CN114195888B - 一种抗新冠抗体药物的核心序列 - Google Patents
一种抗新冠抗体药物的核心序列 Download PDFInfo
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- CN114195888B CN114195888B CN202111484814.3A CN202111484814A CN114195888B CN 114195888 B CN114195888 B CN 114195888B CN 202111484814 A CN202111484814 A CN 202111484814A CN 114195888 B CN114195888 B CN 114195888B
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Abstract
一种抗新冠抗体药物的核心序列。本发明获得了全新的抗新冠病毒S蛋白RBD单克隆抗体的核心序列，与现有技术抗新冠单抗的重链和轻链序列均完全不同。经实验证实，具有本发明公开的抗新冠病毒S蛋白RBD核心序列的单克隆抗体与新冠病毒S蛋白RBD及其突变体均具有高亲和力，可有效抑制新冠病毒S蛋白RBD及其突变体与ACE2的结合，对于新冠假病毒及其突变株假病毒均有明显的中和活性，可用于制备抗新冠病毒抗体药物。
Description
技术领域：
本发明涉及抗体药物制备，特别涉及一种抗新冠抗体药物的核心序列。
背景技术：
抗病毒单克隆抗体特异性的分子基础主要来自于它重链和轻链的高度可变区CDR1、CDR2和CDR3，这些区域是与抗原结合的关键部位。
因此获得单克隆抗体的重链和轻链的CDR1、CDR2和CDR3的序列，是制备抗体药物的关键。因为除了此核心区域外，抗体序列的其它位置都是框架区。在CDR区序列不变以及和抗原结合所需的三维结构不受影响的条件下，对框架区进行适当的突变，可以在不影响抗体的药物活性的同时减弱其免疫原性、提高亲和力从而提高抗体的治疗效果和安全性。
无论何种形式单克隆抗体，包括具有所需特异性的结合结构域的任意特异性结合因子，可以是单价的或是单链抗体、双链抗体、嵌合抗体以及上述抗体的衍生物、功能等同物和同源物，也包括抗体片段和含有抗原结合结构域的任何多肽。其重链和轻链中的高度可变区CDR1、CDR2和CDR3的序列都是制备抗体的核心和基础。
已知SARS-CoV-2的S蛋白是通过受体结合结构域(RBD)与宿主受体血管紧张素转换酶2(ACE2)结合，介导病毒进入宿主细胞。S蛋白所包含两个功能性亚基S1和S2中，S1负责与宿主细胞受体结合。S1由N端结构域(NTD)和受体结合结构域(RBD)组成，对决定组织嗜性和宿主范围至关重要。因此，提供可以有效与ACE2竞争结合的单克隆抗体的高度可变区CDR1、CDR2和CDR3的核心序列，有望制备治疗新型冠状病毒相关疾病的药物。
发明内容
本发明的目的是制备治疗新型冠状病毒相关疾病的药物，具体是提供可以有效与ACE2竞争结合的单克隆抗体的核心序列。
以下所称新冠病毒S蛋白RBD指SARS-CoV-2(COVID-19)S protein RBD。
本发明获得了全新的抗新冠病毒S蛋白RBD单克隆抗体的核心序列，与现有技术抗抗新冠病毒单克隆抗体的重链和轻链序列均完全不同。
本发明提供的抗新冠病毒S蛋白RBD单克隆抗体的核心序列，重链和轻链的CDR1、CDR2和CDR3分别是如下所示氨基酸序列的多肽：
重链：SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3；
轻链：SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6；
根据上述抗新冠病毒S蛋白RBD单克隆抗体的核心序列可以制备有效抑制新冠病毒S蛋白RBD及其突变体的抗体药物。
具体的，本发明通过如下手段得到了上述抗新冠病毒S蛋白RBD单克隆抗体的核心序列：
A新冠病毒S蛋白RBD蛋白作为抗原以30μg/只免疫BALB/C小鼠，三周后同样剂量加强免疫；
