CN117050166A - 中和呼吸道合胞病毒的c-型单域抗体及应用 - Google Patents
中和呼吸道合胞病毒的c-型单域抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种中和呼吸道合胞病毒(respiratory syncytial virus,RSV)的C‑型单域抗体及应用。本发明的C型单域抗体是针对RSV F蛋白的抗体,能够抑制病毒侵入细胞,可用于RSV的预防、治疗和诊断,以及用于进一步研究RSV感染中的机制。相对于全长单抗,本发明的C型单域抗体分子量较小,具有更好的组织渗透性和结合存在位阻效应的抗原表位的能力;它可在包括原核、酵母、哺乳动物细胞在内的多个表达系统内进行表达,生产成本低、周期短;C型单域抗体给药途径广,能够通过滴鼻、喷鼻、喷雾、雾化吸入、肌肉注射、静脉注射、皮下注射等方式给药。
Description
技术领域
本发明涉及生物医药工程技术领域,具体涉及一种中和呼吸道合胞病毒的C-型单域抗体及应用。
背景技术
呼吸道合胞病毒呈季节性流行,可经呼吸道和密切接触传播,主要感染婴幼儿、老人以及免疫力低下人群,导致急性呼吸道感染、肺炎,甚至死亡。然而至今无有效的疫苗和特异性治疗药物,临床则是使用利巴韦林(非特异性、副作用大),或预防性注射Palivizumab单抗(价格昂贵、中和活性较弱)以达到抗病毒的目的,亟待研发新一代药物。
发明内容
针对现有技术中的问题,本申请提供了一种中和呼吸道合胞病毒的C-型单域抗体及应用。
本发明从人源IgG1的CH2结构域改造后的CH2-m01sm2为骨架的噬菌体表面展示文库中,以呼吸道合胞病毒囊膜蛋白F(F蛋白)为抗原,经过筛选和后续改造,得到候选克隆。我们用大肠杆菌原核表达并纯化所得到的克隆蛋白,并对其生物学特异性及中和活性进行了鉴定,最终获得一种能够中和呼吸道合胞病毒的C-型单域抗体BBT-VC001-1(也可称为C-型纳米抗体BBT-VC001-1),可以用于呼吸道合胞病毒的预防和治疗。
BBT-VC001-1具有三个Loop区,三个Loop区Loop 1、Loop 2和Loop 3的氨基酸序列分别如SEQ ID No.1、SEQ ID No.3和SEQ ID No.6所示。
BBT-VC001-1中编码所述三个Loop区Loop 1、Loop 2和Loop 3的核苷酸序列分别如SEQ ID No.39、SEQ ID No.40和SEQ ID No.41所示。
BBT-VC001-1的氨基酸序列如SEQ ID No.10所示,核苷酸序列如SEQ IDNo.42所示。
本发明提供的上述针对RSV F蛋白的C-型单域抗体BBT-VC001-1,是通过以下方法得到:首先构建以CH2(图1)为骨架的噬菌体展示文库,然后以RSV F蛋白为抗原,经过筛选和优化得到克隆BBT-VC001-1。表达纯化该克隆(图2),并对其进行鉴定分析。通过ELISA对BBT-VC001-1的结合活性进行了鉴定,实验结果表明BBT-VC001-1能够与RSV F蛋白特异性相结合(图3)。间接免疫荧光实验表明,BBT-VC001-1同样可以与细胞膜表面呈天然构象的F蛋白相结合(图4)。另外,细胞水平抗病毒实验表明,BBT-VC001-1具有高效的抗呼吸道合胞病毒作用(图5)。动物水平实验表明,通过肺部雾化给与BBT-VC001-1抗体可对RSV感染Balb/c小鼠起到预防和治疗作用(图6)。
本发明中BBT-VC001-1抗体的氨基酸序列点突变体命名为2C、M17、Zh1、B5、M2、M3、M4、M5、M6、M7、M8、M9、M10、M11、KS、2H、2H-KS、F11、F11-KS、2H-F11、2H-F11-KS具有与BBT-VC001-1抗体类似的结合活性及抗病毒活性(图7-10)。同时,将BBT-VC001-1的三个Loop区替换到其他抗体片段的相应部位(所得抗体为hIgG1 CH2-com、hIgG2 CH2-com、hIgG3 CH2-com、hIgG4 CH2-com),依然可以获得具有结合活性和抗病毒活性的组合体(图11)。将BBT-VC001-1与多肽(ABD和16L)或者蛋白(VH)连接做成融合多肽或者融合蛋白,其结合抗原的活性保持不变(图12),以上所有突变体和抗体的序列编号列在文末表1中。
