CN115160434A - 人源化单域抗体及其应用和药物 - Google Patents
人源化单域抗体及其应用和药物 Download PDFInfo
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- CN115160434A CN115160434A CN202210582152.1A CN202210582152A CN115160434A CN 115160434 A CN115160434 A CN 115160434A CN 202210582152 A CN202210582152 A CN 202210582152A CN 115160434 A CN115160434 A CN 115160434A
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Abstract
本发明公开了一种人源化单域抗体及其应用和药物,涉及抗体技术领域。本发明公开的人源化单域抗体具有SEQ ID NO.1所示氨基酸序列。该人源化单域抗体具有较低的免疫源性,并保持或提高了对新型冠状病毒的中和活性,可用于制备新型冠状病毒药物,为新型冠状病毒预防和治疗提供新的策略和手段。
Description
技术领域
本发明涉及抗体技术领域,具体而言,涉及一种人源化单域抗体及其应用和 药物。
背景技术
目前,需要开发针对新型冠状病毒的药物。
发明内容
本发明的目的在于提供一种人源化单域抗体及其应用和药物。该人源化单域 抗体具有较低的免疫源性,并保持或提高了对新型冠状病毒的中和活性,可用于 制备治疗新型冠状病毒感染疾病的药物,为新型冠状病毒预防和治疗提供新的策 略和手段。
本发明是这样实现的:
第一方面,本发明提供一种抗新型冠状病毒(SARS-COV-2)的人源化单域抗体, 其氨基酸序列如SEQ ID NO.1所示:
X1X2QLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGX43X44X45EX47V X49CISPSGTSTNYADSVKGRFTX70SRDX74X75KNX78X79YLQMNSLX87X88EDTAVYYC AAANPSYYYCSGYPHEYDDWGQGTX121VTVSS;
其中,X1为E或Q,X2为V或L,X43为Q或K,X44为G或E,X45为L或R, X47为A或G,X49为A或S,X70为I或V,X74为N或D,X75为S或A,X78为S或 T,X79为L或V,X87为R或K,X88为A或P,X121为L或Q。
本发明提供的人源化单域抗体具有人源化序列,在维持与抗新型冠状病毒刺突蛋白的特异性结合能力的前提下,降低了免疫源性,并保持或提高了对新型冠状病毒 的中和活性。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.12μg/ml的中和活性。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.08μg/ml的中和活性。
其中,IC50的检测采用竞争法检测,可用本文实施例描述的方法进行。
可选地,在一些实施方案中,在SEQ ID NO.1中:(1):X70为I,X74为N,X79为L,X87为R,X88为A,以及X121为L;或(2):X78为S,X79为V,X87为K,X88为A,以及X121为L。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.06458μg/ml的中和活性。
本发明的实施例发现,对部分位点进行人源化序列改造,提高了对刺突蛋白的中和活性,如上述实施方案提供的人源化单域抗体,对新型冠状病毒野生株的刺突蛋白具 有更高的中和活性,IC50低于0.06458μg/ml。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.12μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病毒 Delta变异株的刺突蛋白具有IC50低于0.032μg/ml的中和活性。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.08μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病毒 Delta变异株的刺突蛋白具有IC50低于0.02μg/ml的中和活性。
可选地,在一些实施方案中,在SEQ ID NO.1中:X44为G,X 45为L,X70为I, X74为N,X79为L,X87为R,X88为A,以及X121为L。
可选地,在一些实施方案中,在SEQ ID NO.1中:X2为L,以及X43为K。
可选地,在一些实施方案中,在SEQ ID NO.1中:X47为G,以及X49为S。
可选地,在一些实施方案中,在SEQ ID NO.1中:X2为V,以及X43为Q。
可选地,在一些实施方案中,在SEQ ID NO.1中:X47为A,以及X49为A。
可选地,在一些实施方案中,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.06458μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病 毒Delta变异株的刺突蛋白具有IC50低于0.0119μg/ml的中和活性。