B ELISA法测定免疫后小鼠血清中的抗体滴度，达到理想效果后以50μg剂量冲击免疫；
C取免疫成功的小鼠脾细胞与SP2/0细胞进行电融合，待细胞长成细胞团后进行上清滴度检测，通过三轮亚克隆获得阳性单克隆细胞株；
D单克隆细胞株扩大培养后进行小鼠腹腔注射制备腹水，并从收集的腹水纯化得到相应的抗体；
E利用SPR技术进行单克隆抗体的亲和动力学测定；
F测定单克隆抗体对新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2结合的竞争抑制作用；
G测定单克隆抗体对于新冠假病毒及其突变株(Delta)假病毒的中和活性。
本发明获得的抗新冠病毒S蛋白RBD单克隆抗体命名为B2-33-B10(以下或称“抗体B2-33-B10”)。此抗体特异性的分子基础主要来自于它的高度可变区CDR1、CDR2和CDR3，这些区域是与抗原结合的关键部位。
本发明所述“单克隆抗体”应该解释为涵盖具有所需特异性的结合结构域的任意特异性结合因子，可以是单价的或是单链抗体、双链抗体、嵌合抗体以及上述抗体的衍生物、功能等同物和同源物，也包括抗体片段和含有抗原结合结构域的任何多肽。
本发明所述单克隆抗体的实例是免疫球蛋白IgG亚型及其亚型亚类；
尽管抗体特异性的分子基础主要来自于它的高度可变区CDR1、CDR2和CDR3，为了维持优选的结合特性，上述CDR的序列应尽可能保留。然而，尽管如果有个别氨基酸的改变，也有可能能够达到本发明目的，甚至使结合特性更为优化。但是，个别氨基酸的改变并不能脱离本发明的构思和发明精神。
除了如上所述重链和轻链中的高度可变区CDR1、CDR2和CDR3外，其它为框架区。框架区可在结合所需的三维结构不受影响的条件下被其他序列置换。
本发明的有益效果：
经实验证实，具有本发明公开的抗新冠病毒S蛋白RBD核心序列的单克隆抗体，有以下突出特点：
1、所述抗体与新冠病毒S蛋白RBD及其突变体(L452R，T478K)均具有高亲和力(详见实施例2)；
2、与ACE2结合的竞争抑制实验显示，所述抗体可有效抑制新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2的结合(详见实施例3)；
3、中和实验显示，所述抗体对于新冠假病毒及其突变株(Delta)假病毒均有明显的中和活性(详见实施例4)。
附图说明
图1、图2分别为实施例2中B2-33-B10抗体与新冠病毒S蛋白RBD及其突变体(L452R，T478K)的亲和力的检测结果，图中Ka、Kd和KD分别为结合常数、解离常数以及亲和力常数；
图3、图4分别为实施例3中B2-33-B10抗体对新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2结合的竞争抑制作用的实验结果；
图5、图6分别为实施例4中B2-33-B10抗体对新冠假病毒及其突变株(Detla)假病毒的中和活性检测的实验结果。
以上所述B2-33-B10是本发明得到的抗新冠病毒S蛋白RBD单克隆抗体的名称。
序列信息:
SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3，分别是抗新冠病毒S蛋白RBD单克隆抗体B2-33-B10重链可变区的CDR1、CDR2和CDR3；
SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6，分别是抗新冠病毒S蛋白RBD单克隆抗体B2-33-B10轻链可变区的CDR1、CDR2和CDR3；
SEQ ID NO.7、SEQ ID NO.8，分别是抗新冠病毒S蛋白RBD单克隆抗体B2-33-B10重链可变区和轻链可变区的氨基酸序列；
具体实施方式
为使本发明的目的、技术方案及效果更加清楚、明确，以下实施例对本发明进行了进一步详细说明。应当理解，实施例中所使用的具体方法、试剂等仅用于举例说明本发明，并不能限定本发明的范围；
