WO2022252770A1 - 一种抗SARS-CoV-2流行突变株的单克隆抗体20D8 - Google Patents
一种抗SARS-CoV-2流行突变株的单克隆抗体20D8 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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- C07K2317/515—Complete light chain, i.e. VL + CL
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- the application belongs to the technical field of biomedicine, and in particular, relates to a monoclonal antibody 20D8 against a mutant strain of SARS-CoV-2.
- SARS-CoV-2 is a novel coronavirus with high infection rate and fatality rate. Since its outbreak, it has caused a global pandemic and seriously threatened global public health. Vaccines can effectively prevent infectious diseases, and some new crown vaccines have been approved for use, but they are mainly aimed at healthy people. Neutralizing antibodies against SARS-CoV-2 can effectively block the combination of virus and cells, and have shown positive effects in clinical trials.
- Emerging mutant strains such as P.1 strain (containing mutations such as K417T, E484K and N501Y), B.1.351 (also known as 501Y.V2, containing mutations such as K417N, E484K and N501Y) and B.1.1.7 (containing mutations such as E484K and N501Y and other mutations) multiple point mutations in the amino acid sequence of the spike protein (including RBD and NTD regions) will not only affect the transmission speed and pathogenicity of the mutant strain, but also have a greater effect on the neutralizing activity of the monoclonal antibody. The impact may lead to the occurrence of virus immune escape. Therefore, there is an urgent need to develop monoclonal antibodies with broad-spectrum neutralization ability against SARS-CoV-2 wild strains and mutant strains, which has great applications in the detection and treatment of SARS-CoV-2 wild strains and mutant strains. prospect.
- This application first relates to the monoclonal antibody 20D8 against SARS-CoV-2 virus, characterized in that the six CDR regions of the antibody are:
- VHCDR1 comprises the amino acid sequence shown in SEQ ID NO.1: EYTMY;
- VHCDR2 Heavy chain CDR2 (VHCDR2) comprises the amino acid sequence shown in SEQ ID NO.2: GINPNIGDTGYNQKFKG;
- VHCDR3 comprises the amino acid sequence shown in SEQ ID NO.3: DTGNYPFDY;
- VLCDR1 Light chain CDR1 (VLCDR1) comprises the amino acid sequence shown in SEQ ID NO.4: KSSQSLLYSSNQKNYLA;
- VLCDR2 Light chain CDR2 (VLCDR2) comprises the amino acid sequence shown in SEQ ID NO.5: WASTRES;
- VLCDR3 comprises the amino acid sequence shown in SEQ ID NO.6: QQYYSYPLT.
- the full length of the heavy chain variable region of the monoclonal antibody 20D8 comprises the amino acid sequence shown in SEQ ID NO.7:
- the full length of the light chain variable region of the monoclonal antibody 20D8 comprises the amino acid sequence shown in SEQ ID NO.8:
- the monoclonal antibody 20D8 is an IgG antibody.
- the monoclonal antibody 20D8 can neutralize wild-type SARS-CoV-2 and various SARS-CoV-2 mutant strains, including P.1 strain, B.1.351 strain and B.1.1.7 strain.
- the antigenic structural region bound by the monoclonal antibody 20D8 is the RBD domain in the spike protein S1 of the SARS-CoV-2 virus, and can bind to SARS-CoV-2 containing mutations such as D614G, K417T/N, E484K, and N501Y.
- SARS-CoV-2 containing mutations such as D614G, K417T/N, E484K, and N501Y.
- the present application also relates to the encoding nucleic acid fragment encoding the monoclonal antibody 20D8.
- the present application also relates to an antibody, the light chain of the antibody is:
- the heavy chain of the antibody is:
- the present application also includes the application of the monoclonal antibody 20D8 in the preparation of a reagent for detecting SARS-CoV-2 virus, preferably, the reagent is for detecting SARS-CoV-2 virus P.1 strain and B.1.351 strain , B.1.1.7 strain reagents.
- the present application also includes the application of the monoclonal antibody 20D8 in the preparation of a reagent for inhibiting SARS-CoV-2 virus, preferably, the reagent is for inhibiting SARS-CoV-2 virus P.1 strain and B.1.351 strain , B.1.1.7 strain reagents.
