WO2023168776A1 - 一种广谱新型冠状病毒的人源抗体及其应用 - Google Patents

一种广谱新型冠状病毒的人源抗体及其应用 Download PDF

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WO2023168776A1
WO2023168776A1 PCT/CN2022/085186 CN2022085186W WO2023168776A1 WO 2023168776 A1 WO2023168776 A1 WO 2023168776A1 CN 2022085186 W CN2022085186 W CN 2022085186W WO 2023168776 A1 WO2023168776 A1 WO 2023168776A1
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seq
antibody
antigen
amino acid
human antibody
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French (fr)
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高福
仝舟
仝剑宇
崔庆为
赵欣
王奇慧
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中国科学院微生物研究所
山西高等创新研究院
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    • 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
    • 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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  • the invention belongs to the field of biomedicine technology, and specifically relates to broad-spectrum human antibodies against novel coronavirus and their applications.
  • the S309 antibody was obtained in vitro from a SARS patient who had been infected for 10 years by using the SARS2 target protein.
  • this type of volunteer have extremely high There are few, and even if broad-spectrum protection is achieved across the span of SARS and COVID-19, there is still no relevant conclusion on the ability of the neutral spectrum to resist new mutations of Covid-19. , only looking at Omicron, many pan-sarbecovirus star antibodies including S2H97, 2-36, MW06, etc. have lost efficacy. What’s even more troublesome is that the SARS2 outbreak did not last long.
  • the display library is based on the abstraction of complex immune reactions occurring in the body from a single perspective of the combination of antigens and antibodies in vitro. Compared with single cell sorting technology, display library technology can quickly realize alternating subtraction screening of different antigens in vitro. Broad-spectrum antibody enrichment, and secondly, antibody display library technology can achieve new combinations of VH/VL in different people, improving the availability of new antibodies by orders of magnitude.
  • the present invention uses new antibody amplification primers to construct a phage display library for recovered patient antibodies. Different from phage display screening in the prior art, the present invention uses the original strain RBD and Beta strain RBD antigen sequential screening mode to quickly focus on conserved targets. Multiple strains of fully human antibodies that bind to different epitopes of RBD and can achieve broad-spectrum neutralization of VOCs were successfully isolated in vitro. Among them, the IMCAS-123 antibody has an affinity of nM for the prototype strain, Alpha, Beta, Delta, and Omicron, and the neutralization of the highly transmissible Omicron mutant strain pseudovirus reaches 0.04ug/ml, which is the best against Omicron escape strains reported so far. of neutralizing antibodies.
  • the present invention first provides broad-spectrum new coronavirus human antibodies or antigen-binding fragments thereof that are effective against the prototype strain, Alpha, Beta, Delta, and Omicron strains of SARS-CoV-2.
  • the human antibody or antigen-binding fragment thereof includes a heavy chain variable region and a light chain variable region
  • the amino acid sequence of CDR3 included in the heavy chain variable region is SEQ ID NO: 15, 19, 20, 21, 27, and 28,
  • the preferred amino acid sequences of CDR1 to 2 included in the heavy chain variable region are respectively: SEQ ID NO: 13 and 14
  • the amino acid sequences of CDR1 to 2 included in the light chain variable region are respectively: SEQ ID NO: 16 and 17.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, 23, 24, and 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26.
  • the full-length amino acid sequence of the heavy chain is as shown in SEQ ID NO: 10, or there is I102S, I102M or M104L, and the full-length amino acid sequence of the light chain is as shown in SEQ ID NO: 12.
  • the human antibody is a single chain antibody.
  • its amino acid sequence is as shown in SEQ ID NO: 8, or there are I216S, I216M, M218L mutations, especially L101F, L101F+H111Y mutations.
  • the present invention further provides nucleic acids encoding the human antibodies or antigen-binding fragments thereof.
  • the full-length nucleotide sequence of the heavy chain is shown in SEQ ID NO: 9, and the full-length nucleotide sequence of the light chain is shown in SEQ ID NO: 11.
  • it is a single-chain antibody, and its nucleotide sequence is shown in SEQ ID NO: 7.
  • the present invention also provides the expression vector or recombinant cell encoding the nucleic acid.
  • the heavy chain and light chain encoding nucleic acids are constructed on the same or different expression vectors.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases caused by SARS-CoV-2, containing the human antibody or antigen-binding fragment thereof as an active ingredient. Furthermore, pharmaceutically acceptable auxiliaries are also included.
  • the present invention also provides the use of the human antibody or its antigen-binding fragment, which is characterized in that it is used in the preparation of drugs for the prevention or treatment of diseases caused by SARS-CoV-2.
  • the SARS-CoV-2 is selected from one or more of the prototype strain, Alpha, Beta, Delta, and Omicron strains.
  • the present invention also provides a human antibody for the new coronavirus, which is characterized by a sequence combination of heavy chain VH3-23, IGHJ1: light chain VK1-12, and IGK4 based on the IMGT website classification principle.