本发明提供特异性的抗新冠病毒S蛋白RBD单克隆抗体的重链和轻链序列。所述单克隆抗体由新冠病毒S蛋白RBD蛋白免疫BALB/c小鼠后经杂交瘤筛选得到的对应的单克隆细胞株表达；所述单克隆抗体型别为IgG型；
以下实施例中所用的抗原为购买自北京百普赛斯生物科技股份有限公司的新冠病毒S蛋白RBD(货号：SPD-C52H3)以及其突变体蛋白(货号：SPD-C52Hh)，蛋白C端包含6×His标签；
所使用的免疫佐剂为北京博奥龙免疫技术有限公司的5周快速免疫佐剂(货号：KX0210041)，初次免疫21天后加强免疫一次，细胞融合前使用抗原冲击免疫一次即可进行细胞融合；
所使用的融合方法为电融合法，电融合仪为BTX公司的ECM2001型，所用融合缓冲液为原厂细胞融合液(货号：47-0001)；
融合后的细胞长出细胞团后采用ELISA检测上清抗体表达，酶标板包被新冠病毒S蛋白RBD蛋白或者His蛋白，His蛋白用来排除Anti-His的假阳性孔；
阳性孔采用有限稀释法进行亚克隆，亚克隆共进行三轮，最终获得阳性单克隆细胞株；
阳性单克隆进行腹水制备后纯化，获得对应的单克隆抗体，单克隆抗体进一步用于亲和力测定、竞争结合实验以及假病毒中和实验。
以下各实施例中提到的B2-33-B10，为本发明提供的抗新冠病毒S蛋白RBD单克隆抗体的代号。
实施例1抗原免疫、细胞融合以及阳性克隆的筛选和腹水抗体的制备纯化
实验目的：
采用购买自北京百普赛斯生物科技股份有限公司的新冠病毒S蛋白RBD蛋白作为抗原制备单克隆抗体。
实验方法：
使用杂交瘤技术制备抗新冠病毒S蛋白RBD单克隆抗体。具体方法为：
使用30μg蛋白对4-6周的雌性BALB/C小鼠进行免疫；
首次免疫后第21天采用同样方法加强免疫一次；
首次免疫后第35天通过眼眦采血分离血清进行血清滴度的ELISA测定；
抗体滴度达到要求后采用50μg新冠病毒S蛋白RBD蛋白进行抗原冲击免疫；
冲击免疫3天后取脾细胞与SP2/0细胞进行融合，待细胞成团后ELISA检测杂交瘤上清液中的抗新冠病毒S蛋白RBD抗体。
实验结果：
经过三轮亚克隆，通过亲和力以及生化水平竞争结合实验双重筛选，最终获得了新冠病毒S蛋白RBD抗体高表达单克隆细胞株，命名为B2-33-B10。将单克隆细胞扩增后进行腹水制备纯化抗体，用于后续亲和力测定、竞争结合实验以及假病毒中和实验。
经验证，所得单克隆抗体B2-33-B10具有高亲和力，可以有效竞争抑制新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2的结合，同时，假病毒中和实验显示了抗体对新冠假病毒及其突变株(Detla)假病毒均具有良好的中和活性。
实施例2单克隆抗体B2-33-B10与新冠病毒S蛋白RBD及其突变体(L452R，T478K)的亲和动力学分析
实验目的：
采用Biacore T200系统检测单克隆抗体B2-33-B10的亲和动力学常数。
试剂和方法：
Mouse Antibody Capture Kit为购自GE公司的商品化试剂盒，采用氨基偶联的方法将抗小鼠Fc IgG固定在CM5传感器芯片上，通过偶联的抗小鼠Fc IgG捕获单克隆抗体B2-33-B10，然后注射一系列浓度梯度的新冠病毒S蛋白RBD及其突变体(L452R，T478K)蛋白，每个循环结束后采用试剂盒自带的pH 1.7的Glycine-Hcl再生芯片。
运行缓冲液为HBS-EP+(10mM HEPES，pH7.4，150mM Nacl，3mM EDTA和0.05％P20)，测定温度25℃；
使用Biacore T200评估软件，根据1：1结合模型拟合数据，计算结合(Ka)和解离(Kd)速率常数以及平衡常数(KD)。
实验结果：
根据检测结果，抗体亲和力数据的具体结果见图1、图2和下表：
实验结论：
本发明获得的单克隆抗体B2-33-B10与新冠病毒S蛋白RBD及其突变体(L452R，T478K)均具有高亲和力。
实施例3单克隆抗体B2-33-B10对新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2结合的竞争抑制作用
实验目的：