本发明的实施例发现,对部分位点进行人源化序列改造,提高了对新型冠状病毒Delta变异株的刺突蛋白的中和活性,也如上述实施方案提供的人源化单域抗体,对新 型冠状病毒Delta变异株的刺突蛋白具有更高的中和活性,即IC50低于了0.0119μg/ml。
第二方面,本发明提供抗新型冠状病毒刺突蛋白的人源化单域抗体,其氨基酸序列如SEQ ID NO.2-14中的任意一者所示。
第三方面,本发明提供抗新型冠状病毒的人源化纳米抗体,其包括如上所述的人源化单域抗体。
可选地,在一些实施方案中,所述人源化纳米抗体还包含半衰期延长结构域。
可选地,在一些实施方案中,所述半衰期延长结构域选自免疫球蛋白的Fc区或血清白蛋白结合结构域。
可选地,在一些实施方案中,所述免疫球蛋白选自IgG、IgM、IgE、IgA和IgD。
可选地,在一些实施方案中,所述人源化纳米抗体包括至少两个所述人源化单域抗体。
可选地,在一些实施方案中,所述半衰期延长结构域为免疫球蛋白的Fc区。
可选地,在一些实施方案中,所述人源化单域抗体连接至所述Fc区的N端、或C 端,或二者均有。
第四方面,本发明提供一种融合蛋白,其包括如上所述的人源化单域抗体。
第五方面,本发明提供一种缀合物,其包括如上所述的人源化单域抗体。
第六方面,本发明提供一种核酸分子,其编码如上所述的人源化单域抗体,或编码如上所述的人源化纳米抗体,或编码如上所述的融合蛋白,或编码如上所述的缀合物。
第七方面,本发明提供一种重组载体,其含有如上所述的核酸分子。
第八方面,本发明提供一种重组细胞,其含有如上所述的核酸分子,或含有如上所述的重组载体。
第九方面,本发明提供一种药物组合物,其含有如上所述的人源化单域抗体,如上所述的人源化纳米抗体,如上所述的融合蛋白,如上所述的缀合物,如上所述的核酸 分子,如上所述的重组载体,或如上所述的重组细胞。
药物组合物可还包含可药用载体。作为可药用载体,黏合剂、助流剂、崩解剂、 赋形剂、增溶剂、分散剂、稳定剂、助悬剂、色素、矫味剂等可用于经口施用;缓冲剂、 防腐剂、疼痛减轻剂、增溶剂、等张剂、稳定剂等可用于注射用混合物;以及基质、赋 形剂、润滑剂、防腐剂等可用于表面施用。
可选地,在一些实施方案中,所述药物组合物还含有药学上可接受的辅料。
第十方面,本发明提供如上所述的人源化单域抗体,如上所述的人源化纳米抗体,如上所述的融合蛋白,如上所述的缀合物,如上所述的核酸分子,如上所述的重组载体, 或如上所述的重组细胞在制备用于预防和/或治疗新型冠状病毒感染疾病的药物中的应 用。
第十一方面,本发明提供一种制备如上所述的人源化单域抗体的方法,其包括:培养如上所述的重组细胞,从培养产物中分离纯化获得所述人源化单域抗体。
术语定义
除非另外定义,与本公开结合使用的科学和技术术语应具有本领域普通技术 人员通常理解的含义。
比利时科学家Raymond Hamers等研究发现骆驼血液中除了有普通四条链抗体外,还有一种自然存在的缺失轻链的重链抗体,该重链抗体基因在重组时,剪切掉了重链稳 定区的CH1区的基因,因而表达的重链缺少CH1区,不能与轻链连接形成普通的轻、 重链四条链的抗体,形成了仅有两条重链(无CH1)的重链抗体。将该重链抗体的可变区, 称为VHH,即单域抗体,以与普通抗体重链可变区VH加以区别。
本文中,术语“氨基酸”表示天然存在或非天然存在的羧基α-氨基酸。术语“氨 基酸”用在本申请中可以包括天然存在的氨基酸和非天然存在的氨基酸。天然存在的氨 基酸包括丙氨酸(三字母密码:Ala,单字母密码:A),精氨酸(Arg,R),天冬酰胺(Asn, N),天冬氨酸(Asp,D),半胱氨酸(Cys,C),谷氨酰胺(Gln,Q),谷氨酸(Glu,E),甘 氨酸(Gly,G),组氨酸(His,H),异亮氨酸(Ile,I),亮氨酸(Leu,L),赖氨酸(Lys,K), 甲硫氨酸(Met,M),苯丙氨酸(Phe,F),脯氨酸(Pro,P),丝氨酸(Ser,S),苏氨酸(Thr,T),色氨酸(Trp,W),酪氨酸(Tyr,Y),和缬氨酸(Val,V)。非天然存在的氨基酸包括 但不限于α-氨基己二酸,氨基丁酸,瓜氨酸,高瓜氨酸,高亮氨酸,高精氨酸,羟基 脯氨酸,正亮氨酸,吡啶基丙氨酸,肌氨酸等等。
本文中,术语“氨基酸序列”是指氨基酸相互连接形成肽链(或多肽)的顺序,氨基酸序列只能按照一个方向读取。氨基酸有100多种不同类型,其中20种常用,本发明 不排除氨基酸链上有其他物质,例如糖类、脂类等修饰,本发明也不限于20中常用的 氨基酸。
本文中,术语“人源化”是指抗体的可变区(VH或VHH)的FR区部分或所有全部 由人类抗体基因所编码。人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反 应。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等几类。应该理解,本领域技 术人员根据本发明公开的人源化序列能够制备出其他单域抗体的合适人源化序列,其也 在本发明涵盖的范围之内。
本文中,术语“核酸分子”可以是DNA形式或RNA形式。DNA形式包括cDNA、 基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链 或非编码链。