  • IMCAS-123 Comparing the IMCAS-123 screened by this invention with all 4210 different COVID-19 antibody sequences reported around the world, IMCAS-123 has the following five characteristics: the first heavy chain CDR3 AKDHLITMVQPEYFHH amino acid sequence was discovered for the first time; the light chain CDR3: QQADSFPLT sequence was discovered for the first time ; The heavy chain and light chain pairing coding format is based on the classification principles of the IMGT website: heavy chain VH3-23, IGHJ1: light chain VK1-12, IGK4. This sequence combination was discovered for the first time among all 4188 public sequences of new coronavirus antibodies.
  • IMCAS-123 there is no amino acid mutation in the heavy chain of IMCAS-123 (the first amino acid at the N-terminus is caused by the amplification primer). This is the first report that an antibody without any amino acid mutations has the ability to protect all new coronavirus mutant strains.
  • eight antibodies that have been reported on different epitopes confirmed that the IMCAS-123 epitope competes with ACE2, S309, REGN10933, and REGN10987, and it was determined that IMCAS-123 is a type of antibody that can inhibit ACE2 across three known epitopes at the same time.
  • Figure 1 Molecular sieve chromatography of the RBD antigen expression of the Prototype new coronavirus prototype strain in Example 1 and the SDS-PAGE image of the target peak.
  • Figure 2 shows molecular sieve chromatography of RBD antigen expression of the Beta new coronavirus mutant strain in Example 1 and its SDS-PAGE image of the target peak.
  • Figure 5 293F expresses IMCAS-123 full-antibody cross-over Superdex200pg molecular sieve diagram and SDS-PAGE diagram.
  • Example 1 Obtaining IMCAS-123 antibody with broad-spectrum binding ability
  • RNA Discard the supernatant, add 1 ml of 75% ethanol for washing, vortex to mix, centrifuge at 7500g (4°C) for 5 minutes, and discard the supernatant. Let the precipitated RNA dry naturally at room temperature. Dissolve RNA pellet with RNase-free water.
  • HiScript-TS 5'/3'RACE Kit Vazyme reverse transcription kit
  • 2 ⁇ Taq Master Mix enzyme Vazyme
  • reaction conditions are as follows: 95°C, 2min; 95°C, 15s, 58°C (heavy chain/ ⁇ chain/ ⁇ chain), 15s, 72°C, 30s, 35 cycles of 72°C, 7min. 1.2% agarose gel electrophoresis, separate the PCR products, and cut the 400-500bp band to recover.
  • the overlapping product was ligated with the sfiI-digested pcomb3xss (addgene) plasmid at a ratio of 3:1 to form Phagemid.
  • the ligation product was transformed into Top10 competent cells, coated with ampicillin-resistant plates (1:1000), and cultured overnight at 37°C. , collect and maximally purify plasmids from all colonies (obtain a large number of plasmid libraries first, and then convert them into phage libraries based on the plasmid process each time to ensure library uniformity) to obtain a 5-10mg plasmid library.
  • Use a biorad to transform 20ug of plasmid into TG1 competent cells.
  • the 5' end of the protein coding region is preceded by the signal peptide nucleotide sequence ATGTTTGTGTTTCTTGTGCTTCTTCCTCTTGTGTCATCACAATGC, and the 3' end of the protein coding region is connected to the coding sequence of 6 histidine tags (hexa-His-tag) and the translation termination codon.
  • 293T cells were cultured in DMEM containing 10% FBS. Transfect 293T with plasmid. 4-6 hours after transfection, change the medium of the cells into serum-free DMEM and continue to culture for 3 days. Collect the supernatant, add DMEM, culture for another 4 days, and collect the supernatant.
  • Dissolve 100ul of the purified phage into 900ul of dissolution buffer (0.5% BSA, 0.05% PBST), mix well, add 100ul to each well with a volley gun, and incubate at room temperature for 2 hours. Discard the phage solution in the Elisa plate and wash the plate 10 times with 0.05% PBST. Add 100ul of eluent (pH 2.2, 0.1M HCL) into each well using a volute gun, and shake at 400rpm for 20min. Mix the eluates from every 10 wells together to obtain 1000ul of phage.
  • helper phage M13KO7 (9 ⁇ 10 12 pfu/ml) at a ratio of 1:1000, and shake at 220 rpm for half an hour at 37°C. Then transfer it to an Erlenmeyer flask containing 30 ml of 2YT medium, and add ampicillin and kanamycin at a ratio of 1:1000. Shake the culture at 220 rpm for 4 hours at 37°C, add IPTG 1:1000 to the Erlenmeyer flask, and then incubate overnight at 30°C or screen the 5E12pfu phage library. After repeating the above steps three times, the wild-type novel coronavirus RBD protein was changed into the mutant novel coronavirus beta-RBD protein for the fourth round of screening.
