CN111647076B - 抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用 - Google Patents
抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用 Download PDFInfo
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Abstract
本发明涉及抗新型冠状病毒SARS‑Cov‑2的中和性单域抗体及其应用。该抗体至少具有重链CDR1、重链CDR2、重链CDR3之一。该抗体可用于制备针对COVID‑19的诊断试剂或诊断试剂盒、抗体药物或药物组合物。本发明通过噬菌体展示技术获得了抗新型冠状病毒SARS‑Cov‑2的中和性单域抗体,该抗体能够阻断SARS‑Cov‑2‑RBD与ACE2阳性细胞的结合,对SARS‑Cov‑2伪病毒具有显著的病毒中和作用,为COVID‑19的预防和治疗提供了有效的备选抗体药物。
Description
技术领域
本发明涉及一种抗新型冠状病毒SARS-Cov-2的中和性单域抗体及其应用,属于生物医药技术领域。
背景技术
COVID-19由新型冠状病毒(Severe Acute Respiratory Syndrome Coronavirus2,SARS-CoV-2)感染诱发。SARS-CoV-2为正链单股RNA病毒,直径约80-120nm,是β冠状病毒属(Betacoronavirus)的成员[1],与SARS-CoV同属于Sarbecovirus亚属,核酸同源性为79.5%[2]。目前SARS-CoV-2病毒的来源和天然宿主尚未明确,无法采取有效的措施来预防和阻止此病毒传播。
现有研究表明,与其他冠状病毒一样,SARS-Cov-2的宿主受体为血管紧张素转换酶2(ACE2)[3],在肺部组织和小肠组织表达丰富[4]。SARS-CoV-2感染人体时,会通过病毒外壳上Spike蛋白S1亚基的受体识别结构域(receptor binding domain,RBD)与ACE2结合,诱发S2亚基结构的改变,进而促进病毒与宿主细胞膜的融合,介导病毒入侵宿主细胞[5]。
Spike蛋白以三聚体形式表达于病毒外壳上,每个单体结构由一个S1亚基和一个S2亚基组成。已经有研究发现,Spike蛋白三聚体上的S1蛋白的RBD存在“关闭”和“开放”的不同状态。当处于“开放”状态时,三聚体中的一个RBD处于伸展状态[6],这种构象的精细改变介导了Spike蛋白与ACE2的识别与结合。
单域抗体(single domain antibody,sdAb),即重链单域抗体VHH,仅包括抗体重链的可变区。单域抗体分子量小,蛋白表达水平较高,免疫原性弱、易于生产制备[7]等优势。更重要的是,单域抗体能识别抗原表面隐蔽的精细结构,可精确地瞄准、捕获目标靶点,特异性地与靶分子结合[8]。
目前,尚无SARS-CoV-2病毒特异性的疫苗和中和性抗体应用于临床。因此,通过快速、高效的SARS-Cov-2中和性抗体筛选,获得具有中和作用的人源单克隆抗体,是预防和治疗COVID-19的迫切需要。
发明内容
本发明的主要目的是:克服现有技术存在的问题,提供一种抗新型冠状病毒SARS-Cov-2的中和性单域抗体,具有针对新型冠状病毒SARS-Cov-2的高效抗病毒能力。同时,还提供该抗体的应用。
本发明解决其技术问题的技术方案如下:
一种抗新型冠状病毒SARS-Cov-2的中和性单域抗体,所述单域抗体由重链构成,其特征是,所述抗体至少具有以下技术特征之一:
i、所述重链包括重链CDR1,其氨基酸序列为:X1-F-X2-F-X3-X4-Y,其中,X1为D或S,X2为A、D或Y,X3为S或A,X4为S或D;
ii、所述重链包括重链CDR2,其氨基酸序列为:I-G-X5-I-X6-H-S-G-S-T-Y-Y-N-P-S-L-K-S-X7-V,其中,X5为E或S,X6为H或Y,X7为L或R;
iii、所述重链包括重链CDR3,其氨基酸序列为:VKDFGHLGQMAS、VKDLGFADH、VKDFVVGETAEFSY、或AREWHSGYDY。
优选地,所述重链CDR1的氨基酸序列为:DFAFSSY、SFDFSSY、SFDFSDY、或DFYFADY;
所述重链CDR2的氨基酸序列为:IGEIHHSGSTYYNPSLKSLV、IGEIHHSGSTYYNPSLKSRV、IGEIHHSGSTYYNPSLKSRV、或IGSIYHSGSTYYNPSLKSRV。