本发明还揭示了上述中和呼吸道合胞病毒的C-型单域抗体在制备针对呼吸道合胞病毒的预防和治疗药物、检测探针、融合多肽、融合蛋白、偶联抗体中的应用。本发明的中和呼吸道合胞病毒的C-型单域抗体可在制备呼吸道合胞病毒防治相关的滴鼻剂、喷鼻剂、雾化药、肌肉注射制剂、静脉注射制剂中应用,优选地,各配方中的浓度范围在0.01ng/mL至1g/mL。
本发明使用的术语“C-型单域抗体”指以抗体恒定区CH2结构域为骨架的一类抗体。相对于全长单克隆抗体,单域抗体具有更好的组织渗透性,且能够优势结合空间上存在位阻效应的抗原表位。
本发明的有益效果为:本发明的C-型单域抗体是针对RSV F蛋白的抗体,能够抑制病毒侵入细胞,可用于RSV的预防、治疗和诊断,以及用于进一步研究RSV感染中的机制。相对于全长单抗(分子量~150kD),本发明的C-型单域抗体分子量较小(12~15kD),具有更好的组织渗透性和结合存在位阻效应的抗原表位的能力;它可在包括原核、酵母、哺乳动物细胞在内的多个表达系统内进行表达,生产成本低、周期短;C-型单域抗体给药途径广,能够通过滴鼻、喷鼻、喷雾、雾化吸入、肌肉注射、皮下注射、静脉注射等方式给药。
附图说明
图1:C-型单域抗体结构示意图。C-型单域抗体骨架来源于抗体IgG1恒定区的CH2结构域,含有三个柔性的Loop区(Loop 1、Loop 2和Loop 3),对应于抗体可变区中的三个互补决定区(complementarity-determining region,CDR)。
图2:C-型单域抗体BBT-VC001-1的表达纯化。目的蛋白经聚丙烯酰胺凝胶电泳(SDS-PAGE)检测,泳道由左至右依次为蛋白分子量标准(Marker)和纯化后的BBT-VC001-1。
图3:ELISA测定BBT-VC001-1和人呼吸道合胞病毒囊膜蛋白F的结合。BBT-VC001-1与F蛋白结合活性EC50为28nM,牛血清白蛋白(BSA)为阴性对照。
图4:间接免疫荧光鉴定BBT-VC001-1与人呼吸道合胞病毒表面的天然F蛋白结合。BBT-VC001-1与病毒粒子表面的囊膜蛋白F结合后,经荧光二抗染色呈绿色荧光。
图5:BBT-VC001-1中和人呼吸道合胞病毒实验。BBT-VC001-1抑制呼吸道合胞病毒A型(RSV A)的IC50为8.326ng/mL;对呼吸道合胞病毒B型(RSV B)的IC50为8.419ng/mL。
图6:BBT-VC001-1在Balb/c小鼠体内对人呼吸道合胞病毒感染的预防和治疗作用。预防性肺部雾化给BBT-VC001-1可保护小鼠体重的下降(图A)及降低肺部病毒载量(RNA拷贝数)(图B),治疗性肺部雾化给BBT-VC001-1可显著降低肺部病毒载量(病毒滴度)(图C)。
图7:抗体BBT-VC001-1的Loop区和/或骨架区进行点突变后得到的突变体的结合活性,EC50值与BBT-VC001-1相当。
图8:图7所示的抗体BBT-VC001-1的突变体中的2C、M17、Zh1、B5、M2、M3、M4的抗病毒活性,IC50值与BBT-VC001-1相当。
图9:图7所示的抗体BBT-VC001-1的突变体中的M5-M11的抗病毒活性,IC50值与BBT-VC001-1相当。
图10:图7所示的抗体BBT-VC001-1的突变体中的KS、2H、2H-KS、F11、F11-KS、2H-F11、2H-F11-KS的抗病毒活性,IC50值与BBT-VC001-1相当。
图11:BBT-VC001-1中的三个Loop区序列替换到不同类型抗体CH2骨架的Loop区上获得的组合体的结合活性(11A)和抗病毒活性(11B)。
图12:BBT-VC001-1与多肽或者蛋白偶联后的结合活性。
具体实施方式
下面结合实施例和附图对本发明作详细说明,以下实施例是在以本发明技术方案为前提下进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