  • Sequence and compare the sequences Transform the phage plasmids with a ScFv sequence repeat number greater than 2 into SS320 cells using electroporation technology. Add anti-resistant medium and shake the bacteria at 37°C for 1 hour. Spread 1 ⁇ ampicillin-resistant plate and select single clones. Colonize into 100ul of culture medium containing ampicillin and culture in a 37°C incubator overnight. The next day, add 1.5 ml of SB medium containing 1 ⁇ ampicillin and 20 ml of 1 M Mgcl2, continue to culture in a 37°C incubator at 400 rpm/min for 8 hours, and then add 1 M IPTG at 1:1000 for overnight induction at 37°C.
  • the secondary antibody was goat anti-rabbit IgG-HRP, incubated for 1 hour, and eluted three times with 100ul of 0.1% PBST solution.
  • the scfv with the optimal broad-spectrum binding ability was confirmed to be IMCAS-123, and the SCFV form of the antibody (the amino acid sequence is shown in SEQ ID NO: 8, and the nucleotide sequence is shown in SEQ ID NO: 7).
  • Sequencing analysis shows that the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 10, the full-length amino acid sequence of the light chain is shown in SEQ ID NO: 12, and the corresponding full-length nucleotide sequence of the heavy chain is shown in SEQ ID NO:9 is shown, and the full-length nucleotide sequence of the light chain is shown as SEQ ID NO:11.
  • CDR1 GTFFSSYA (SEQ ID NO: 13)
  • CDR2 ISGSGGST (SEQ ID NO: 14)
  • CDR3 AKDHLITMVQPEYFHHW (SEQ ID NO: 15).
  • Light chain CDR1-QGISRW SEQ ID NO: 16
  • CDR2 AAG (SEQ ID NO: 17)
  • CDR3 CQQADSF (SEQ ID NO: 18).
  • the supernatant was combined with HisTrapTM HP affinity column overnight, and the target protein was eluted from the His column with 10% (20mM Tris, 150mM NaCl, pH 8.0, 300mM imidazole), and 10KD protein concentration tube with buffer ( 20mM Tris, 150mM NaCl, pH 8.0) for liquid change to remove the imidazole concentration in the protein solution and concentrate it to a volume of less than 500 ⁇ l.
  • AKTA-purifier (GE) and superdex75 Increase 10/300 GL molecular sieve (GE) to balance the concentrated protein solution, balance the molecular sieve with (20mM Tris, 150mM NaCl, pH 8.0), load the 500 ⁇ l loop, and monitor the UV at 280nm. Absorption value, collect the target protein, and identify the protein purity through SDS-PAGE.
  • the molecular sieve pattern and SDS-PAGE pattern of the target protein showed that the purified IMCAS 123 scfv protein was obtained (as shown in Figure 3).
  • the surface plasmon resonance phenomenon is used to detect intermolecular interactions, which is completed on the biomacromolecule interaction analysis system Biacore 8K produced by GE Healthcare Group.
  • Biacore 8K biomacromolecule interaction analysis system
  • SA chip biotin-streptavidin coupling method
  • the antibody IMCAS-123 with concentration gradients of 6.25nM, 12.5nM, 25nM, 50nM and 100nM was injected into the chip.
  • the analysis was performed at a constant temperature of 25°C, and the buffer used was 0.05% PBST.
  • the binding curve is as shown in the figure.
  • the curves of different concentrations form the kinetic curve shown in the figure. Calculation of binding kinetic constants was performed using BIA evaluation software version 3.2 (Biacore, Inc.). The results are shown in Figure 4.
  • the affinity constant of antibody IMCAS-123 and SARS-CoV-2 Prototype RBD protein is 0.18nM, and the affinity constant of antibody IMCAS-123b and SARS-CoV-2 Alpha variant RBD protein is 0.15nM.
  • the affinity constant of antibody IMCAS-123 and SARS-CoV-2 Beta variant RBD protein is 0.32nM; the affinity constant of antibody IMCAS-123 and SARS-CoV-2 Delta variant RBD protein is 0.48nM, the affinity constant of antibody IMCAS-123 and SARS-CoV
  • the affinity constant of -2 Lamda variant RBD protein is 0.45nM, and the affinity constant of antibody IMCAS-123 and SARS-CoV-2 Omicron variant RBD protein is 1.41nM.
  • the above data shows that antibody IMCAS-123 has a strong affinity with SARS-CoV-RBD. Strong affinity.
  • Heavy chain H CMV promoter-EcoRI-signal peptide (SP)-heavy chain variable region (VH)-heavy chain constant region (CH)-Xhol;
  • Light chain kappa CMV promoter-EcoRI-signal peptide (SP)-light chain variable region (VK)-light chain constant region (CL ⁇ )-Xhol;
  • the light and heavy chain variable region sequences and the corresponding expression vector pCAGGS containing the constant regions of the heavy chain CH and the light chain CL ⁇ were connected through homologous recombination and cloned into the expression vector pCAGGS to obtain the light and heavy chain codes containing the specific antibody.