优选地,所述重链包括重链CDR1、重链CDR2以及重链CDR3;
当重链CDR1为DFAFSSY时,重链CDR2为IGEIHHSGSTYYNPSLKSLV,且重链CDR3为VKDFGHLGQMAS;
当重链CDR1为SFDFSSY时,重链CDR2为IGEIHHSGSTYYNPSLKSRV,且重链CDR3为VKDLGFADH;
当重链CDR1为SFDFSDY时,重链CDR2为IGEIHHSGSTYYNPSLKSRV,且重链CDR3为VKDFVVGETAEFSY;
当重链CDR1为DFYFADY时,重链CDR2为IGSIYHSGSTYYNPSLKSRV,且重链CDR3为AREWHSGYDY。
优选地,所述单域抗体的氨基酸序列如SEQ ID NO:5至SEQ ID NO:8之一所示。
优选地,所述重链具有标记,所述标记包括荧光标记、酶标记、以及放射性标记。
本发明还提供:
编码前文所述抗新型冠状病毒SARS-Cov-2的中和性单域抗体的核酸。
优选地,所述核酸的序列如SEQ ID NO:1至SEQ ID NO:4之一所示。
本发明还提供:
前文所述抗新型冠状病毒SARS-Cov-2的中和性单域抗体用于制备诊断试剂或诊断试剂盒、药物或药物组合物的用途。
前文所述核酸用于制备抗新型冠状病毒SARS-Cov-2的中和性单域抗体、药物或药物组合物的用途。
其中,所述药物或药物组合物具有针对新型冠状病毒SARS-Cov-2的中和性抗病毒作用。
本发明通过噬菌体展示技术获得了抗新型冠状病毒SARS-Cov-2的中和性单域抗体,该抗体能够阻断SARS-Cov-2-RBD与ACE2阳性细胞的结合,对SARS-Cov-2伪病毒具有显著的病毒中和作用,为COVID-19的预防和治疗提供了有效的备选抗体药物,具有潜在的临床应用前景。
附图说明
图1为本发明实施例1中ELISA检测富集噬菌体对抗原蛋白的结合情况图。
图2为本发明实施例2中噬菌体对SARS-Cov-2-RBD-hFc蛋白的特异性结合检测(ELISA)图。
图3为本发明实施例3的表达载体示意图。
图4为本发明实施例3的SDS-PAGE结果图。
图5为本发明实施例4的亲和力分析结果图。
图6为本发明实施例5的单域抗体阻断效应分析图。
图7为本发明实施例6的单域抗体抗伪病毒中和效应评估图。
具体实施方式
下面结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。所用方法如无特别说明均为常规方法,所用试剂和材料如无特别说明均为市售品。
实施例1、筛选靶向SARS-Cov-2-RBD的全人源单域抗体
以Tomlinson I&J噬菌体文库(Genservice Ltd.,Cambridge,UK,文库大小为1.47×108)的抗体序列为模板,通过PCR扩增该文库的抗体重链序列,构建单域抗体噬菌体文库。
采用噬菌体展示技术,以SARS-Cov-2-RBD his(Arg330-Val524)蛋白为阳性抗原,以SARS-Cov-2-RBD mut-hFC为阴性抗原进行筛选。
分别使用50μg/ml的上文所述SARS-Cov-2-RBD his抗原、SARS-Cov-2-RBD mut-hFc于4℃包被免疫板过夜;用含有5%脱脂奶粉,0.1%Tween-20的PBS溶液室温封闭免疫板1小时;将上文获得的单域抗体噬菌体文库以1012pfu与10%脱脂奶粉PBS溶液1:1混合后室温孵育2小时,加入封闭好的SARS-Cov-2-RBD mut-hFc抗原免疫板中(100μl/孔),室温孵育1小时,进行阴性抗原预吸附;预吸附后上清转移至加入封闭好的SARS-Cov-2-RBD his抗原免疫板中(100μl/孔),室温孵育1小时。用0.1%Tween-20的PBS溶液洗涤免疫板20次;100μl100mM Triethylamine室温洗脱30分钟;洗脱的噬菌体感染对数生长期的TG1细胞,扩增和回收后用于下一轮淘选。淘选后ELISA分析阳性噬菌体富集情况。
ELISA检测的具体过程为:以5μg/ml的上文所述SARS-Cov-2-RBD his抗原和阴性对照蛋白GPC5 his分别于4℃包被免疫板过夜;用含有3%脱脂奶粉,0.1%Tween-20的PBS溶液室温封闭免疫板1小时;将每一轮富集的噬菌体扩增并回收,以1:1比例与6%脱脂奶粉PBS室温孵育2小时,加入封闭好的免疫板中(100μl/孔),室温孵育1小时;用0.1%Tween-20的PBS溶液洗涤免疫板5次;将HRP/Anti-M13 Monoclonal conjugate以1:4000比例与含5%脱脂奶粉,0.05%Tween-20的PBS溶液混合,加入洗涤好的免疫板中(50μl/孔),室温孵育1小时;用0.05%Tween-20的PBS溶液洗涤免疫板5次;将TMB显色液加入免疫板中(100μl/孔),室温显色3分钟后,加入0.5M硫酸终止显色(100μl/孔);用酶联免疫检测仪在450nm波长下检测吸光值,并分析每轮扩增后噬菌体的亲和力。