- the present application also includes the application of monoclonal antibody 20D8 in the preparation of medicines, the medicines are medicines for preventing and/or treating diseases caused by SARS-CoV-2 virus infection, preferably, the medicines are medicines for preventing and/or treating Or a medicine for treating diseases caused by SARS-CoV-2 virus P.1 strain, B.1.351 strain and B.1.1.7 strain infection.
- the currently developed vaccines and some antibodies approved for emergency use are mainly directed against the unmutated wild strain of SARS-CoV-2.
- Mutant strains of SARS-CoV-2 such as P.1 strain, B.1.351 strain and B.1.1.7 strain showed varying degrees of tolerance to some antibodies at different stages of development, convalescent plasma, and post-vaccine plasma .
- the experimental results of the 20D8 monoclonal antibody involved in this application show that the antibody 20D8 has a strong neutralizing effect on SARS-CoV-2 wild strains and mutant strains such as P.1 strain, B.1.351 strain, and B.1.1.7 strain. and activity. Therefore, the broad-spectrum neutralizing activity monoclonal antibody 20D8 obtained in this application has important research and application value in the specific prevention, treatment and diagnosis of diseases caused by the wild strain of SARS-CoV-2 and its mutant strains.
- Fig. 3 Detection results of the binding activity of the monoclonal antibody 20D8. is the binding activity against SARS-CoV-2 wild strain spike protein S1;
- the eluate was collected and further dialyzed in PBS buffer.
- the purified antibody was taken for SDS-PAGE and HPLC-SEC detection and analysis.
- sequence structure of the six CDR regions of the 20D8 antibody is:
- VHCDR1 Heavy chain CDR1 (VHCDR1), SEQ ID NO.1: EYTMY;
- VHCDR2 Heavy chain CDR2 (VHCDR2), SEQ ID NO.2: GINPNIGDTGYNQKFKG;
- VHCDR3 Heavy chain CDR3 (VHCDR3), SEQ ID NO.3: DTGNYPFDY;
- VLCDR1 Light chain CDR1 (VLCDR1), SEQ ID NO.4: KSSQSLLYSSNQKNYLA;
- VLCDR2 Light chain CDR2
- SEQ ID NO.5 WASTRES
- VLCDR3 Light chain CDR3
- SEQ ID NO.6 QQYYSYPLT.
- sequences of the light chain variable region and the heavy chain variable region of the 20D8 antibody are respectively: SEQ ID NO.8, SEQ ID NO.7;
- the ELISA method was used to determine the binding ability of the 20D8 antibody to the spike protein S1 of the SARS-CoV-2 virus. The steps are briefly described as:
- the neutralizing activity of 20D8 against SARS-CoV-2 wild strain (WT) and its mutant strains was detected by a pseudovirus detection system, and the pseudoviruses were purchased from Beijing Tianyao Biotechnology Development Company: wild strain (article number: 80033), P. Neutralizing activity of pseudoviruses corresponding to 1 strain (Cat. No.: 80045), B.1.351 strain (Cat. No.: 80044) and B.1.1.7 strain (Cat. No.: 80043).
- the procedure is briefly described as follows: the serially diluted antibody 20D8 was mixed with the pseudovirus and incubated at 37°C for 1 hour, and no antibody or virus was added to the control group.
- B.1.351 strain and B.1.1.7 strain pseudovirus neutralizing activity IC50 are respectively:
- this monoclonal antibody has neutralizing activity against both wild and mutant strains of SARS-CoV-2.
