WO2023102692A1 - 协同中和新型冠状病毒的人源抗体和抗体组合及其应用 - Google Patents

协同中和新型冠状病毒的人源抗体和抗体组合及其应用 Download PDF

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WO2023102692A1
WO2023102692A1 PCT/CN2021/135785 CN2021135785W WO2023102692A1 WO 2023102692 A1 WO2023102692 A1 WO 2023102692A1 CN 2021135785 W CN2021135785 W CN 2021135785W WO 2023102692 A1 WO2023102692 A1 WO 2023102692A1
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antibody
amino acid
variable region
chain variable
acid sequence
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French (fr)
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庾蕾
管永军
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广州医科大学附属市八医院
抗体生物制药有限公司
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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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

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  • the invention belongs to the field of biotechnology, and in particular relates to human antibodies and antibody combinations for synergistically neutralizing novel coronaviruses and applications thereof.
  • coronavirus pneumonia COVID-19
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Monoclonal antibodies are undoubtedly the most potential class of biological agents for treatment, and have been used in the treatment of tumors, autoimmune diseases and infectious diseases.
  • mAbs monoclonal antibodies
  • the rapid isolation of human monoclonal antibodies, high-throughput sequencing technology, and the development of structural biology have made antibodies an important means of rapidly responding to emerging infectious diseases.
  • SARS-CoV-2 uses the spike protein on the surface of the virus, the S protein, to mediate the fusion of the virus and the cell membrane, and then invade the host cell.
  • S protein includes two subunits, S1 and S2, and S1 is divided into N-terminal (NTD) and receptor binding (RBD) regions.
  • NTD N-terminal
  • RBD receptor binding
  • S1-RBD is considered to be the main protein that induces the production of host neutralizing antibodies.
  • the present invention aims to provide a human antibody and antibody combination that synergistically neutralize the novel coronavirus and its application, specifically to isolate a group of human monoclonal antibodies that can efficiently neutralize the novel coronavirus from patients who have recovered from novel coronavirus pneumonia, including 3 1 against RBD and 1 against NTD (825, 843, 826 and 846).
  • a single antibody (825 or 843) can effectively neutralize the currently popular variant strains of the virus, including British strains, South African strains, Brazilian strains, and Indian strains; the three antibody combinations (825/843/826 and 825/843 /846) can effectively neutralize the currently popular mutant strains of the virus, and its neutralizing activity IC50 in the pseudovirus system can reach a level of about 10 ng/ml.
  • the first object of the present invention is to provide a human antibody against the novel coronavirus, which is 825 monoclonal antibody, 843 monoclonal antibody, 826 monoclonal antibody or 846 monoclonal antibody;
  • the 825 monoclonal antibody contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region of the 825 monoclonal antibody contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.1 , the light chain variable region contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.2;
  • the 843 monoclonal antibody contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region of the 843 monoclonal antibody contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.3 , the light chain variable region contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.4;
  • the 826 monoclonal antibody contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region of the 826 monoclonal antibody contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.5 , the light chain variable region contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.6;
  • the 846 monoclonal antibody contains a heavy chain variable region and a light chain variable region, and the heavy chain variable region of the 846 monoclonal antibody contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.7 , the light chain variable region contains an amino acid sequence at least 90% homologous to the amino acid sequence shown in SEQ ID NO.8.
  • amino acid sequence of the heavy chain variable region of the 825 monoclonal antibody is shown in SEQ ID NO.1
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO.2;
  • amino acid sequence of the heavy chain variable region of the 843 monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4;
  • amino acid sequence of the heavy chain variable region of the 826 monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6;
  • amino acid sequence of the heavy chain variable region of the 846 monoclonal antibody is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.
  • nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 825 monoclonal antibody is shown in SEQ ID NO.9, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 825 monoclonal antibody The acid sequence is shown in SEQ ID NO.10;
  • nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 843 monoclonal antibody is shown in SEQ ID NO.11, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 843 monoclonal antibody is as follows Shown in SEQ ID NO.12;
  • nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 826 monoclonal antibody is shown in SEQ ID NO.13, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 826 monoclonal antibody is as follows Shown in SEQ ID NO.14;
  • nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of the antibody 846 monoclonal antibody is shown in SEQ ID NO.15, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region of the antibody 846 monoclonal antibody is as follows Shown in SEQ ID NO.16.
  • the human antibody combination contains at least one of the above-mentioned antibodies.
  • the antibody combination comprises at least one of antibody combination 1, antibody combination 2, antibody combination 3 and antibody combination 4, wherein,
  • Antibody combination consisting of 825 monoclonal antibodies, 843 monoclonal antibodies, 826 monoclonal antibodies and 846 monoclonal antibodies;
  • Antibody combination 2 consisting of 825 monoclonal antibodies, 843 monoclonal antibodies and 826 monoclonal antibodies;
  • Antibody combination 3 consisting of 825 monoclonal antibodies, 843 monoclonal antibodies and 846 monoclonal antibodies;
  • Antibody combination 4 consists of 825 monoclonal antibodies and 843 monoclonal antibodies.