实施例1:表达并纯化人呼吸道合胞病毒囊膜蛋白recF
根据呼吸道合胞病毒的基因序列(GenBank No.M11486.1),将呼吸道合胞病毒囊膜蛋白F的编码基因与抗体IgG1的Fc片段编码基因融合,制备重组F蛋白(recF)基因。将基因与载体pSecTag2A连接、转化,克隆构建真核表达质粒pSecTag2A-recF。转染前1天将293F细胞(控制细胞密度为5~10×105个/mL)40mL接种于125mL悬浮细胞培养瓶。将40μg质粒(pSecTag2A-recF)稀释于4mL培养基中轻轻浑匀,再将120μL PEI(polyethylenimine)稀释于培养液中,轻轻浑匀。室温孵育20分钟后将它们逐滴加入细胞中。将细胞放入悬浮培养箱中,250rpm/min,37℃悬浮培养。144小时后收集培养基上清,用HRP-anti-human Fc通过蛋白免疫印迹(Western Blot)检测蛋白的表达。
检测到目的蛋白表达后,扩大细胞培养与转染规模,大量表达蛋白recF。收集培养基上清,用Protein A填料纯化目的蛋白,随后用截留分子量为10kD的超滤离心管超滤置换缓冲液。最终浓缩后经SDS-PAGE验证其纯度。
实施例2:噬菌体展示库的构建以及筛选
以人源CH2为骨架,按照已有的文献构建噬菌体展示库(Gong R.,et al.,PLoSOne,2012,7:e42288),以真核表达的抗原recF进行筛选。将纯化的抗原偶联磁珠后,用噬菌体展示库中进行筛选。recF特异性的噬菌体可被抗原所捕获,得到候选克隆。对候选克隆进一步亲和力成熟(Wang R.,et al.,Virol Sin.,2021,11:1-11),得到C-型单域抗体BBT-VC001-1。
测序可知,BBT-VC001-1编码基因序列。BBT-VC001-1的三个Loop区为:Loop 1、Loop 2和Loop 3,其各自的编码基因序列和氨基酸序列在后表1中列出。
实施例3:BBT-VC001-1的表达纯化
按照已有文献(Gong R.,et al.,Methods Mol Biol.,2012,899:85-102)对BBT-VC001-1进行表达和纯化。将BBT-VC001-1原核表达载体转入E.coli HB2151中。挑取单克隆菌落接种于含100μg/mL氨苄青霉素的SB培养基(1L培养基中含30g胰蛋白胨、20g酵母提取物和10g MOPS,pH值用NaOH调至7.0)中,待OD600达到0.7~1.0时加入IPTG至终浓度为200μg/ml,于37℃、220rpm的条件下进行诱导表达14~16h。于4℃、6000rpm、15min离心收集菌体,弃培养基,沉淀重悬于PBS(pH 7.0)中,再经多粘菌素B(polymyxin B)处理1小时后离心收集上清。用Ni-NTA填料纯化,经SDS-PAGE验证其纯度,结果如图2所示。随后用截留分子量为3kD的超滤离心管超滤浓缩。所得到的BBT-VC001-1的C-末端含6×His标签和FLAG标签。
实施例4:ELISA测定BBT-VC001-1和重组囊膜蛋白recF的结合
将recF(4μg/mL)包被在ELISA板上,4℃孵育过夜后用PBS+3%milk于37℃封闭1h。加入梯度稀释的BBT-VC001-1,37℃孵育2小时后用PBST(PBS+0.05% Tween 20)洗四次,再用HRP-Anti-FLAG单克隆抗体于37℃孵育1小时后,用PBST洗四次,再加入ABTS进行显色。牛血清白蛋白(BSA)作为阴性对照。BBT-VC001-1和recF结合的EC50为28nM,且不与BSA结合,结果如图3所示。
实施例5:间接免疫荧光测定BBT-VC001-1和人呼吸道合胞病毒粒子表面的天然F蛋白的结合
将100μL的Hep-2(2×105个/mL)细胞接种96孔板,培养14~16h。PBS洗涤细胞,加入呼吸道合胞病毒(100PFU/孔)感染1h,弃除病毒并补加100μL含2%胎牛血清的DMEM继续37℃培养细胞。约48h后,4%甲醛溶液固定细胞30min,5% BSA封闭1h。依次孵育BBT-VC001-1作一抗,FITC-Anti-His作二抗。使用荧光显微镜观察染色结果(图4)。