  • the plasmids encoding the IMCAS-123 light and heavy chain genes were co-transfected into 293F cells at a density of 3*10 ⁇ 6 at a heavy chain: light chain ratio of 1:1.5. Dilute the plasmid with 150mM NaCl and add 1ug of plasmid to 1ml of cells. Dilute 1mg/ml of PEI with 150mM NaCl and add 3ul of PEI to 1ml of cells. Let stand for 5 minutes. Mix the above two and let stand for 20min. Add 293F cells drop by drop. 24 hours after transfection, add 0.035 ml of feeding solution to 1 ml, and then add feeding solution every 48 hours.
  • pipette tip sterile
  • round-bottom 96-well plate 10cm cell culture dish
  • flat-bottom 12-well plate flat-bottom 96-well plate
  • flow cytometry fixative flow cytometry tube
  • each mutant plasmid of pCAGGS-SARS-CoV-2-S was transfected into a 10cm2 dish of 293T cells (cell volume 80%-90%). After 4 hours, the medium was changed to DMEM (10% FBS). 24 hours after transfection, VSV was added.
  • - ⁇ G-GFP pseudovirus 5ml change the medium after 2 hours, add DMEM 10% FBS, containing VSVG antibody 1:1000 (10mg/ml by I1Hybridoma CRL2700 TM cell expression, final concentration 10ug/ml), collect the supernatant 20h after adding pseudovirus, centrifuge at 3000rpm for 10min, and pass through a 0.45 filter membrane. Freeze-80 in aliquots. For cells that were not transfected with S protein, the VSV- ⁇ G-GFP pseudovirus and antibody were subsequently added as a pseudovirus packaging control.
  • Pseudovirus was treated with 0.5U/ ⁇ l BaseMuncher endonuclease (Abcam, ab270049) for 1.5 hours.
  • DMEM 10% FBS 044 to dilute the pseudovirus 3 times in a gradient (2*, 6*), add 100ul/well to a 96-well plate, and make three parallel wells for each sample (6 holes for each pseudovirus and each cell, A total of 8 kinds of fake poisons) CQ1 takes pictures and reads after 15 hours to calculate the titer.
  • IMCAS-123 has a very good neutralizing effect on pseudoviruses of all VOCs virus strains proposed by the WHO. It is currently the only known antibody that can neutralize all three Omicron mutants.
  • the biomolecule interaction analyzer Octet red96 was used to detect whether the binding of IMCAS-123 to SARS-CoV-2 Prototype RBD competes with hACE2 and other antibodies.
  • Another antibody at 400nM is passed through the tip in the presence of 400nM IMCAS-123 antibody.
  • IMCAS123 binding site site-directed mutagenesis was carried out for the HCDR3 region through a primer array, and it was converted into scFv protein expression through Example 4 (Small-scale expression and identification method of candidate antibody SS320 prokaryotic cells), and Example 5 (Octet detection of IMCAS-123 Competitive experiments with other confirmed target-binding antibodies) were used to verify the result characteristics after mutation.
  • the results are shown in Figure 8. The results showed that three mutations, I102S, I102M, and M104L, showed an increase in antibody binding speed.
  • the corresponding HCDR3 sequences after mutation are AKDHL S TMVQPEYFHHW, AKDHL M TMVQPEYFHHW, and AKDHLIT L VQPEYFHHW.
  • Further research screened out two mutations, L101F and L101F+H111Y (the corresponding HCDR3 sequences after the mutation are AKDHFITMVQPEYFHHW and AKDHFITMVQPEYFYHW respectively). The results showed that the neutralizing ability of the antibody against the four subtypes of pseudoviruses of the Omicron mutant strain was more significant. The improvements are shown in Figures 9 and 10 respectively.