  • Another object of the present invention is to provide an application of the above-mentioned human antibody or a combination of the above-mentioned human antibodies in the preparation of a drug for neutralizing novel coronavirus and its variant strains.
  • the novel coronavirus strains include, but are not limited to, virus variant British strains, South African strains, Brazilian strains, and Indian strains.
  • Another object of the present invention is to provide an application of the above-mentioned human antibody in the preparation of a drug or diagnostic reagent or kit for preventing or treating infection by the novel coronavirus SARS-CoV-2.
  • Another object of the present invention is to provide an application of the above-mentioned combination of human antibodies in the preparation of drugs or diagnostic reagents or kits for the prevention or treatment of novel coronavirus SARS-CoV-2 infection.
  • the medicine also includes pharmaceutical excipients.
  • the present invention adopts the patented technology of B cell culture combined with antibody cloning, and screens out antibodies against different epitopes of viral antigens with high neutralization activity without offset, including three antibodies against RBD and one against NTD (825, 843, 826 and 846).
  • a single antibody (825 or 843) can effectively neutralize the currently circulating virus variants, including British strains, South African strains, Brazilian strains and Indian strains; screened antibody combinations (825/843/826 and 825/843/846) It can effectively neutralize the currently popular mutant strains of the virus, and its neutralizing activity IC50 in the pseudovirus system can reach a level of about 10ng/ml.
  • the drug or preparation prepared by the antibody combination can be used for high-risk groups infected by SARS-CoV-2 passive prevention and treatment of novel coronavirus pneumonia.
  • the present invention has the following beneficial effects:
  • the present invention adopts the screening strategy of non-biased B cell culture combined with antibody clones, which can isolate conformation-dependent functional antibodies that exist in vivo but are difficult to imitate in vitro.
  • This antibody isolation strategy does not need to label memory B with antigens Cells, thus not limited by labeled antigens, can simultaneously screen antibodies that bind to different target proteins;
  • the 2 sets of three antibody combinations screened out by the present invention can effectively neutralize the currently popular virus mutant strains, including British strains, South African strains, Brazilian strains and Indian strains, and their neutralizing activity IC50 in the pseudovirus system All can reach the level of about 10ng/ml;
  • the human antibodies and antibody combinations that synergistically neutralize the novel coronavirus provided by the present invention can be used in the preparation of medicaments or diagnostic reagents or kits for preventing or treating SARS-CoV-2 infection.
  • Fig. 1 is the sequence feature diagram of the new antibody obtained
  • Figure 2 is a diagram showing the results of antibody binding to viral S protein and truncated S protein
  • Figure 3 is the obtained new antibody protein reduction SDS-PAGE gel
  • Fig. 4 is the binding figure of combined antibody and RBD protein
  • Figure 5 is a diagram of the binding of the combined antibody to the S1 protein
  • Fig. 7 is the IC50 result of antibody neutralization experiment to various mutant pseudoviruses
  • Figure 8 is a graph showing the results of an antibody neutralization experiment against a Lambda pseudovirus.
  • the flow cytometer is a Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cytometry sorting system, which can be purchased from Shanghai Zequan Instrument Equipment Co., Ltd.;
  • the cDNA synthesis kit is SuperScript III First Strand Synthesis System, which can be purchased from Invitrogen Company of the United States, and the number is #18080051;
  • the fluorescently labeled antibody is a group of fluorescent antibodies, including IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO fluorescent antibodies, purchased from Beckman Coulter;
  • the cell growth factors CpG, IL21, IL2, and irradiated healthy PBMC can be purchased from Guangzhou Haojin Biotechnology Co., Ltd.;
  • the Phusion ultra-fidelity DNA polymerase is Phusion High-Fidelity PCR Master Mix with GC Buffer, which can be purchased from NEB Co., Ltd., number #M0532s.
  • Peripheral blood mononuclear cell isolation Take peripheral venous EDTA anticoagulant blood from COVID-19-infected patients during recovery period, and use density gradient centrifugation to separate peripheral blood mononuclear cells, aliquot 5 ⁇ 10 6 /tube, and freeze in liquid nitrogen.
  • fluorescently labeled antibody staining Place peripheral blood mononuclear cells in a water bath at 37°C to dissolve, wash with PBS buffer for 3 times, then add antibodies for staining, incubate at room temperature in the dark for 15 minutes, wash with PBS buffer, then add 400 ⁇ L of PBS buffer Suspended cells on a flow cytometer, the flow cytometer is the Beckman Coulter MoFlo Astrios EQ ultra-high-speed flow cell sorting system, fluorescently labeled antibody staining: 9 analysis tubes are set, and the corresponding individual tubes are added to tubes 1-7 Fluorescence-labeled antibodies, add 7 kinds of mixed fluorescent-labeled antibodies to the ninth tube, the eighth tube is a blank tube with only cells, and the sample tube is stained the same as the ninth tube.