实施例6:在Hep-2细胞中BBT-VC001-1抗体抑制人呼吸道合胞病毒复制
将100μL的Hep-2(2×105个/mL)细胞接种96孔板,培养14~16h。将呼吸道合胞病毒(A型和B型)与不同浓度的BBT-VC001-1抗体混匀孵育1h,之后加入PBS洗涤后的细胞中,继续37℃培养细胞。24h-48h后,4%甲醛溶液固定细胞30min,5% BSA封闭1h。依次孵育IgG-mpe8作一抗,FITC-Anti-human作二抗,使用高内涵细胞分析仪拍照分析。根据检测结果,参照阴阳性对照孔计算各孔病毒感染抑制率。BBT-VC001-1抑制呼吸道合胞病毒A型的IC50为8.326ng/mL;抑制B型的IC50为8.419ng/mL(图5)。
实施例7:在小鼠模型中,BBT-VC001-1对人呼吸道合胞病毒感染的保护作用
预防性抗病毒:病毒感染前麻醉Balb/c小鼠,将50μL BBT-VC001-1按照2.5mg/kg剂量肺部雾化给抗体(预防组),对照组肺部雾化给50μL PBS(空白组和PBS组)。6小时后,预防组和PBS组中每只小鼠滴鼻1×107FFU的RSV A2型病毒进行感染。随后,每天称量小鼠体重。攻毒后第四天麻醉处死部分小鼠,取肺组织测定病毒载量。结果如图6A所示,相较于PBS组,预防组体重明显回升,并且肺组织病毒载量降低。
治疗性抗病毒:首先,麻醉小鼠,每只小鼠滴鼻1×107FFU的RSV A2进行感染。3小时后,将不同剂量(5mg/kg和20mg/kg)的BBT-VC001-1以50μL每只的体积肺部雾化给抗体,对照组肺部雾化给50μL PBS,之后第一天和第二天各给抗体一次。攻毒后第四天麻醉处死小鼠,取肺组织测定病毒滴度。结果如图6C所示,给抗体组小鼠肺组织的病毒滴度显著性降低。
实施例8:BBT-VC001-1抗体的点突变体、组合体及融合体的构建与活性
点突变体的构建与活性:在BBT-VC001-1的Loop区和/或骨架区进行单点或者多点突变,获得不同的序列2C(L4S,L14F)、M17(V67I)、Zh1(L4S,L14F,V67I)、B5(V15M,I37V,D49H,V53T,S65R)、M2(S2T,V15M,D49H,V53T,S65R,K81R,K114N)、M3(V15M,D49H,A51G,V53T,S65R)、M4(V15M,D49H,V53T,S65R)、M5(V15M,I37V,D49H,V53T,S65R,E91V,S111A)、M6(V3I,V15M,S27F,I37V,V48M,D49H,V53T,S65R,E91V)、M7(K11N,V15M,I37V,V46E,D49H,V53T,D57E,S65R,L73P,E91V)、M8(V15M,I37V,D49H,V53T,K54N,S65R,N79S,D101N)、M9(V15M,I37V,K38N,D49H,V53T,S65R,E97D)、M10(K9E,V15M,I37V,D49H,V53T,S65R,T71I,K112I)、M11(E91V)、KS(K112I,A113K,K114S)、2H(L4H,L6H,L73K)、2H-KS(L4H,L6H,L73K,K112I,A113K,K114S)、F11(P10S,K11N,T13A,V72I)、F11-KS(P10S,K11N,T13A,V72I,K112I,A113K,K114S)、2H-F11(L4H,L6H,P10S,K11N,T13A,V72I,L73K)、2H-F11-KS(L4H,L6H,P10S,K11N,T13A,V72I,L73K,K112I,A113K,K114S),经基因合成公司合成后构建到表达载体中进行蛋白表达,蛋白表达方法参照实施例3。获得的点突变蛋白进行ELISA和抗病毒实验检测,检测方法分别参照实施例4和实施例6。检测结果如图7~10所示,这些点突变蛋白与RSV的重组囊膜蛋白recF的结合活性在20nM-150nM之间,对RSV A2毒株的抗病毒活性在3ng/mL-55ng/mL之间。结果表明在BBT-VC001-1蛋白的Loop区和/或骨架区进行一些点突变获得的突变体仍然具有活性。