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Abstract
本申请涉及一种抗SARS-CoV-2流行突变株的单克隆抗体20D8,该抗体能中和野生型SARS-CoV-2和多种SARS-CoV-2突变株,包括P.1株、B.1.351株和B.1.1.7株。
Description
相关申请的交叉引用
本申请要求于2021年06月04日提交中国专利局的申请号为202110625569.7、名称为“一种抗SARS-CoV-2流行突变株的单克隆抗体20D8”的中国专利申请的优先权,其全部内容通过引用结合在本申请中。
本申请属于生物医药技术领域,具体的,涉及一种抗SARS-CoV-2流行突变株的单克隆抗体20D8。
SARS-CoV-2是一种新型冠状病毒,具有高传染率和致死率,自爆发以来,已引起全球大流行,严重威胁到全球公共健康。疫苗能有效预防传染病,且目前已有部分新冠疫苗被批准使用,但其主要是针对健康人群。针对SARS-CoV-2的中和抗体能有效阻断病毒与细胞的结合,且在临床试验上显示出积极效果。
目前,有部分抗SARS-CoV-2的单克隆抗体已被批准紧急使用,且有大量处于不同的开发阶段。然而,现有的大部分抗体主要是针对未突变的SARS-CoV-2野生株(Wild-type,WT)。新出现的突变株如P.1株(含K417T、E484K和N501Y等突变)、B.1.351(也称为501Y.V2,含K417N、E484K和N501Y等突变)和B.1.1.7(含E484K和N501Y等突变)在刺突蛋白(包括RBD和NTD区域)氨基酸序列上的多个点突变不仅会影响突变株的传播速度、致病性,还会对单克隆抗体的中和活性产生较大的影响,可能导致病毒免疫逃逸的发生。因此亟需开发针对SARS-CoV-2野生株及突变株具有广谱中和能力的单克隆抗体,这在针对包括SARS-CoV-2野生株和突变株的检测及治疗中具有较大的应用前景。
发明内容
本申请首先涉及针对SARS-CoV-2病毒的单克隆抗体20D8,其特征在于,该抗体的六个CDR区为:
(1)重链CDR1(VHCDR1)包含如SEQ ID NO.1所示的氨基酸序列:EYTMY;
(2)重链CDR2(VHCDR2)包含如SEQ ID NO.2所示的氨基酸序列:GINPNIGDTGYNQKFKG;
(3)重链CDR3(VHCDR3)包含如SEQ ID NO.3所示的氨基酸序列:DTGNYPFDY;
(4)轻链CDR1(VLCDR1)包含如SEQ ID NO.4所示的氨基酸序列:KSSQSLLYSSNQKNYLA;
(5)轻链CDR2(VLCDR2)包含如SEQ ID NO.5所示的氨基酸序列:WASTRES;
(6)轻链CDR3(VLCDR3)包含如SEQ ID NO.6所示的氨基酸序列:QQYYSYPLT。
进一步的,
所述的单克隆抗体20D8的重链可变区全长包含如SEQ ID NO.7所示的氨基酸序列:
所述的单克隆抗体20D8的轻链可变区全长包含如SEQ ID NO.8所示的氨基酸序列:
进一步的,所述的单克隆抗体20D8为IgG型抗体。
进一步的,所述的单克隆抗体20D8能中和野生型SARS-CoV-2和多种SARS-CoV-2突变株,包括P.1株、B.1.351株和B.1.1.7株。
具体的,所述的单克隆抗体20D8结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1中的RBD结构域,且能结合含D614G、K417T/N、E484K和N501Y等突变的SARS-CoV-2的刺突蛋白S1。
本申请还涉及编码所述单克隆抗体20D8的编码核酸片段。
本申请还涉及一种抗体,所述的抗体的轻链为:
(1)SEQ ID NO.8所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.8所示的氨基酸序列具有95%以上同源性的氨基酸序列;
所述的抗体的重链为:
(1)SEQ ID NO.7所示的氨基酸序列经替换、缺失或添加一个或几个氨基酸后,形成的具有同等功能的序列;
或(2)与SEQ ID NO.7所示的氨基酸序列具有95%以上同源性的氨基酸序列。
本申请还包括所述的单克隆抗体20D8在制备检测SARS-CoV-2病毒的试剂中的应用,优选的,所述的试剂为检测SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株的试剂。