  • fluorescent labels include IgD-FITC, CD19-ECD, CD27-PC7, CD38-APC A750, IgM-PB and CD45-KO fluorescent antibodies.
  • Sorting of memory B cells The sample tubes were analyzed on the machine, live CD45-positive white blood cells were circled according to 7-AAD and CD45, and B cells were circled according to CD19. Define the IgD - IgM - CD27 + CD38 low population as memory B cells in the IgM and IgD double-negative B cell population, and sort the memory B cells into a 96-well cell culture plate containing cell culture medium, 100 cells/well .
  • Example 2 Memory B cell culture and screening of culture supernatant antibodies
  • the heavy chain and light chain variable region PCR products of the antibody were digested (VH, AgeI/SalI, VK, AgeI/Xhol, VL, AgeI/BsiWI) and cloned into an antibody expression vector containing a human IgG1 constant region.
  • the sequence information of monoclonal antibodies 825, 843, 826 and 846 obtained by analyzing the heavy chain and light chain variable region gene sequences through IMGT/V-Quest is shown in Table 1 below:
  • the antibody-containing culture supernatant was purified on a protein A affinity column, and the protein content was determined;
  • Capture the S protein in the supernatant with an anti-tag antibody establish an ELISA method, and use the capture ELISA method to analyze the binding of the antibody to the S protein;
  • Biofilm light interferometry to analyze the binding of antibodies or combined antibodies to RBD or S1; specifically, use Protein A sensor to sequentially capture anti-tag antibody, tagged RBD or S1 protein to detect the binding of antibody to RBD or S1;
  • BLI technology tandem method is used to detect the binding of antibodies to RBD or S1 by competing with each other; specifically, Protein A sensor is used to sequentially capture anti-tag antibody, tagged RBD or S1 protein, primary antibody and competing antibody to A decrease in detection signal or no detection signal is judged as competition;
  • tags are all D7; the biomolecular interaction analyzer used is purchased from ForteBio Company, model Octet K2.
  • Fig. 1 is the obtained monoclonal antibodies 825, 843, 826 and 846 and their sequence characteristics
  • Fig. 2 is the binding diagram of monoclonal antibodies 825, 843, 826 and 846 with viral S protein and truncated S protein
  • experiment The results showed that monoclonal antibodies 825, 843 and 826 were RBD antibodies, and monoclonal antibody 846 was NTD antibody. Mab 846 does not bind the RBD.
  • monoclonal antibody 825 binds to S and S1 proteins, with EC50 of 0.012 ⁇ g/mL and 0.039 ⁇ g/mL, respectively; monoclonal antibody 843 binds to S and S1 proteins, with EC50 of 0.002 ⁇ g/mL and 0.006 ⁇ g/mL, respectively; Monoclonal antibody 826 binds to S and S1 proteins with EC50 of 0.008 ⁇ g/mL and 0.030 ⁇ g/mL; monoclonal antibody 846 binds to S and S1 proteins with EC50 of 0.136 ⁇ g/mL and 0.049 ⁇ g/mL, respectively.
  • the reducing SDS-PAGE gel images show the heavy chain and light chain sizes of monoclonal antibodies 825, 843, 826 and 846.
  • the antibody combination 825/843/826 or 825/843/846 three antibodies can simultaneously bind to RBD or S1 protein.
  • antibodies 825, 843 and 826 respectively bind to different epitopes on RBD.
  • Expi293F was co-transfected with pPAX2/pLV-EGFP (1:1) expression plasmid containing SARS-CoV-2 spike protein S gene, and pseudovirus was packaged.
  • Pseudovirus microneutralization experiment draw 50 ⁇ L sample, add 800 TCID50 pseudovirus, incubate for 1 hour, add 50,000/well HEK293T cells; check the plate after 48 hours of continuous culture. A decrease in fluorescence value, that is, an inhibition rate greater than 50%, was considered to have neutralizing activity.
  • the antibodies 825, 843, 846 and 826 provided by the present invention and their antibody combinations 825/843/826 and 825/843/846 can effectively neutralize the activity of the new coronavirus; and in the case of the same amount of antibodies, Antibody combinations 825/843/826 and 825/843/846 achieved better neutralization than antibodies 825, 843, 826 and 846 alone.