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Abstract

本发明涉及广谱新型冠状病毒的人源抗体及其应用。利用原型株RBD与Beta毒株RBD抗原递次筛选模式，快速聚焦保守靶位，成功在体外分离到结合在RBD不同表位的能对VOC实现广谱中和的全人源抗体。其中，IMCAS-123抗体对原型株、Alpha、Beta、Delta、Omicron亲和力达到nM水平，对高传播力Omicron突变株假病毒中和达到0.04ug/ml，是目前已有报道中唯一对Omicron三种逃逸株BA.1 BA.2 BA.3均可中和的抗体。

Description

一种广谱新型冠状病毒的人源抗体及其应用 技术领域
本发明属于生物医药技术领域，具体涉及广谱新型冠状病毒的人源抗体及其应用。
背景技术
筛选广谱高中和活性的新冠病毒抗体，对于临床预防与治疗具有重要的现实意义。
新冠暴发后全人类通过疫苗构筑了最大规模预存免疫，然而随着病毒在人群持续传播，产生的多种突变给全球的疫苗免疫防线带来极大压力，目前Omicron出现不但跨越了多种疫苗的免疫防线，全球8个上市抗体仅存S309尚可以继续抵御，Omicron更具备了跨种传播感染小鼠等啮齿类动物的可能性。值得警惕的是，目前虽然Omicron表现出对人致病力降低，但是长远看高传播力的病毒一旦进入非人类宿主，是否免疫压力改变而带来的新型突变将不在适应人类，进而产生对人类免疫逃逸与致病力双加强的新毒株。这些科学问题都迫使科学家加快在人类免疫群体中，阐明冠状病毒交叉保护能力机制，同时对抗突变广谱抗体研究重要再一次被突出到显著位置。
然而目前研究的技术手段存在局限，全球利用新冠康复病人外周血细胞测序产出的2988抗体主要针对的是缺乏抗突变能力的免疫优势表位，鲜有针对保守表位的广谱中和能力，多个顶级期刊归纳分类文章已经关注到这个问题。从进化选择来说保守表位只有弱免疫源性才能在宿主免疫对抗中适应生存，针对保守位点免疫原性低的特点，在流感广谱抗体研究中，科学家进一步通过不同抗原的免疫聚焦、续贯免疫的策略提升“抗体的有效调动”继而获得广谱抗体，例如S309抗体是从一位感染10年的SARS康复患者中体外利用SARS2靶蛋白获得的，但是这类志愿者的不但存在极少，而且即便在SARS与COVID-19跨度上实现广谱保护，其中和谱对Covid-19新生突变的抵御能力尚缺相关性定论。，仅以Omicron来看包括S2H97、2-36、MW06等多个pan-sarbecovirus明星抗体出现效能丧失。更棘手的是，SARS2暴发事件不长，对于同一人先后感染不同VOC病毒株的机率或是对同一人交替免疫不同VOC疫苗的可能性极小，这驱使研究人员必须另辟蹊径挖掘这种弱免疫原性的广谱抗体。体外操作的抗体展示库技术的独特优势，可在应对Covid-19突变株广谱抗体的难题上发挥重要作用。本质上来说，展示库是基于体外从抗原与抗体的结合的单一角度抽象体内发生的复杂免疫反应，展示库技术与单细胞分选技术相比可以在体外快速实现不同抗原的交替差减筛选实现广谱抗体富集，其次抗体展示库技术可以实现不同人中VH/VL的全新组合从数量级水平提升了新抗体可获得性。
发明内容
本发明利用全新抗体增扩引物构建康复病人抗体噬菌体展示库，与现有技术中噬菌体展示筛选不同，本发明利用原性株RBD与Beta毒株RBD抗原递次筛选模式，快速聚焦保守靶位，成功在体外分离到多株结合在RBD不同表位的能对VOC实现广谱中和的全人源抗体。其中IMCAS-123抗体对原型株、Alpha、Beta、Delta、Omicron亲和力达到nM水平，对高传播力Omicron突变株假病毒中和达到0.04ug/ml，是目前已有报道中对Omicron逃逸株最好的中和抗体。
因此，本发明首先提供针对SARS-CoV-2的原型株、Alpha、Beta、Delta、Omicron株均有效的广谱新型冠状病毒的人源抗体或其抗原结合片段。
在个优选实施方式中，所述人源抗体或其抗原结合片段包括重链可变区和轻链可变区，重链可变区包括的CDR3的氨基酸序列是SEQ ID NO：15、19、20、21、27、28所示，优选地重链可变区包括的CDR1至2的氨基酸序列分别是：SEQ ID NO：13和14所示；其轻链可变区包括的CDR3的氨基酸序列是：SEQ ID NO：18，优选地，包括轻链可变区包括的CDR1至2的氨基酸序列分别是：SEQ ID NO：16、17所示。
更优选地，其重链可变区氨基酸序列如SEQ ID NO：22、23、24、25所示，轻链可变区氨基酸序列如SEQ ID NO：26所示。
进一步优选地，重链的全长氨基酸序列如SEQ ID NO：10所示，或者存在I102S、I102M或M104L，轻链的全长氨基酸序列如SEQ ID NO：12所示。