组合体的构建与活性:将BBT-VC001-1中的三个Loop区序列即Loop 1、Loop 2和Loop 3替换到不同类型抗体CH2骨架的Loop区上:人类的IgG1 CH2区、IgG2 CH2区、IgG3CH2区、IgG4 CH2区。获得的组合体分别命名为hIgG1CH2-com、hIgG2 CH2-com、hIgG3 CH2-com、hIgG4 CH2-com。将这些组合体进行基因合成、蛋白表达、活性检测(方法同上)。检测结果如图11所示,hIgG1CH2-com、hIgG2 CH2-com、hIgG3 CH2-com、hIgG4 CH2-com同样具有结合活性和细胞水平的抗病毒活性。结果提示,BBT-VC001-1的三个Loop区可以放在不同的骨架上发挥作用。
融合体的构建与活性:将BBT-VC001-1与多肽(16L和ABD)或者蛋白(VH)偶联,分别命名为BBT-VC001-1-16L、BBT-VC001-1-ABD、BBT-VC001-1-VH。之后进行基因合成、蛋白表达、活性检测(方法同上)。检测结果如图12所示,融合多肽或者蛋白后的BBT-VC001-1依然保持结合活性。结果表明,BBT-VC001-1可在制备针对人呼吸道合胞病毒的预防和治疗融合多肽、融合蛋白中的应用。
最后附上述实施例中所涉及的所有抗体及其片段的序列信息表。
表1.抗体及其Loop的序列信息表
本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是,本发明的保护和权利要求范围不限于所提供的案例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例,都属于本发明保护的范围。
Claims (10)
1.一种中和呼吸道合胞病毒的C-型单域抗体,其特征在于:所述C-型单域抗体以CH2结构域为骨架,它具有三个Loop区:Loop 1、Loop 2和Loop 3,
Loop 1的氨基酸序列选自SEQ ID No.1或SEQ ID No.2;
Loop 2的氨基酸序列选自SEQ ID No.3-5中任一项;
Loop 3的氨基酸序列选自SEQ ID No.6-9中任一项。
2.根据权利要求1所述中和呼吸道合胞病毒的C-型单域抗体,其特征在于:Loop 1、Loop 2和Loop 3的氨基酸序列分别如SEQ ID No.1、SEQ ID No.3和SEQ ID No.6所示。
3.根据权利要求1所述中和呼吸道合胞病毒的C-型单域抗体,其特征在于:所述C-型单域抗体的氨基酸序列选自SEQ ID No.10-35中任一项。
4.权利要求1~3所述的中和呼吸道合胞病毒的C-型单域抗体在制备针对呼吸道合胞病毒的预防和治疗药物、检测探针、融合多肽、融合蛋白、偶联抗体中的应用。
5.权利要求1~3所述的中和呼吸道合胞病毒的C-型单域抗体在制备呼吸道合胞病毒防治相关的滴鼻剂、喷鼻剂、雾化药、注射制剂中的应用。
6.一种呼吸道合胞病毒抗体融合多肽,其特征在于,所述抗体融合多肽的氨基酸序列如SEQ ID No.36所示。
7.一种呼吸道合胞病毒抗体融合多肽,其特征在于,所述抗体融合多肽的氨基酸序列如SEQ ID No.37所示。
8.一种呼吸道合胞病毒抗体融合蛋白,其特征在于,所述抗体融合蛋白的氨基酸序列如SEQ ID No.38所示。
9.根据权利要求2所述中和呼吸道合胞病毒的C-型单域抗体,其特征在于:编码所述Loop 1、Loop 2和Loop 3的核苷酸序列分别如SEQ ID No.39、SEQ IDNo.40和SEQ ID No.41所示。
10.根据权利要求3所述中和呼吸道合胞病毒的C-型单域抗体,其特征在于:编码所述C-型单域抗体的核苷酸序列如SEQ ID No.42所示。
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