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Abstract

涉及抗新型冠状病毒的人源抗体、抗体组合及其应用。所述抗体分离自新型冠状病毒肺炎康复者，为3个针对RBD的825单抗、843单抗、826单抗，以及1个针对NTD的846单抗。825或843单抗能够有效中和目前流行的新型冠状病毒变异株，例如英国株、南非株、巴西株和印度株等。825/843/826及825/843/846三种抗体组合能够有效中和目前流行的新型冠状病毒变异株，在假病毒体系中的中和活性IC50均可达10ng/mL。所述抗体、抗体组合能够用于制备预防或治疗新型冠状病毒感染的药物或诊断试剂或试剂盒。

Description

协同中和新型冠状病毒的人源抗体和抗体组合及其应用 技术领域
本发明属于生物技术领域，具体涉及协同中和新型冠状病毒的人源抗体和抗体组合及其应用。
背景技术
目前正在全球范围爆发流行的新型冠状病毒肺炎(COVID-19)是由可引起严重急性呼吸道综合征冠状病毒2(SARS-CoV-2)所导致。对人类健康及经济造成严重危害。截止目前为止全球已有确诊病例近2亿，死亡病例约420多万。而我们仍没有找到有效的特异抗病毒药物。
单克隆抗体(monoclonal antibodies，mAbs)无疑是最具潜力的一类用于治疗的生物制剂，已应用于肿瘤、自身免疫性疾病及感染性疾病的治疗。人单抗的快速分离、高通量测序技术及结构生物学的发展等使得抗体成为快速应对新发传染性疾病的重要手段。
目前人源单克隆抗体主要采用如下策略获得(参见：Immunol Res.2020 Dec；68(6):325-339.)：(1)以病毒抗原免疫转基因鼠。这种转基因鼠由于转入了人的抗体重链和轻链基因，经抗原免疫可产生来源于人抗体序列的抗体，即人源抗体。但这种方法需要先准备免疫用的病毒抗原，且抗体的产生是在小鼠的免疫环境，不能展示人自然感染中和抗体的产生。(2)噬菌体展示技术。采用该技术从来自免疫或非免疫单链抗体可变区片段(scFv)库筛选。由于来源非免疫cDNA文库，抗体没有经历亲和力成熟过程，或组成的抗体重链与轻链不是原始配对，难以获得亲和力高的抗体。(3)直接从感染康复者记忆B细胞克隆抗体。随着抗体技术的发展人们可以直接从单个B细胞获得抗体。多数采用从感染康复者PBMC经流式细胞分选出单个病毒抗原特异的记忆B细胞，进行抗体克隆的策略。该法虽然可快速拿到抗体，但只能拿到可以和病毒蛋白结合的抗体，而丢失了机体具有重要功能的构象依赖的抗体。
SARS-CoV-2借助病毒表面的刺突蛋白即S蛋白介导病毒与细胞膜的融合，进而入侵宿主细胞。S蛋白包括S1和S2两个亚单位，S1分为N-端(NTD)和受体结合(RBD)区域。病毒感染时，首先是S1-RBD与细胞膜上的受体结合激发S构象变化，导致病毒与细胞膜融合而进入靶细胞。S1-RBD被认为是诱导宿主中和抗体产生的主要蛋白。因此，面对新型冠状病毒肺炎疫情，大多研究者多采用表达RBD重组蛋白，进而分选出RBD特异B细胞以获得中和抗体。目前已陆续报道了这类RBD抗体的获得，有些具有很强的中和病毒的能力(参见：1、Science.2020Aug21；369(6506):956-963.和2、Nature.2020 Aug；584(7821):450-456.)。但采取这种策略会丢失一些不针对S1-RBD的抗体，甚至会错过机体产生的具有重要抗病毒作用的抗体。
因此研究人员改变策略，采用S蛋白筛选抗体。国内外已有研究团队从感染康复者获得针对S1-NTD中和抗体(参见：Cell.2021 Apr 29；184(9):2332-2347.)。
采用针对单一表位的单个单抗作为抗病毒治疗手段容易出现病毒逃逸现象，即新出现的变异病毒株可使相应抗体失去中和病毒的能力，甚至使2种联用抗体都失去活性(参考文献：Cell Rep Med.2021 Apr 20；2(4):100255.)。例如礼来(Lily)的两个新冠中和抗体药物(LY-CoV555和LY-CoV016)组成的鸡尾酒已经对南非及巴西突变病毒失效了。因此，联用针对不同表位的多种抗体作为抗病毒治疗策略，才能更有效地防止逃逸突变病毒的产生。要解决这个问题首先需要获得具有高效中和活性的针对病毒抗原不同表位的抗体，筛选出具有协同作用的抗体组合，而且单个抗体或抗体组合要有效中和目前流行的病毒变异株。
发明内容
本发明旨在提供一种协同中和新型冠状病毒的人源抗体和抗体组合及其应用，具体为从新型冠状病毒肺炎康复者分离出可高效中和新型冠状病毒一组人单抗，包括3个针对RBD和1个针对NTD的抗体(825，843，826和846)。其 中，单个抗体(825或843)可有效中和目前流行的病毒变异株包括英国株、南非株、巴西株和印度株等；筛选出的3种抗体组合(825/843/826及825/843/846)可有效中和目前流行的病毒变异株，其在假病毒体系中的中和活性IC50均可达10ng/ml左右水平。
为了达到上述目的，本发明采用以下技术方案：
本发明的第一目的是提供了新型冠状病毒的人源抗体，所述人源抗体为825单克隆抗体、843单克隆抗体、826单克隆抗体或846单克隆抗体；
所述825单克隆抗体含重链可变区和轻链可变区，所述825单克隆抗体的重链可变区含与SEQ ID NO.1所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.2所示氨基酸序列至少90％同源的氨基酸序列；