本发明中的核苷酸分子全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列,尤其是片段 长度较短时。通常,通过先合成多个小片段,然后再进行连接可获得序列很长的片段。 此外,还可将编码序列和表达标签(如6His),或其他功能性蛋白片段等融合在一起,形 成融合蛋白。
目前,已经可以完全通过化学合成来得到编码本发明蛋白的核苷酸分子序列。
在本文中,术语“载体”包括含有本发明的核酸分子或其片段的、能够携带遗传 信息并且可以将遗传信息递送到细胞中的分子或试剂。典型的载体包括质粒、病毒、噬 菌体、黏粒和微型染色体。载体可以是克隆载体(即用于将遗传信息转移到细胞中的载 体,可以繁殖所述细胞并且可以选择存在或不存在所述遗传信息的所述细胞)或表达载 体(即包含必要的遗传元件从而允许所述载体的遗传信息在细胞中表达的载体)。因此, 克隆载体可以包含选择标记,以及与所述克隆载体所指定的细胞类型相匹配的复制起 点,而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件。本文中,本发明 的核酸分子或其片段可以插入到合适的载体中以形成携带本发明核酸分子的克隆载体 或表达载体。
本文中,术语“Fc区”是指免疫球蛋白的C端区域,该区域是由重链恒定 结构域中仅CH2和CH3组成的功能性结构单元。Fc没有结合抗原的能力,然 而其具有半衰期延长的性质,并且具有恒定的氨基酸序列。
本文中,术语“缀合物”是指本发明单域抗体与酶相(如辣根过氧化物酶、 碱性磷酸酶等)、放射性同位素、荧光化合物或化学发光化合物、治疗剂等中的 一种或多种相偶联得到的缀合物,这些缀合物可用于检测新冠病毒刺突蛋白或治 疗新冠病毒刺突蛋白感染疾病。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使 用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因 此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性 劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例3中抗体表达质粒的图谱。
图2为实施例4中人源化纳米抗体对野生株新型冠状病毒SARS-COV-2 RBD蛋白的中和活性结果。
图3为实施例4中人源化纳米抗体对Delta变异株新型冠状病毒 SARS-COV-2RBD蛋白的中和活性结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施 例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规 条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通 过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
抗新型冠状病毒刺突蛋白纳米抗体人源化改造
通过CDR-grafting的方法进行人源化改造。首先使用MOE(Molecular OperatingEnvironment)软件对COV2-171-11(参见中国发明专利 CN111647077B)进行抗体同源建模。根据模型结构,结合发明人的经验,分析 能影响抗原结合区域构象稳定的关键组成氨基酸残基。随后检索人免疫球蛋白数 据库,搜索人种系抗体库中与COV2-171-11同源性高的人IGVH序列作为人源 化改造模板。分别将COV2-171-11与匹配的人IGVH序列进行比对,结合发明 人的经验重点分析原始纳米单抗上能影响抗原结合区域构象稳定的关键组成氨 基酸残基与人IGVH序列不一致的位点,并在模型中观察是否能进行人源化的替 换。
根据不同的人源化替换程度,基于母本序列可以同时产生多条人源化后的序 列。此外,还以NbBcII10-FGLA(PDB编号:3EAK)为模板进行人源化改造, NbBcII10-FGLA是人源化纳米抗体,使用NbBcII10-FGLA作为框架,直接将 COV2-171的CDR区移植完成改造,得到一系列人源化VHH序列,如下SEQ ID NO.1:
X1X2QLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGX43X44X45EX47V X49CISPSGTSTNYADSVKGRFTX70SRDX74X75KNX78X79YLQMNSLX87X88EDTAVYYC AAANPSYYYCSGYPHEYDDWGQGTX121VTVSS;
其中,X代表氨基酸残基可替换位点,具体地,X1为E或Q,X2为V或L,X43为Q或K,X44为G或E,X45为L或R,X47为A或G,X49为A或S,X70为I或V, X74为N或D,X75为S或A,X78为S或T,X79为L或V,X87为R或K,X88为A或 P,X121为L或Q。
实施例2
抗新型冠状病毒刺突蛋白人源化单域抗体
在实施例1的基础上,本实施例提供具体的人源化VHH序列,具体氨基酸 序列如下表1和表2:
表1
表2
实施例3
抗新型冠状病毒刺突蛋白人源化纳米抗体
将实施例2的VHH序列连接至hIgG1-Fc(SEQ ID NO:15)的N端,得到抗新 型冠状病毒人源化纳米抗体。在其他实施例中,也可以VHH序列连接至hIgG1-Fc 的C端。