从生化水平检测单克隆抗体B2-33-B10对新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2结合的竞争抑制。
实验方法：
将ACE2蛋白以2μg/mL，100μL/孔的量包被至酶标板中，4℃包被过夜；次日，将酶标板中的包被液弃掉，PBST洗涤3次后用2％BSA封闭液于37℃微孔板恒温振荡器中孵育1.5h；1.5h后弃掉封闭液，PBST洗涤3次，加入配好的0.25μg/mL新冠病毒S蛋白RBD或其突变体(L452R，T478K)和2倍浓度的单克隆抗体B2-33-B10梯度液(4、2、1、0.5、0.25、0.125、0.0625、0μg/mL)至酶标板中，每孔各加50μL，用封口膜封好，37℃恒温孵育箱中孵育1h；1h后弃掉液体，PBST洗涤4次，将Anti His-HRP抗体按照1：4000的比例稀释后加入到酶标板中，每孔100μL，用封口膜封好，37℃恒温孵育箱中孵育1h；1h后弃掉液体，PBST洗涤4次，每孔加入100μL显色液，显色15-20min；显色完成后加入终止液，每孔50μL；450nm的波长下测量酶标板各孔的OD值。并将实验结果用GraphPad Prism 6软件进行处理分析。
实验结果：见图3、图4；
各抗体竞争抑制效应IC50汇总见下表：
| 抗体名称 | S-RBD | S-RBD(L452R-T478K) | |
| EC50(μg/mL) | B2-33-B10 | 0.09627 | 0.1015 |
实验结论：
本发明获得的单克隆抗体B2-33-B10对新冠病毒S蛋白RBD及其突变体(L452R，T478K)与ACE2结合具有明显的竞争抑制作用。
实施例4单克隆抗体B2-33-B10对新冠假病毒及其突变株(Detla)假病毒中和活性检测
实验目的：测试单克隆抗体B2-33-B10对新冠假病毒及其突变株(Detla)假病毒的中和活性。
新冠原始假病毒购自北京博奥龙免疫技术有限公司(货号BDAA0026),
新冠假病毒Delta突变株购自复百澳(苏州)生物科技有限公司(货号：FNV3718)
实验方法：
取对数生长期的HEK293-ACE2细胞，以6×105/孔的量铺至96孔白板中，37℃，5％CO2培养箱中培养过夜；第二天，取样品，按照实验设计稀释样品，样品稀释完成后从液氮罐取出假病毒，37℃水浴快速轻柔解冻后置于冰上；稀释好的单克隆抗体B2-33-B10样品与假病毒在96孔板混合，然后室温孵育1h；孵育完成后将混合液加至前一天预先铺好HEK293-ACE2细胞的96孔白板中，放入37℃，5％CO2培养箱中继续培养48h；48h后弃去孔板内的培养液，每孔加入50μL荧光素酶显色液，室温孵育5min，多功能酶标仪化学发光法读取数值。
实验结果：见图5、图6；
各抗体新冠假病毒中和效应IC50汇总见下表：
| 抗体名称 | FNV-SARS-CoV-2-S | B.1.617.2(Delta) | |
| EC50(ng/mL) | B2-33-B10 | 43.00 | 283.6 |
实验结论：
本发明获得的单克隆抗体B2-33-B10对新冠假病毒及其突变株(Detla)假病毒均具有明显的中和抑制活性。
序列表
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Claims (2)
1.一种抗SARS-CoV-2 (COVID-19) S protein RBD单克隆抗体，其特征在于：重链可变区中的CDR1、CDR2 和CDR3分别是如下氨基酸序列的多肽：SEQ ID NO.1、SEQ ID NO.2和SEQID NO.3；轻链可变区中的CDR1、CDR2 和CDR3分别是如下氨基酸序列的多肽：SEQ ID NO.4、SEQ ID NO.5和SEQ ID NO.6。
2.权利要求1所述的单克隆抗体在制备新型冠状病毒SARS-CoV-2 (COVID-19)及其Detla突变株中和抗体药物中的应用。
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