结果如图1所示,在四轮富集后,富集的噬菌体群对SARS-Cov-2-RBD his抗原的亲和力显著升高。
在第四轮富集的噬菌体群中随机挑取单克隆,检测它们对SARS-Cov-2-RBD his抗原的结合特性,结果发现,有4条抗体序列发生了富集,分别为4A12,4D5,4A10和4C5。
经测序鉴定,4个抗体的DNA序列分别如SEQ ID NO:1至4所示,氨基酸序列分别如SEQ ID NO:2至8所示。
DNA序列:
抗体4A12,SEQ ID NO:1,
gaggtgcagctgttggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctctgatttcgctttctcttcttatgaaatgagctgggtccgccaggctccagggaagggcctagagtggattggggaaatccatcatagtgggagcacctactacaacccgtccctcaagagtctagtcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccgtatattactgtgtgaaagatttcgggcacctcggtcaaatggcctcctggggccagggaaccctggtcaccgtctcctca。
抗体4D5,SEQ ID NO:2,
gaggtgcagctgttggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctcttctttcgatttctcttcttatgaaatgagctgggtccgccaggctccagggaaggccctggagtggattggggaaatccatcatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccatgtattactgtgtgaaggatttggggtttgcggaccactggggccagggaaccctggtcaccgtctcctca。
抗体4A10,SEQ ID NO:3,
gaggtgcagctgttggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctcttctttcgatttctctgattatgaaatgagctgggtccgccaggctccagggaagggtctagagtggattggggaaatccatcatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccacgtattactgtgtgaaagattttgtagtgggagaaaccgcggagttttcgtattggggccagggaaccctggtcaccgtctcctca。
抗体4C5,SEQ ID NO:4,
gaggtgcagctgttggagtctgggggaggcttggtacagcctggagggtccctgagactctcctgtgcagcctctgatttctatttcgctgattatgaaatgagctgggtccgccaggctccagggaaggggctagagtggattgggagtatctatcatagtgggagcacctactacaacccgtccctcaagagtcgagtcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacagccatgtattactgtgcaagagaatggcatagtggctatgactactggggccagggaaccctggtcaccgtctcctca。
氨基酸序列:
抗体4A12,SEQ ID NO:5,
EVQLLESGGGLVQPGGSLRLSCAASDFAFSSYEMSWVRQAPGKGLEWIGEIHHSGSTYYNPSLKSLVTISRDNSKNTLYLQMNSLRAEDTAVYYCVKDFGHLGQMASWGQGTLVTVSS。