在一个具体实施方式中，所述人源抗体为单链抗体。优选地，其氨基酸序列如SEQ ID NO：8所示，或者存在I216S、I216M、M218L突变，尤其是L101F、L101F+H111Y突变。
本发明进一步提供所述的人源抗体或其抗原结合片段的编码核酸。优选地，重链的全长核苷酸序列如SEQ ID NO：9所示，轻链的全长核苷酸序列如SEQ ID NO：11所示。或者其为单链抗体，其核苷酸序列如SEQ ID NO：7所示。
由此，本发明还提供所述的编码核酸的表达载体或重组细胞。具体地，对于全抗而言，所述重链和轻链编码核酸构建在同一个或不同的表达载体上。
进而，本发明提供含有所述的人源抗体或其抗原结合片段作为有效成分的预防或治疗SARS-CoV-2引起的疾病的药物组合物。进一步地，还包括药学上可接受助剂。
同时，本发明还提供所述的人源抗体或其抗原结合片段的用途，其特征在于，在制备治疗SARS-CoV-2引起的疾病的预防或治疗的药物中应用。其中，所述SARS-CoV-2选自原型株、Alpha、Beta、Delta、Omicron株中一种或多种。
此外，本发明还提供一种新型冠状病毒的人源抗体，其特征在于，基于IMGT网站分类原则由重链VH3-23，IGHJ1：轻链VK1-12，IGK4的序列组合。
本发明筛选到的IMCAS-123与全球已经报道的全部4210条不同新冠抗体序列比较，IMCAS-123具有以下五个特点：第一重链CDR3 AKDHLITMVQPEYFHH氨基酸序列首次发现；轻链CDR3：QQADSFPLT序列首次发现；重链轻链配对编码形式基于IMGT网站分类原则属于：重链VH3-23，IGHJ1：轻链VK1-12，IGK4这个序列组合方式在新冠抗体全部4188条公开序列中为首次发现。其次，IMCAS-123的重链未发生氨基酸突变(N端首个氨基酸是由于增扩引物导致)，是首次报道未有任何氨基酸突变的抗体实现对全部新冠突变株的保护能力。此外，通过8个已报道在不同表位的抗体确认IMCAS-123表位与ACE2、S309、REGN10933、REGN10987均发生竞争，判定IMCAS-123是一类跨三个已知表位间同时可以抑制ACE2结合抗体，从2022年2月1日前全部已发表抗体数据来看，123是首次被证明可以在假病毒水平中和Omicron新冠突变株BA.1、BA.2、BA.3三种分型的抗体。因此，本发明提供广谱新型冠状病毒的人源抗体具有重大的应用价值。
附图说明
图1实施例1中Prototype新冠原型株RBD抗原表达的分子筛层析及其目的峰的SDS-PAGE图。
图2实施例1中Beta新冠突变株RBD抗原表达的分子筛层析及其目的峰的SDS-PAGE图。
图3纯化的IMCAS 123 scfv蛋白的分子筛图谱和SDS-PAGE图。
图4 IMCAS-123与SARS-CoV-RBD亲和力检测(biacore-8k)。
图5 293F表达IMCAS-123全抗过Superdex200pg分子筛图和SDS-PAGE图。
图6 IMCAS-123对WHO提出的全部VOCs病毒株的假病毒中和实验。
图7 IMCAS-123与ACE2/其他抗体竞争性结合验证(Octet)。
图8 IMCAS-123三种点突变与新冠不同突变株RBD结合验证(Octet)。
图9 L101F点突变与新冠不同突变株RBD结合验证(Octet)。
图10 L101F+H111Y点突变与新冠不同突变株RBD结合验证(Octet)。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,第四版)中所述的条件，或按照制造厂商所建议的条件。
实施例1：广谱结合能力IMCAS-123抗体的获得
1.康复病人外周血淋巴细胞RNA提取与人源单链抗体噬菌体展示文库的建立
在12名感染新型冠状病毒且痊愈出院的人员知情同意下，各采集3-10mL的血液，分离PBMCs，转入1.5mlEP管中。加入700mltrizol并放置5分钟，在上述EP管中，加入0.14ml氯仿，盖上EP管盖子，剧烈震荡15秒，室温静置3分钟，12000g(4℃)离心15分钟。取上层水相置于新EP管中，加入0.5ml异丙醇，在室温下静置10分钟，12000g(4℃)离心10分钟。弃上清，加入1ml 75％乙醇进行洗涤，涡旋混合，7500g(4℃)离心5分钟，弃上清。让沉淀的RNA在室温在自然干燥。用Rnase-free water溶解RNA沉淀。