所述843单克隆抗体含重链可变区和轻链可变区，所述843单克隆抗体的重链可变区含与SEQ ID NO.3所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.4所示氨基酸序列至少90％同源的氨基酸序列；
所述826单克隆抗体含重链可变区和轻链可变区，所述826单克隆抗体的重链可变区含与SEQ ID NO.5所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.6所示氨基酸序列至少90％同源的氨基酸序列；
所述846单克隆抗体含重链可变区和轻链可变区，所述846单克隆抗体的重链可变区含与SEQ ID NO.7所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.8所示氨基酸序列至少90％同源的氨基酸序列。
优选的，所述825单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.1所示，轻链可变区氨基酸序列如SEQ ID NO.2所示；
Figure PCTCN2021135785-appb-000001
所述843单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.3所示，轻链 可变区氨基酸序列如SEQ ID NO.4所示；
Figure PCTCN2021135785-appb-000002
所述826单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.5所示，轻链可变区氨基酸序列如SEQ ID NO.6所示；
Figure PCTCN2021135785-appb-000003
所述846单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.7所示，轻链可变区氨基酸序列如SEQ ID NO.8所示。
Figure PCTCN2021135785-appb-000004
优选的，编码所述抗体825单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.9所示，编码所述抗体825单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.10所示；
Figure PCTCN2021135785-appb-000005
Figure PCTCN2021135785-appb-000006
编码所述抗体843单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.11所示，编码所述抗体843单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.12所示；
Figure PCTCN2021135785-appb-000007
编码所述抗体826单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.13所示，编码所述抗体826单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.14所示；
Figure PCTCN2021135785-appb-000008
Figure PCTCN2021135785-appb-000009
编码所述抗体846单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.15所示，编码所述抗体846单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.16所示。
Figure PCTCN2021135785-appb-000010
优选的，所述人源抗体组合含有上述所述抗体中的至少一个。
优选的，所述抗体组合含抗体组合1、抗体组合2、抗体组合3和抗体组合4中的至少一种，其中，
抗体组合1，由825单克隆抗体、843单克隆抗体、826单克隆抗体和846单克隆抗体组成；
抗体组合2，由825单克隆抗体、843单克隆抗体和826单克隆抗体组成；
抗体组合3，由825单克隆抗体、843单克隆抗体和846单克隆抗体组成；
抗体组合4，由825单克隆抗体、843单克隆抗体组成。
本发明的又一个目的是提供了一种上述所述人源抗体或上述所述人源抗体组合在制备中和新型冠状病毒及其变异株药物中的应用。
优选的，所述新型冠状病毒株包括但不限于病毒变异英国株、南非株、巴西株、印度株。
本发明的又一个目的是提供了一种如上述所述人源抗体在制备预防或治疗新型冠状病毒SARS-CoV-2感染的药物或诊断试剂或试剂盒中的应用。
本发明的又一个目的是提供了一种如上述所述的人源抗体组合在制备预防或治疗新型冠状病毒SARS-CoV-2感染的药物或诊断试剂或试剂盒中的应用。
优选的，所述药物还包括药用辅料。
目前针对新型冠状病毒肺炎尚缺乏有效的预防和治疗策略。本发明采用B细胞培养结合抗体克隆的专利技术，无偏移地筛选出具有高效中和活性的针对病毒抗原不同表位的抗体包括3个针对RBD和1个针对NTD的抗体(825，843，826和846)。其中，单个抗体(825或843)可有效中和目前流行的病毒变异株包括英国株、南非株、巴西株和印度株；筛选出的抗体组合(825/843/826及825/843/846)可有效中和目前流行的病毒变异株,其在假病毒体系中的中和活性IC50均可达10ng/ml左右水平，所述抗体组合制备的药物或制剂可用于SARS-CoV-2感染高危人群的被动预防及新型冠状病毒肺炎的治疗中。