本申请还包括所述的单克隆抗体20D8在制备抑制SARS-CoV-2病毒的试剂中的应用,优选的,所述的试剂为抑制SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株的试剂。
本申请还包括单克隆抗体20D8在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物,优选的,所述的药物为预防和/或治疗因SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株感染导致的疾病的药物。
本申请的有益效果:目前已开发的疫苗和部分被批准紧急使用的抗体主要是针对未突变的SARS-CoV-2野生株。SARS-CoV-2突变株如P.1株、B.1.351株和B.1.1.7株显示出对部分在不同开发阶段的抗体、康复者血浆和疫苗免疫后血浆具有不同程度的耐受性。本申请所涉及的20D8单克隆抗体的实验结果表明,该 抗体20D8对SARS-CoV-2野生株和突变株如P.1株、B.1.351株、B.1.1.7株具有较强的中和活性。因此,本申请获得的广谱中和活性单克隆抗体20D8为应对SARS-CoV-2野生株及其突变株引起的疾病的特异性预防、治疗和诊断中具有重要的研究和应用价值。
图1、单克隆抗体20D8的SDS-PAGE电泳检测结果:泳道1为非还原SDS-PAGE电泳,泳道2为还原SDS-PAGE电泳;
图2、单克隆抗体20D8的SEC-HPLC检测结果;
图3、单克隆抗体20D8结合活性检测结果。为针对SARS-CoV-2野生株刺突蛋白S1的结合活性;
图4、单克隆抗体20D8对SARS-CoV-2假病毒的中和活性检测结果,和含有SARS-CoV-2突变株P.1、B.1.351和B.1.1.7突变位点的假病毒的中和活性检测结果。
若未特别说明,以下实施例中所用的技术手段均为本领域技术人员所熟知的常规手段,所有试剂耗材均为市售商品。
实施例1、单克隆抗体20D8的制备及纯化
1、制备:在提前7天注射弗氏不完全佐剂后,将能够稳定分泌20D8抗体的单克隆杂交瘤细胞株(0.5mL,3×10
6cells/mL)注射至小鼠腹腔,继续培养7~10d。
2、纯化:收获腹水,37℃静置2h后,5000rpm离心30min,收集中层上清液并过滤,随后用Protein G亲和层析柱进行纯化。纯化步骤简述为:
用pH为7.0的0.1M Tris缓冲液平衡;
上样后,用pH为7.0的0.1M Tris缓冲液淋洗;
随后用pH为8.0的1.0M Tris缓冲液进行洗脱。
收集洗脱液进一步于PBS缓冲液中透析。取纯化后的抗体进行SDS-PAGE和HPLC-SEC检测分析。
3、结果分析:SDS-PAGE结果见图1,显示在非还原条件下,20D8抗体呈现分子量约为150kDa的条带;在还原条件下呈现分子量约为50kDa和25kDa的两条带,分别对应抗体的重链和轻链。SEC-HPLC结果见图2,显示纯化后的单克隆抗体纯度达到99%以上。纯化后的单克隆抗体经肽图分析后得到的氨基酸序列与预期的氨基酸序列一致。
具体的,所述的20D8抗体的六个CDR区的序列结构为:
重链CDR1(VHCDR1),SEQ ID NO.1:EYTMY;
重链CDR2(VHCDR2),SEQ ID NO.2:GINPNIGDTGYNQKFKG;
重链CDR3(VHCDR3),SEQ ID NO.3:DTGNYPFDY;
轻链CDR1(VLCDR1),SEQ ID NO.4:KSSQSLLYSSNQKNYLA;
轻链CDR2(VLCDR2),SEQ ID NO.5:WASTRES;
轻链CDR3(VLCDR3),SEQ ID NO.6:QQYYSYPLT。
所述的20D8抗体的轻链可变区和重链可变区的序列分别为:SEQ ID NO.8、SEQ ID NO.7;
SEQ ID NO.7:
SEQ ID NO.8:
实施例2、抗体的功能分析
1、抗SARS-CoV-2单克隆抗体20D8与抗原的结合活性检测
采用ELISA法测定20D8抗体与SARS-CoV-2病毒的刺突蛋白S1结合能力。步骤简述为:
(1)以SARS-CoV-2刺突S1-His重组蛋白(Sino公司,货号:40591-V08H)为包被抗原,用碳酸盐缓冲液将2.0μg/mL抗原包被于酶标板,37℃孵育2h;
(2)用酪蛋白缓冲液于37℃封闭2h;加入系列稀释后的待检抗体,37℃孵育1h;