人源化纳米抗体的制备方法如下:
(1)蛋白瞬转表达:
将含有目的基因的表达质粒(如图1所示)通过与转染试剂PEI形成阳离子 复合物后,导入到宿主细胞Expi293,质粒在细胞内期间,质粒上的外源基因在 细胞内发生转录翻译,从而得到目的蛋白。
Expi293在37℃、8%二氧化碳、130rpm条件培养,并在转染前通过细胞计 数,将2E6的细胞接种至1L摇瓶中,培养体系约为300ml。配制转染复合物准 备转染:首先将750μg目标质粒加入到含有15ml Opti-MEM试剂的50ml离心 管中,轻轻混匀,标记为A管;将1.5mg转染试剂PEI加入到含有15mlOpti-MEM 试剂的50ml离心管中,轻轻混匀后,室温孵育5min,标记为B管;将B管PEI 稀释液逐滴加入到A管DNA稀释液中,轻轻混匀后,室温孵育15min,孵育结 束后,将PEI-目标质粒复合物加入到Expi293细胞,置于37℃摇床中继续培养。 直到D7-D10后收样。
(2)复合物样品的纯化:
瞬转细胞表达液经过9000rpm/20min离心,收集上清,再经过0.22μm滤膜 除菌过滤。纯化采用ProA亲和层析。过程如下,使用AKTA avant 150层析设备, 用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX,GE), 加载样品至层析柱,使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样 后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱,随后使用洗脱缓 冲液(20mM NaAc,pH=3.4)洗脱目标蛋白,收集管中预先加入中和缓冲液(1M Tris,pH8.0),中和缓冲液的加入体积根据洗脱样品的预估含量而定,一般加入 10%洗脱体积量。
样品使用Biotek-Epoch-Take-3进行浓度测定,利用A280方法检测抗体浓 度,即以消光系数E.C.=1.37(根据氨基酸序列预测),光程=0.05mm(Take-3板 不同孔光程有细微差异,会自动校正),通过设备检测样品吸光值,按照 Lambert-Beer law计算待测抗体的浓度。样品浓度过低则需要进行超滤浓缩,使 用超滤浓缩管( Ultra-15Centrifugal Filter Devices,30kD)按照说明书 提供的通用操作方法,将样品浓度浓缩至>0.5mg/ml;收集浓缩端样品,0.22μm 无菌针头滤器除菌过滤(科百特,PES,0.22μm,直径13mm)后分装冻存备用;
抗新型冠状病毒人源化纳米抗体的制备数据如下表3。
表3
实施例4
人源化纳米抗体对新型冠状病毒的中和活性检测
使用竞争法检测人源化设计抗体对不同变种新型冠状病毒SARS-COV-2 RBD蛋白中和活性。实验条件如下:
包被条件:
病毒蛋白:不同变种新型冠状病毒SARS-COV-2RBD蛋白,2μg/ml;
包被液:50mm pH9.51 CB;
包被体积:100μl/孔;
包被温度:2-8度;
包被时间:18小时;
封闭液:含1%BSA+1×PBS;
封闭温度:37度;
封闭时间:3小时;
加样:加50μl待测抗体(所有抗体起始浓度是5μg/mL,并按照5倍进行系 列梯度稀释),孵育30min,使用洗板液(1xPBS)清洗5次后,加入50μl ACE2 抗原蛋白(起始浓度0.5mg/ml,稀释400倍后使用),使用洗板液(1xPBS)清 洗3次后,显色检测。
表4不同人源化纳米抗体对不同毒株新型冠状病毒RBD蛋白的IC50 (μg/ml)
结果见图2-图3,以及表4。从上述结果可以看出,所有人源化纳米抗体均 能有效阻断野生型SARS-COV-2RBD蛋白、Delta SARS-COV-2RBD蛋白与人 ACE2受体蛋白的结合,显示出显著的新型冠状病毒中和活性,其中,R1376、 R1374、和R1373对两种毒株都具有较显著的中和活性。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域 的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内, 所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 广东菲鹏制药股份有限公司
<120> 人源化单域抗体及其应用和药物
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 126
<212> PRT
<213> 人工序列
<220>
<221> X1
<222> (1)..(1)
<223> Xaa 为 Glu 或 Gln
<220>
<221> X2
<222> (2)..(2)
<223> Xaa 为Val或Leu
<220>
<221> X43
<222> (43)..(43)
<223> Xaa 为Gln或Lys
<220>
<221> X44
<222> (44)..(44)
<223> Xaa 为Gly或Glu
<220>
<221> X45
<222> (45)..(45)
<223> Xaa 为Leu或Arg
<220>
<221> X47
<222> (47)..(47)
<223> Xaa为Ala或Gly
<220>
<221> X49
<222> (49)..(49)