与现有技术相比，本发明具有以下有益效果：
(1)本发明采用无偏移的B细胞培养结合抗体克隆的筛选策略，可分离在 体内存在而在体外很难效仿的构象依赖的功能性抗体，该抗体分离策略不需要用抗原标记记忆B细胞，因而不受标记抗原的限制，可同时筛选与不同靶标蛋白结合的抗体；
(2)采用本发明技术获得4株针对SARS-CoV-2表面刺突蛋白不同区域的中和抗体，抗体的重链和轻链配对是自然产生抗体原有的，亲和力高，更适合于实际应用；
(3)本发明筛选出的2套三种抗体组合均可有效中和目前流行的病毒变异株，包括英国株、南非株、巴西株和印度株，其在假病毒体系中的中和活性IC50均可达10ng/ml左右水平；
(4)本发明提供的协同中和新型冠状病毒的人源抗体和抗体组合可用于制备预防或治疗SARS-CoV-2感染的药物或诊断试剂或试剂盒中的应用。
附图说明
图1为获得的新抗体的序列特征图；
图2为抗体与病毒S蛋白及截短S蛋白结合结果图；
图3为获得的新抗体蛋白还原SDS-PAGE胶图；
图4为组合抗体与RBD蛋白结合图；
图5为组合抗体与S1蛋白结合图；
图6抗体相互竞争与RBD或S1蛋白结合；
图7为抗体对各种突变假病毒中和实验IC50结果；
图8为抗体对Lambda假病毒中和实验结果图。
具体实施方式
以下通过实施例形式的具体实施方式，对本发明的上述内容作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下实施例。
所述流式仪为贝克曼库尔特MoFlo Astrios EQ超高速流式细胞分选系统，可购自上海泽权仪器设备有限公司；
所述cDNA合成为试剂盒为SuperScript III First Strand Synthesis System，可购自美国invitrogen公司，编号为#18080051；
所述荧光标记抗体为一组荧光抗体，包括IgD-FITC、CD19-ECD、CD27-PC7、CD38-APC A750、IgM-PB和CD45-KO荧光抗体，购自贝克曼库尔特公司；
所述细胞生长因子CpG、IL21、IL2、放射照射过的健康人PBMC可购自广州豪晋生物科技有限公司；
所述Phusion超保真DNA聚合酶为Phusion High-Fidelity PCR Master Mix with GC Buffer，可购自NEB有限公司，编号#M0532s。
实施例1 记忆B细胞的识别和分选
1.1外周血单个核细胞分离：取新冠感染者恢复期外周静脉EDTA抗凝血，利用密度梯度离心方法分离外周血单个核细胞，分装5×10 6/管，置于液氮中冻存。
1.2荧光标记抗体染色：外周血单个核细胞放置于37℃中水浴溶化，使用PBS缓冲液洗涤3次，接着加入抗体进行染色，室温避光孵育15min，使用PBS缓冲液洗涤后，加入400μL PBS缓冲液悬浮细胞上流式仪，所述流式仪为贝克曼库尔特MoFlo Astrios EQ超高速流式细胞分选系统，荧光标记的抗体染色：设9个分析管，1-7管加入相应的单个荧光标记的抗体，第9管加入7种混合的荧光标记的抗体，第8管为只有细胞的空白管，样品管同第9管一样染色。其中，荧光标记包括IgD-FITC、CD19-ECD、CD27-PC7、CD38-APC A750、IgM-PB和CD45-KO荧光抗体。
表1记忆B细胞分选各管中荧光标记抗体组合信息表
分析管组别 组别 抗体及荧光标记 体积 荧光通道
1 单染 IgD-FITC 10 FL1
2 单染 CD19-ECD 5 FL3
3 单染 7-AAD 10 FL4
4 单染 CD27-PC7 5 FL5
5 单染 CD38-APCA750 2 FL8
6 单染 IgM-PB 5 FL9
7 单染 CD45-KO 5 FL10
8 空白组 不加抗体 - -
9 样品组 7种抗体混合液 - -
1.3记忆B细胞的分选：样品管上机分析，依据7-AAD及CD45圈出活的CD45阳性的白细胞，依据CD19圈出B细胞。在IgM和IgD双阴的B细胞群体里定义IgD -IgM -CD27 +CD38 low群体为记忆B细胞，将记忆B细胞分选至含细胞培养液的96孔细胞培养板中，100细胞/每孔。
实施例2 记忆B细胞培养及培养上清抗体的筛选
向含记忆B细胞的96孔细胞培养板中加入细胞生长因子CpG、IL21、IL2、放射照射过的健康人PBMC及含有EBV的B95.8细胞培养上清培养7-10天。用捕获ELISA筛选B细胞培养上清中针对SARS-CoV-2 S蛋白抗体的存在。
实施例3 抗体的克隆
3.1 cDNA合成：对于筛选出上清中有针对SARS-CoV-2 S蛋白抗体存在的B细胞，先提RNA再逆转录成cDNA。
3.2巢式PCR扩增抗体的重链和轻链可变区基因：PCR引物序列及具体PCR扩增过程详见参考文献：J Immunol Methods.2008Jan 1；329(1-2):112-24.，反应使用Phusion超保真DNA聚合酶(Phusion High-Fidelity PCR Master Mix with GC Buffer,NEB,#M0532s)。
3.3抗体的重链及轻链可变区PCR产物经酶切(VH，AgeI/SalI,VK,AgeI/Xhol,VL,AgeI/BsiWI)克隆到含有人IgG1恒定区的抗体表达载体上。将测得重链和轻链可变区基因序列经IMGT/V-Quest分析得单克隆抗体825、843、826和846的序列信息如下表1所示：
表2单克隆抗体825、843、826和846的序列信息表
Figure PCTCN2021135785-appb-000011