该氨基酸序列中,氨基酸残基26-32(即DFAFSSY)为重链CDR1,氨基酸残基48-67(即IGEIHHSGSTYYNPSLKSLV)为重链CDR2,氨基酸残基96-107(即VKDFGHLGQMAS)为重链CDR3。
抗体4D5,SEQ ID NO:6,
EVQLLESGGGLVQPGGSLRLSCAASSFDFSSYEMSWVRQAPGKALEWIGEIHHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCVKDLGFADHWGQGTLVTVSS。
该氨基酸序列中,氨基酸残基26-32(即SFDFSSY)为重链CDR1,氨基酸残基48-67(即IGEIHHSGSTYYNPSLKSRV)为重链CDR2,氨基酸残基96-104(即VKDLGFADH)为重链CDR3。
抗体4A10,SEQ ID NO:7,
EVQLLESGGGLVQPGGSLRLSCAASSFDFSDYEMSWVRQAPGKGLEWIGEIHHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTATYYCVKDFVVGETAEFSYWGQGTLVTVSS。
该氨基酸序列中,氨基酸残基26-32(即SFDFSDY)为重链CDR1,氨基酸残基48-67(即IGEIHHSGSTYYNPSLKSRV)为重链CDR2,氨基酸残基96-109(即VKDFVVGETAEFSY)为重链CDR3。
抗体4C5,SEQ ID NO:8,
EVQLLESGGGLVQPGGSLRLSCAASDFYFADYEMSWVRQAPGKGLEWIGSIYHSGSTYYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAMYYCAREWHSGYDYWGQGTLVTVSS。
该氨基酸序列中,氨基酸残基26-32(即DFYFADY)为重链CDR1,氨基酸残基48-67(即IGSIYHSGSTYYNPSLKSRV)为重链CDR2,氨基酸残基96-105(即AREWHSGYDY)为重链CDR3。
实施例2、单域抗体的抗原特异性分析
本实施例采用ELISA检测实施例1的4A12、4D5、4A10和4C5噬菌体与SARS-Cov-2-RBD-hFc蛋白的结合情况。
具体过程为:分别使用5μg/ml的SARS-Cov-1-RBD-hFc、SARS-Cov-2-RBD-hFc、SARS-Cov-2-RBD mut-hFc于4℃包被免疫板过夜;用含有3%脱脂奶粉,0.05%Tween-20的PBS溶液室温封闭免疫板1小时;分别将4A12,4D5,4A10,4C5的噬菌体加入各封闭好的免疫板中(50μl/孔),室温孵育1小时;用0.05%Tween-20的PBS溶液洗涤免疫板3次(340ul/孔);将HRP/Anti-M13 Monoclonal conjugate以1:4000比例与含5%脱脂奶粉,0.05%Tween-20的PBS溶液混合,加入洗涤好的免疫板中(50μl/孔),室温孵育1小时;用0.05%Tween-20的PBS溶液洗涤免疫板5次;将TMB显色液加入免疫板中(100μl/孔),室温显色3分钟后,加入0.5M硫酸终止显色(100μl/孔);用酶联免疫检测仪在450nm波长下检测吸光值,并分析每轮扩增后噬菌体的亲和力。
如图2所示,结果显示4A12、4D5、4A10、4C5抗体特异性识别SARS-Cov-2-RBD-hFc蛋白,但不识别SARS-Cov-1-RBD-hFc以及SARS-Cov-2-RBD mut-hFc蛋白。
实施例3、单域抗体的表达与纯化
构建实施例1各抗体的真核细胞表达载体pFUSE-4A12-hFC,pFUSE-4D5-hFC,pFUSE-4A10-hFC,pFUSE-4C5-hFC(Invivigen,San Diego,CA),如图3所示。在293T细胞中转染后收集上清,并用protein A-Agarose分离柱进行纯化,SDS-PAGE检测抗体的纯度,如图4所示。
具体过程为:将抗体4A12,4D5,4A10,4C5的VH序列克隆到表达载体pFUSE-4A12-hFc,pFUSE-4D5-hFc,pFUSE-4A10-hFc,pFUSE-4C5-hFc中(Invivigen,San Diego,CA)中,制备质粒。用添加10%胎牛血清、100U/ml青霉素、0.1mg/ml链霉素的DMEM培养基在细胞培养皿中种5百万个HEK293T细胞,置于5%CO2,37℃培养箱中培养。当细胞密度到达60-80%时,利用PEI将10μg pFUSE-4A12-hFc,pFUSE-4D5-hFc,pFUSE-4A10-hFc,pFUSE-4C5-hFc质粒转染入HEK293T细胞;收集上清。