<223> Xaa为Ala或Ser
<220>
<221> X70
<222> (70)..(70)
<223> Xaa为Ile或Val
<220>
<221> X74
<222> (74)..(74)
<223> Xaa为Asn或Asp
<220>
<221> X75
<222> (75)..(75)
<223> Xaa为Ser或Ala
<220>
<221> X78
<222> (78)..(78)
<223> Xaa为Ser或Thr
<220>
<221> X79
<222> (79)..(79)
<223> Xaa为Leu或Val
<220>
<221> X87
<222> (87)..(87)
<223> Xaa为Arg或Lys
<220>
<221> X88
<222> (88)..(88)
<223> Xaa为Ala或Pro
<220>
<221> X121
<222> (121)..(121)
<223> Xaa为Leu或Gln
<400> 1
Xaa Xaa Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Xaa Xaa Xaa Glu Xaa Val
35 40 45
Xaa Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Xaa Ser Arg Asp Xaa Xaa Lys Asn Xaa Xaa Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Xaa Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 126
<212> PRT
<213> 人工序列
<400> 2
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 126
<212> PRT
<213> 人工序列
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 4
<211> 126
<212> PRT
<213> 人工序列
<400> 4
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala Val
35 40 45
Ala Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 5
<211> 126
<212> PRT
<213> 人工序列
<400> 5
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 126
<212> PRT
<213> 人工序列
<400> 6
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 7
<211> 126
<212> PRT
<213> 人工序列
<400> 7
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 8
<211> 126
<212> PRT
<213> 人工序列
<400> 8
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 126
<212> PRT
<213> 人工序列
<400> 9
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 126
<212> PRT
<213> 人工序列
<400> 10
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 11
<211> 126
<212> PRT
<213> 人工序列
<400> 11
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 126
<212> PRT
<213> 人工序列
<400> 12
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 13
<211> 126
<212> PRT
<213> 人工序列
<400> 13
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 14
<211> 126
<212> PRT
<213> 人工序列
<400> 14
Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Pro Ser Gly Thr Ser Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Asn Pro Ser Tyr Tyr Tyr Cys Ser Gly Tyr Pro His Glu
100 105 110
Tyr Asp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 15
<211> 232
<212> PRT
<213> 人工序列
<400> 15
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
Claims (19)