通过HiScript-TS 5'/3'RACE Kit(Vazyme)逆转录试剂盒，按说明书分别增扩VH、VL的DNA模板后，用2×Taq Master Mix酶(Vazyme)进行PCR，通过引物组合扩增抗体可变区序列，反应条件如下：95℃，2min；95℃，15s，58℃(重链/κ链/λ链)，15s，72℃，30s，35个循环，72℃，7min。1.2％的琼脂糖凝胶电泳，分离PCR产物，将400-500bp的条带切胶回收。等摩尔比将12个人VH混合一起，同法混合VL片段后，将混合后VH与VL进行等摩尔比混合后用PCR搭桥引物连接抗体基因的重链轻链可变区，使用2×Taq Master Mix酶(Vazyme)进行PCR，反应条件如下：95℃，2min；95℃，15s，67℃，15s，72℃，30s，30个循环，72℃，7min增扩出完整的单链抗体scfv(VH-VL)，1.2％的琼脂糖凝胶电泳，分离PCR产物，将750-800bp的条带切胶回收。将Overlapping后的产物与sfiI酶切后的pcomb3xss(addgene)质粒按照3：1的比例连接形成Phagemid，连接产物转化Top10感受态细胞，涂氨苄抗性平板(1：1000)，37℃过夜培养后，将所有菌落收集大提质粒(先获得大量质粒文库，然后基于质粒每次按流程转换为噬菌体文库，以保证库均一性)，得到5-10mg质粒文库。将20ug质粒使用电转仪(biorad)转化入TG1感受态细胞，1mlSOC37度1小时慢摇后，加入5ml氨苄抗性培养基，40分钟后加入5E7个辅助噬菌体，1小时后，转入125ml锥形瓶，加入氨苄西林和卡那霉素双抗LB，25ml，30度过夜，可增扩得到5E12pfu/ml的噬菌体文库。
2.关键抗原制备
合成野生型新型冠状病毒刺突蛋白受体结合域段(RBD)蛋白(氨基酸序列如SEQIDNO：1所示、核苷酸序列如SEQIDNO：2所示)Beta型新型冠状病毒刺突蛋白受体结合域段(RBD) 蛋白(氨基酸序列如SEQIDNO：3所示、核苷酸序列如SEQIDNO：4所示)通过EcoRI与XhoI两个酶切位点构建到pCAGGS质粒载体上。其中蛋白编码区5‘端前置信号肽核苷酸序列ATGTTTGTGTTTCTTGTGCTTCTTCCTCTTGTGTCATCACAATGC，同时蛋白编码区的3’端连上6个组氨酸标签(hexa-His-tag)的编码序列及翻译终止密码子。用含10％FBS的DMEM培养293T细胞。用质粒转染293T。转染4-6小时后给细胞换液成无血清的DMEM继续培养3天，收集上清，并补加DMEM，再培养4天，收集上清。收集的上清经过5000rpm离心30min后，与含有20mM磷酸钠(pH 8.0)的缓冲液等体积混合，经过0.22μm滤膜过滤后，与His-trapExcel预装柱结合(5mL，GE Healthcare)。以10mM咪唑洗脱结合的蛋白。收集此蛋白浓缩后进行分子筛层析。目的峰通过SDS-PAGE确定，结果如图1说明Prototype新冠原型株RBD抗原目标蛋白得到正常表达，图2说明Beta新冠突变株RBD抗原正常表达。
3.人源单链抗体噬菌体展示文库筛选
取纯化的野生型新型冠状病毒RBD蛋白，用PBS(Ph7.4)溶液按5ng/ul，每孔100ul包被96孔板，置于4℃冰箱中过夜包被。过夜包被后，弃去各孔内的包被液用0.05％的PBST溶液洗去未吸附的抗原，每孔加入65ul浓度为0.5％的BSA封闭半小时，随后加入40ul 10％吐温-20室温下静置半小时30min后，弃去封闭液，0.05％的PBST洗板3次。取100ul纯化后的前述噬菌体溶入900ul溶解buffer(0.5％的BSA0.05％的PBST)中混匀，用排枪向每孔中加入100ul，室温下孵育2h。弃去Elisa板中的噬菌体溶液，用0.05％的PBST洗板10次。用排枪向每孔内加入100ul洗脱液(pH＝2.2,0.1M HCL)，400rpm摇20min。将每10个孔内的洗脱液混合在一起，得到1000ul噬菌体。每管噬菌体加入200ul终止液(1M Tris和0.5％BSA 1:1混合)，收集在2ml离心管内。取1管XLI-Blue感受态细胞(约100ul)接入5ml液体LB培养基中，摇至OD600介于0.6至0.8时停止。向5ml菌液中加入600ul前述噬菌体，转移至50ml无菌离心管中，37℃下220rpm摇菌半小时。向50ml管中加入1/1000的比例氨苄西林37℃下220rpm接菌，摇至OD值0.8左右。按1：1000的比例加入辅助噬菌体M13KO7(9×10 12pfu/ml)，37℃下220rpm摇菌半小时。随后转入到含有30ml 2YT培养基的锥形瓶，1：1000加入氨苄西林和卡那霉素。37℃下220rpm摇菌4小时，向锥形瓶中1:1000加入IPTG 30℃下过夜培养后或的5E12pfu噬菌体筛选文库。重复上述步骤三次后，将野生型新型冠状病毒RBD蛋白变化为突变新型冠状病毒beta-RBD蛋白开展第四轮筛选。将第四轮洗脱的噬菌体，20ul接入摇至OD600介于0.6至0.8时的XLI-Blue感染20分钟后，涂布LB平板，37度过夜静置后，挑选384个单克隆进行菌落PCR，使用2×Taq Master Mix酶(Vazyme)进行PCR，反应条件如下：95℃，2min；95℃，15s，67℃，15s，72℃，30s，30个循环，72℃，7min 增扩。1.2％的琼脂糖凝胶电泳，分离PCR产物，将850-1000bp的阳性条带切胶回收。
