBD特异结合的。
表3.单抗6G2与RBD的结合活性
| 抗原 | RBD |
| EC<sub>50</sub>(μg/mL) | 0.02834 |
实施例3SARS-CoV-2假病毒细胞模型上中和活性分析
3.1假病毒包装：将编码全长SARS-CoV-2S蛋白的基因插入pDC316载体，得到质粒pDC316-SARS-CoV-2-S。野生型及带有突变的假病毒参考序列如表4所示。将ACE2 基因稳定转染进HEK293T细胞，构建稳定表达人ACE2的细胞ACE2-293T细胞。将 5×106ACE2-293T细胞接种在10cm细胞培养皿中，并在37℃，5％CO2下生长过夜。用 Lipo 2000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48h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℃。
表4野生型及突变的假病毒参考序列
3.2稀释抗体：将抗体6G2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。
3.3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。
3.4将ACE2-293T细胞稀释到2×105cells/mL，每孔加入100μL，于37℃、5％CO2培养箱培养48h。
3.5检测Luciferase读值：弃每孔培养基，用PBS洗两遍，加入细胞裂解液40μL裂解30min，用枪头吹吸3次后吸取20μL转移到白色96孔板中，加入10μL底物，避光孵育30sec，使用Varioskan LUX multimode microplate reader(ThermoFisher Scientific)测Luciferase读值。
3.6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。
3.7结果：见图5(图5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％) 和表5。本发明公开的单抗6G2对SARS-CoV-2野生型及突变株假病毒都具有较好的中和活性，在野生型假病毒模型上EC50是33.95μg/ml(224.7nM)。
表5.单抗6G2在SARS-CoV-2假病毒细胞模型上中和活性分析
| Variants | WT | B.1.1.7 | B.1.525 | P.1 | B.1.617.2 | B.1.351 |
| EC<sub>50</sub>(μg/mL) | 33.95 | 124.1 | >150 | >150 | 6.569 | 4.12 |
实施例4SARS-CoV-2真病毒感染的细胞模型中和活性分析
4.1将Vero E6细胞用0.25％的胰酶消化后，用培养基(DMEM+10％FBS)稀释至 2×105cells/mL浓度，接种到96孔细胞培养板中，每孔100μL，置于37℃、5％的CO2细胞培养箱培养过夜。
4.2次日，将纯化单抗6G2用培养基DMEM+2％FBS自初始浓度，3倍稀释，加入 96孔培养板，每孔体积120μL；随即每孔加入120μL SARS-CoV-2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。
4.3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的细胞培养箱继续培养72h。
4.4 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，每孔加入200μL纯水，重复洗涤6次。
4.5弃洗液，用吸水纸拍干板孔中残留液体，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。
4.6单抗6G2在细胞模型上的保护效果见图6(图6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6G2在真病毒感染的细胞模型中 EC50为17.13μg/ml(114.5nM)。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 浙江安维珞诊断技术有限公司
<120> 一种抗SARS-CoV-2全人源化单克隆抗体及应用
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Claims (10)
1.一种靶向RBD的抗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26-33，51-57，96-113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27-33，51-53，89-98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：重链可变区的氨基酸序列如SEQ IDNO: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：重链恒定区的氨基酸序列如SEQ IDNO: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2或3所述的靶向RBD的抗SARS-CoV-2全人源单克隆抗体的多核苷酸，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所示。
6.一种表达权利要求2或3所述的靶向RBD的抗SARS-CoV-2全人源单克隆抗体的表达载体，其特征在于，所述载体含有权利要求4所述编码所述抗体重链的可变区的多核苷酸以及编码所述抗体轻链的可变区的多核苷酸。
7.一种表达权利要求2或3所述的靶向RBD的抗SARS-CoV-2全人源单克隆抗体的宿主细胞，其特征在于，所述宿主细胞含有权利要求6所述的表达载体。
8.根据权利要求7所述的宿主细胞，其特征在于，所述宿主细胞为HEK293T细胞。
9.一种用于治疗或预防新冠病毒感染的药物组合物，其包括权利要求1-3任一项所述的靶向RBD的抗SARS-CoV-2全人源单克隆抗体。
10.权利要求1-3任一项所述靶向RBD的抗SARS-CoV-2全人源单克隆抗体或权利要求9所述的药物组合物在制备检测新冠病毒产品或制备防治新冠病毒感染药物中的应用。
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| CN113512113A (zh) * | 2021-08-03 | 2021-10-19 | 浙江大学医学院附属第一医院 | 人源化广谱高中和活性抗新型冠状病毒单克隆抗体及应用 |
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