(3)加入1:10000稀释后的羊抗鼠IgG-HRP(Biodragon公司,货号:BF03001X),37℃孵育1h;
(4)用显色液显色后,2M的HCl终止反应;酶标仪检测吸光度A450nm值。结果:
20D8抗体与抗原结合活性检测结果见图3,
单克隆抗体20D8针对S1蛋白的结合活性为:EC50=0.050nM。
2、抗SARS-CoV-2单克隆抗体20D8对(突变型)假病毒的中和试验
采用假病毒检测体系检测20D8对SARS-CoV-2野生株(WT)及其突变株的中和活性,假病毒均购自北京天药物生物技术开发公司:野生株(货号:80033)、P.1株(货号:80045)、B.1.351株(货号:80044)及B.1.1.7株(货号:80043)所对应的假病毒的中和活性。步骤简述为:将系列稀释的抗体20D8与假病毒混合后37℃孵育1h,对照组不加抗体或加病毒。然后加入到预先准备好的含Huh7单层细胞的检测平板中培养24h,随后将100μL培养上清替换为等体积荧光底物,室温孵育2min后将150μL裂解液转移到新的96孔板测定荧光值,并计算假病毒中和活性(以IC
50表示)。
结果:
抗体20D8对假病毒中和实验检测结果见图4,
对野生株(WT)和突变株P.1株、B.1.351株及B.1.1.7株假病毒中和活性IC
50分别为:
野生株(WT):1.37ng/mL(图4A);
突变株P.1株:0.56ng/mL(图4A);
突变株B.1.351株:0.47ng/mL(图4B);
突变株B.1.1.7株:0.95ng/mL(图4C)。
因此,该单克隆抗体对SARS-CoV-2野生株和突变株都具有中和活性。
最后需要说明的是,以上实施例仅用作帮助本领域技术人员理解本申请的实质,不用于限定本申请的保护范围。
Claims (9)
- 一种抗SARS-CoV-2病毒的单克隆抗体20D8,其特征在于,该抗体的六个CDR区为:(1)重链CDR1(VHCDR1):包含如SEQ ID NO.1所示的氨基酸序列;(2)重链CDR2(VHCDR2):包含如SEQ ID NO.2所示的氨基酸序列;(3)重链CDR3(VHCDR3):包含如SEQ ID NO.3所示的氨基酸序列;(4)轻链CDR1(VLCDR1):包含如SEQ ID NO.4所示的氨基酸序列;(5)轻链CDR2(VLCDR2):包含如SEQ ID NO.5所示的氨基酸序列;(6)轻链CDR3(VLCDR3):包含如SEQ ID NO.6所示的氨基酸序列。
- 根据权利要求1所述的抗体,其特征在于,所述的单克隆抗体20D8的重链可变区包含如SEQ ID NO.7所示的氨基酸序列:所述的单克隆抗体20D8的轻链可变区包含如SEQ ID NO.8所示的氨基酸序列。
- 根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体20D8为IgG型抗体。
- 根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体20D8能中和野生型SARS-CoV-2和多种SARS-CoV-2突变株,包括P.1株、B.1.351株和B.1.1.7株。
- 根据权利要求1或2所述的抗体,其特征在于,所述的单克隆抗体20D8结合的抗原结构区域为SARS-CoV-2病毒的刺突蛋白S1,且能结合含如下任意一个或多个突变的SARS-CoV-2的刺突蛋白S1:D614G、K417T/N、E484K、N501Y。
- 编码权利要求1-5任一所述的抗体的核酸片段。
- 权利要求1-5任一所述的单克隆抗体20D8在制备检测SARS-CoV-2病毒的试剂中的应用;优选的,所述的试剂为检测SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株的试剂。
- 权利要求1-5任一所述的单克隆抗体20D8在制备抑制SARS-CoV-2病毒的试剂中的应用;优选的,所述的试剂为抑制SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株的试剂。
- 权利要求1-5任一所述的单克隆抗体20D8在制备药物中的应用,所述的药物为预防和/或治疗因SARS-CoV-2病毒感染导致的疾病的药物;优选的,所述的药物为预防和/或治疗因SARS-CoV-2病毒P.1株、B.1.351株、B.1.1.7株感染导致的疾病的药物。
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