1.一种抗新型冠状病毒的人源化单域抗体,其特征在于,其氨基酸序列如SEQ ID NO.1所示:
X1X2QLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGX43X44X45EX47VX49CISPSGTSTNYADSVKGRFTX70SRDX74X75KNX78X79YLQMNSLX87X88EDTAVYYCAAANPSYYYCSGYPHEYDDWGQGTX121VTVSS;
其中,X1为E或Q,X2为V或L,X43为Q或K,X44为G或E,X45为L或R,X47为A或G,X49为A或S,X70为I或V,X74为N或D,X75为S或A,X78为S或T,X79为L或V,X87为R或K,X88为A或P,X121为L或Q。
2.根据权利要求1所述的人源化单域抗体,其特征在于,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.12μg/ml的中和活性;
优选地,所述IC50低于0.08μg/ml。
3.根据权利要求1或2所述的人源化单域抗体,其特征在于,在SEQ ID NO.1中:(1):X70为I,X74为N,X79为L,X87为R,X88为A,以及X121为L;或(2):X78为S,X79为V,X87为K,X88为A,以及X121为L;
优选地,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.06458μg/ml的中和活性。
4.根据权利要求1所述的人源化单域抗体,其特征在于,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.12μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病毒Delta变异株的刺突蛋白具有IC50低于0.032μg/ml的中和活性;
优选地,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.08μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病毒Delta变异株的刺突蛋白具有IC50低于0.02μg/ml的中和活性。
5.根据权利要求1或4所述的人源化单域抗体,其特征在于,在SEQ ID NO.1中:
X44为G,X45为L,X70为I,X74为N,X79为L,X87为R,X88为A,以及X121为L。
6.根据权利要求5所述的人源化单域抗体,其特征在于,在SEQ ID NO.1中:X2为L,以及X43为K;
优选地,在SEQ ID NO.1中:X47为G,以及X49为S。
7.根据权利要求5所述的人源化单域抗体,其特征在于,在SEQ ID NO.1中:X2为V,以及X43为Q;
优选地,在SEQ ID NO.1中:X47为A,以及X49为A。
8.根据权利要求5所述的的人源化单域抗体,其特征在于,所述人源化单域抗体对新型冠状病毒野生株的刺突蛋白具有IC50低于0.06458μg/ml的中和活性;以及所述人源化单域抗体对新型冠状病毒Delta变异株的刺突蛋白具有IC50低于0.0119μg/ml的中和活性。
9.抗新型冠状病毒刺突蛋白的人源化单域抗体,其特征在于,其氨基酸序列如SEQ IDNO.2-14中的任意一者所示。
10.抗新型冠状病毒的人源化纳米抗体,其特征在于,其包括权利要求1-9中任一项所述的人源化单域抗体。
11.根据权利要求10所述的人源化纳米抗体,其特征在于,所述人源化纳米抗体还包含半衰期延长结构域;
优选地,所述半衰期延长结构域选自免疫球蛋白的Fc区或血清白蛋白结合结构域;
优选地,所述免疫球蛋白选自IgG、IgM、IgE、IgA和IgD;
优选地,所述人源化纳米抗体包括至少两个所述人源化单域抗体。
12.一种融合蛋白,其特征在于,其包括权利要求1-9中任一项所述的人源化单域抗体。
13.一种缀合物,其特征在于,其包括权利要求1-9中任一项所述的人源化单域抗体。
14.一种核酸分子,其特征在于,其编码权利要求1-9中任一项所述的人源化单域抗体,或编码权利要求10-11中任一项所述的人源化纳米抗体,或编码权利要求12所述的融合蛋白,或编码权利要求13所述的缀合物。
15.一种重组载体,其特征在于,其含有权利要求14所述的核酸分子。
16.一种重组细胞,其特征在于,其含有权利要求14所述的核酸分子,或含有权利要求15所述的重组载体。
17.一种药物组合物,其特征在于,其含有权利要求1-9中任一项所述的人源化单域抗体,权利要求10-11中任一项所述的人源化纳米抗体,权利要求12所述的融合蛋白,权利要求13所述的缀合物,权利要求14所述的核酸分子,权利要求15所述的重组载体,或权利要求16所述的重组细胞。
18.权利要求1-9中任一项所述的人源化单域抗体,权利要求10-11中任一项所述的人源化纳米抗体,权利要求12所述的融合蛋白,权利要求13所述的缀合物,权利要求14所述的核酸分子,权利要求15所述的重组载体,或权利要求16所述的重组细胞在制备用于预防和/或治疗新型冠状病毒感染疾病的药物中的应用。
19.一种制备如权利要求1-9中任一项所述的人源化单域抗体的方法,其包括:培养权利要求16所述的重组细胞,从培养产物中分离纯化获得所述人源化单域抗体。
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