实施例4 抗体的特性分析
4.1抗体的生产：将构建的序列确定的抗体的重链和轻链载体共转染293T细胞，5天后收获含抗体的培养上清；
4.2抗体的纯化将含抗体的培养上清进行protein A亲和柱纯化，并测定蛋白含量；
4.3抗体与S蛋白的结合反应及中和反应：
用抗标签的抗体捕获上清液中的S蛋白，建立ELISA法，利用捕获ELISA法分析抗体与S蛋白的结合；
利用生物膜光干涉技术(BLI)分析抗体或组合抗体与RBD或S1的结合；具体地，使用Protein A sensor依次捕获抗标签抗体、带标签RBD或S1蛋白以检测抗体与RBD或S1的结合；
采用BLI技术(串联法)检测抗体两两之间相互竞争与RBD或S1的结合； 具体地，使用Protein A sensor依次捕获抗标签抗体、带标签RBD或S1蛋白、第一抗体及竞争抗体，以检测信号减少或无检测信号判定为竞争；
其中，所述标签均为D7；所用生物分子相互作用分析仪购自ForteBio公司，型号Octet K2。
实验结果：图1为获得的单克隆抗体825、843、826和846及其序列特征图；图2为单克隆抗体825、843、826和846与病毒S蛋白及截短S蛋白结合图；实验结果显示，单克隆抗体825、843和826为RBD抗体，单克隆抗体846为NTD抗体。单克隆抗体846不与RBD结合。其中，单克隆抗体825与S及S1蛋白结合，EC50分别为0.012μg/mL和0.039μg/mL；单克隆抗体843与S及S1蛋白结合，EC50分别为0.002μg/mL和0.006μg/mL；单克隆抗体826与S及S1蛋白结合，EC50分别为0.008μg/mL和0.030μg/mL；单克隆抗体846与S及S1蛋白结合，EC50分别为0.136μg/mL和0.049μg/mL。
如图3所示，还原SDS-PAGE胶图显示单克隆抗体825、843、826和846的重链和轻链大小。
如图4和5所示，，抗体组合825/843/826或825/843/846三个抗体可同时结合到RBD或S1蛋白上。
如图6显示，在抗体相互竞争与RBD或S1蛋白结合实验中，抗体825、843和826分别结合在RBD上不同表位。
4.4抗体中和新冠假病毒的活性
假病毒中和体系的建立：利用含有SARS-CoV-2棘突蛋白S基因的表达质粒与pPAX2/pLV-EGFP(1:1)共转染Expi293F，包装假病毒。
假病毒微量中和实验：吸取50μL样品，加入800 TCID50假病毒，孵育1h后，加入5万/孔HEK293T细胞；继续培养后48h检板。以荧光值减少即抑制率 大于50％为有中和活性。
实验结果：如图7所示，根据抗体对各种突变假病毒中和实验IC50结果可知：抗体825、843、846和826，抗体组合825/843、825/843/826和825/843/846均可有效中和各种新冠突变假病毒；如图8所示为抗体对Lambda假病毒中和实验结果图，由图8A及图8B中可以看出，抗体825、843、826和846中和新冠变异株Lambda株其IC50分别为0.004μg/mL、0.011μg/mL、0.4538μg/mL和>10μg/ml，抗体组合825/843/826和825/843/846其IC50均<0.0004μg/mL，由此可见，抗体825、843、846和826及其抗体组合825/843/826和825/843/846均能高效中和Lambda株。
因此，本发明提供的抗体825、843、846和826及其抗体组合825/843/826和825/843/846均能有效的中和新冠假病毒的活性；且在等量抗体的情况下，相比于单独使用抗体825、843、826和846，抗体组合825/843/826和825/843/846所能达到的中和效果更加好。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 新型冠状病毒的人源抗体，其特征在于，所述人源抗体为825单克隆抗体、843单克隆抗体、826单克隆抗体或846单克隆抗体；
    所述825单克隆抗体含重链可变区和轻链可变区，所述825单克隆抗体的重链可变区含与SEQ ID NO.1所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.2所示氨基酸序列至少90％同源的氨基酸序列；
    所述843单克隆抗体含重链可变区和轻链可变区，所述843单克隆抗体的重链可变区含与SEQ ID NO.3所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.4所示氨基酸序列至少90％同源的氨基酸序列；
    所述826单克隆抗体含重链可变区和轻链可变区，所述826单克隆抗体的重链可变区含与SEQ ID NO.5所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.6所示氨基酸序列至少90％同源的氨基酸序列；
    所述846单克隆抗体含重链可变区和轻链可变区，所述846单克隆抗体的重链可变区含与SEQ ID NO.7所示氨基酸序列至少90％同源的氨基酸序列，所述轻链可变区含与SEQ ID NO.8所示氨基酸序列至少90％同源的氨基酸序列。
  2. 根据权利要求1所述人源抗体，其特征在于，
    所述825单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.1所示，轻链可变区氨基酸序列如SEQ ID NO.2所示；