将收集的上清液在3500rpm、4℃条件下离心20分钟,并用0.45μm的微孔滤膜抽滤,进一步去除碎片;将上清液通过Protein A-Agarose(GE Healthcare,Piscataway,NJ)亲和柱分离纯化4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc重组蛋白。通过BCA法测定蛋白浓度,并将3μg 4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc重组蛋白进行聚丙烯酰胺凝胶电泳,得到4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc重组蛋白的条带。
实施例4、单域抗体亲和力分析
利用ELISA实验,测定4A12、4D5、4A10、4C5抗体与SARS-Cov-2-RBD his蛋白的亲和力。
具体过程为:使用5μg/ml的SARS-Cov-2-RBD his蛋白于4℃包被免疫板过夜;用含有3%脱脂奶粉,0.05%Tween-20的PBS溶液室温封闭免疫板1小时;将4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc蛋白用含有3%脱脂奶粉,0.05%Tween-20的PBS溶液分别稀释成20,10,5,2.5,1.25,0.625,0.3125,0.15625,0.078125,0.0390625,0.01953125,0.009765625ug/ml(倍比稀释),加入封闭好的免疫板中(50μl/孔),室温孵育1小时;用0.05%Tween-20的PBS溶液洗涤免疫板3次(340ul/孔);将goat anti-human Fcγ-HRP以1:2000比例与3%脱脂奶粉,0.05%Tween-20的PBS溶液混合,加入洗涤好的免疫板中(50ul/孔),室温孵育1小时;用0.05%Tween-20的PBS溶液洗涤免疫板3次;将TMB显色液加入免疫板中(100ul/孔),室温显色3分钟后,加入0.5M硫酸终止显色(100ul/孔);使用酶联免疫检测仪在450nm波长下检测吸光值并拟合亲和力曲线分析4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc重组蛋白亲和力。
如图5所示,结果显示4A12、4D5、4A10、4C5抗体与SARS-Cov-2-RBD his蛋白的亲和力分别为2.69nM、2.20nM、1.65nM、1.58nM。
实施例5、单域抗体对SARS-Cov-2-RBD与ACE2-CHO细胞结合的阻断
提前将4A12-hFc,4D5-hFc,4A10-hFc,4C5-hFc,Ctrl,M396抗体(此为SARS-Cov-1中和性抗体)与SARS-Cov-2-RBD-hFc蛋白室温预孵育1小时后,将106个ACE2-CHO细胞加入到混合体系中,冰上孵育1小时。PBS洗涤细胞,1:200加入Goat anti-human PE,冰上孵育1小时。PBS洗涤细胞,BD FACS Calibur上机检测SARS-Cov-2-RBD-hFc与ACE2-CHO细胞结合情况。
结果如图6所示,各单域抗体与SARS-Cov-2-RBD蛋白孵育后,能够显著抑制SARS-Cov-2-RBD与ACE2-CHO细胞的结合。
实施例6、病毒中和实验
在慢病毒包装系统中将VSV-G蛋白基因置换为SARS-Cov-2spike基因,与pLVX-EGFP-Luciferase报告基因共转染293T细胞(即以伪病毒转染),收取48小时病毒上清,1:1稀释后待用。将ACE2-CHO细胞按104/孔接种于96孔板中培养过夜。预先将梯度稀释的各单域抗体与病毒上清37℃共孵育1小时后,加入到ACE2-CHO细胞培养板中。48小时后,检测Luciferase活性。
如图7所示,结果表明4A12、4D5、4A10、4C5抗体能够显著抑制伪病毒对ACE2-CHO细胞的感染,IC50分别为:0.19μg/ml,1.13μg/ml,0.66μg/ml,2.32μg/ml。注:图中的31A2是指发明人已申请发明专利的抗Galectin-3的全人源化单域抗体31A2。
本发明抗体可具有标记,如,荧光标记、酶标记、放射性标记等。
本发明的各单域抗体能特异性结合/识别SARS-Cov-2-RBD his抗原,对SARS-Cov-2-RBD his蛋白有较好的亲和力,能够有效抑制SARS-Cov-2入侵细胞,具有成为COVID-19预防和治疗药物的重要应用价值。