    所述843单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.3所示，轻链可变区氨基酸序列如SEQ ID NO.4所示；
    所述826单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.5所示，轻链可变区氨基酸序列如SEQ ID NO.6所示；
    所述846单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.7所示，轻链可变区氨基酸序列如SEQ ID NO.8所示。
  3. 根据权利要求1所述人源抗体，其特征在于，编码所述抗体825单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.9所示，编码所述抗体825单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.10所示；
    编码所述抗体843单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.11所示，编码所述抗体843单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.12所示；
    编码所述抗体826单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.13所示，编码所述抗体826单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.14所示；
    编码所述抗体846单克隆抗体重链可变区氨基酸序列的核苷酸序列如SEQ ID NO.15所示，编码所述抗体846单克隆抗体轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO.16所示。
  4. 协同中和新型冠状病毒的人源抗体组合，其特征在于，所述人源抗体组合含有权利要求1-3任一项所述抗体中的至少一个。
  5. 根据权利要求4所述人源抗体组合，其特征在于，所述抗体组合含抗体组合1、抗体组合2、抗体组合3和抗体组合4中的至少一种，其中，
    抗体组合1，由825单克隆抗体、843单克隆抗体、826单克隆抗体和846单克隆抗体组成；
    抗体组合2，由825单克隆抗体、843单克隆抗体和826单克隆抗体组成；
    抗体组合3，由825单克隆抗体、843单克隆抗体和846单克隆抗体组成；
    抗体组合4，由825单克隆抗体、843单克隆抗体组成。
  6. 一种如权利要求1-3任一项所述人源抗体或权利要求4-5任一项所述人源抗体组合在制备中和新型冠状病毒及其变异株药物中的应用。
  7. 根据权利要求6所述应用，其特征在于，所述新型冠状病毒株包括但不限于病毒变异英国株、南非株、巴西株、印度株。
  8. 一种如权利要求1-3任一项所述人源抗体在制备预防或治疗新型冠状病毒SARS-CoV-2感染的药物或诊断试剂或试剂盒中的应用。
  9. 一种如权利要求4-5任一项所述的人源抗体组合在制备预防或治疗新型冠状病毒SARS-CoV-2感染的药物或诊断试剂或试剂盒中的应用。
  10. 根据权利要求6-9任一项所述的应用，其特征在于，所述药物还包括药用辅料。
PCT/CN2021/135785 2021-12-06 2021-12-06 协同中和新型冠状病毒的人源抗体和抗体组合及其应用 WO2023102692A1 (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021174594A1 (zh) * 2020-03-06 2021-09-10 深圳市第三人民医院 一种抗新型冠状病毒的单克隆抗体及其应用
CN113512113A (zh) * 2021-08-03 2021-10-19 浙江大学医学院附属第一医院 人源化广谱高中和活性抗新型冠状病毒单克隆抗体及应用
WO2021212785A1 (zh) * 2020-04-20 2021-10-28 迈威(上海)生物科技股份有限公司 一种全人源单克隆抗体及其应用
CN113563464A (zh) * 2021-08-01 2021-10-29 中国疾病预防控制中心性病艾滋病预防控制中心 人源化高中和活性抗新型冠状病毒单克隆抗体及应用

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Publication number Priority date Publication date Assignee Title
WO2021174594A1 (zh) * 2020-03-06 2021-09-10 深圳市第三人民医院 一种抗新型冠状病毒的单克隆抗体及其应用
WO2021212785A1 (zh) * 2020-04-20 2021-10-28 迈威(上海)生物科技股份有限公司 一种全人源单克隆抗体及其应用
CN113563464A (zh) * 2021-08-01 2021-10-29 中国疾病预防控制中心性病艾滋病预防控制中心 人源化高中和活性抗新型冠状病毒单克隆抗体及应用
CN113512113A (zh) * 2021-08-03 2021-10-19 浙江大学医学院附属第一医院 人源化广谱高中和活性抗新型冠状病毒单克隆抗体及应用

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