除上述实施例外,本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案,均落在本发明要求的保护范围。
参考文献
[1].Zhu,N.,et al.,A Novel Coronavirus from Patients with Pneumonia inChina,2019.N Engl J Med,2020.382(8):727-733.
[2].Zhou,P.,et al.,A pneumonia outbreak associated with a newcoronavirus of probable bat origin.Nature,2020.579(7798):270-273.
[3].Lan,J.,et al.,Structure of the SARS-CoV-2 spike receptor-bindingdomain bound to the ACE2 receptor.Nature,2020.
[4].Zou,X.,et al.,Single-cell RNA-seq data analysis on the receptorACE2 expression reveals the potential risk of different human organsvulnerable to 2019-nCoV infection.Front Med,2020.
[5].Hoffmann,M.,et al.,SARS-CoV-2 Cell Entry Depends on ACE2 andTMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.Cell,2020.181(2):271-280 e278.
[6].Walls,A.C.,et al.,Structure,Function,and Antigenicity of theSARS-CoV-2 Spike Glycoprotein.Cell,2020.181(2):281-292.e286.
[7].Muyldermans,S.,Nanobodies:natural single-domain antibodies.AnnuRev Biochem,2013.82:775-797.
[8].Ingram,J.R.,F.I.Schmidt,and H.L.Ploegh,Exploiting Nanobodies'Singular Traits.Annu Rev Immunol,2018.36:695-715.
序列表
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Claims (6)
1.一种抗新型冠状病毒SARS-Cov-2的中和性单域抗体,所述单域抗体由重链构成,其特征是,所述抗体具有以下全部技术特征:
所述重链包括重链CDR1、重链CDR2以及重链CDR3;
重链CDR1为DFAFSSY,重链CDR2为IGEIHHSGSTYYNPSLKSLV,且重链CDR3为VKDFGHLGQMAS。
2.根据权利要求1所述的中和性单域抗体,其特征是,所述单域抗体的氨基酸序列如SEQ ID NO:5所示。
3.根据权利要求1所述的中和性单域抗体,其特征是,所述重链具有标记,所述标记包括荧光标记、酶标记、以及放射性标记。
4.编码权利要求1至3任一项所述抗新型冠状病毒SARS-Cov-2的中和性单域抗体的核酸。
5.根据权利要求4所述的核酸,其特征是,所述核酸的序列如SEQ ID NO:1所示。
6.权利要求1至3任一项所述抗新型冠状病毒SARS-Cov-2的中和性单域抗体用于制备诊断试剂或诊断试剂盒、药物的用途。
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WO2023076881A1 (en) * | 2021-10-26 | 2023-05-04 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Single domain antibodies targeting the s2 subunit of sars-cov-2 spike protein |
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