4.候选抗体SS320原核细胞小规模表达与鉴定
测序并进行序列比对，将所ScFv序列重复数大于2的噬菌体质粒，利用电转技术转化进入SS320细胞，加入无抗培养基在37℃摇菌1h，涂1‰氨苄抗性平板，挑单克隆菌落到100ul含有氨苄的培养基中，37℃培养箱中过夜培养。次日，接入到1.5ml的含有1‰氨苄和20ml 1M Mgcl2的SB培养基中,继续在400rpm/min的37℃培养箱中培养8h后1：1000加入1M的IPTG37℃过夜诱导。次日，将诱导完成的菌液收集，6500rpm，4℃，离心30min，收集上清后用0.22μm滤膜进行推滤。上清加入包被有野生型和Beta突变型、Omicron新型冠状病毒RBD蛋白(氨基酸序列如SEQIDNO：5所示、核苷酸序列如SEQIDNO：6所示)的96孔板中进行ELISA实验，每孔按顺序加入100ul试表达溶液，每个样品加3个孔，室温下静置1h。100ul 0.1％PBST溶液洗脱洗3次。按照1：2500的比例在0.1％的PBST中稀释一抗(兔抗HA)，注意避光保存。用排枪向每孔中加入100ul一抗稀释液，室温下孵育1h。100ul 0.1％PBST溶液洗脱洗3次。二抗为羊抗兔IgG-HRP，孵育1h，100ul 0.1％PBST溶液洗脱洗3次。向每孔中加入50ul显色液TMB，37℃反应10-20min至显色适当时，立即加入50ul 2M浓HCl终止显色反应。
确认最优广谱结合能力的scfv为IMCAS-123，所述抗体SCFV形式(氨基酸序列如SEQIDNO：8所示、核苷酸序列如SEQIDNO：7所示)。
测序分析表明，其中重链的全长氨基酸序列如SEQ ID NO：10所示，轻链的全长氨基酸序列如SEQ ID NO：12所示，对应的重链的全长核苷酸序列如SEQ ID NO：9所示，轻链的全长核苷酸序列如SEQ ID NO：11所示。
进一步分析可知，重链的CDR1：GFTFSSYA(SEQ ID NO：13)、CDR2：ISGSGGST(SEQ ID NO：14)、CDR3：AKDHLITMVQPEYFHHW(SEQ ID NO：15)。轻链CDR1-QGISRW(SEQ ID NO：16)、CDR2：AAG(SEQ ID NO：17)、CDR3：CQQADSF(SEQ ID NO：18)。
与截至2022年1月全球已经报道的全部4210条不同新冠抗体序列比较具有以下三个首次特点，第一重链CDR3(AKDHLITMVQPEYFHH)氨基酸序列首次发现；轻链CDR3(QQADSFPLT)序列首次发现；重链轻链配对形式：VH3-23，IGHJ1：VK1-12，IGK4序列首次发现。其次，IMCAS-123的重链未发生氨基酸突变(N端首个氨基酸是由于增扩引物导致)，是首次报道未有任何氨基酸突变的抗体实现对全部新冠突变株的保护能力。
5.抗体SS320原核细胞大规模表达
挑IMCAS-123单克隆SS320菌落到1ml含有氨苄的培养基中，37℃培养箱中过夜培养。次 日，接入到10ml的含有1‰氨苄和20ml 1M Mgcl2的SB培养基中,继续在180rpm/min的37℃培养箱中培养8h后1：1000加入1M的IPTG 37℃过夜诱导。次日，将诱导完成的菌液收集，6500rpm，4℃，离心30min，收集上清后用0.22μm滤膜进行真空抽滤。接着将上清用HisTrapTM HP亲和柱结合过夜，将目的蛋白用10％的(20mM Tris,150mM NaCl，pH 8.0,300mM imidazole)从His柱上洗脱下来，并以10KD蛋白浓缩管用缓冲液(20mM Tris,150mM NaCl，pH 8.0)进行换液，以去除蛋白溶液里的咪唑浓度，并浓缩到体积小于500μl。将浓缩完成的蛋白溶液使用AKTA-purifier(GE)和superdex75 Increase 10/300 GL分子筛(GE)，用(20mM Tris,150mM NaCl，pH 8.0)平衡分子筛，500μl loop环上样，同时监测280nm的紫外吸收值，收取目的蛋白，并通过SDS-PAGE鉴定蛋白纯度。
目的蛋白的分子筛图谱和SDS-PAGE图表明获得了纯化后的IMCAS 123 scfv蛋白(如图3所示)。
实施例2、表面等离子共振技术检测蛋白与抗体亲和力(biacore 8k)
利用表面等离子共振现象检测分子间相互作用，在GE Healthcare集团生产的生物大分子相互作用分析系统Biacore 8K上完成。使用生物素-链霉亲和素偶联法(SA芯片)捕获Prototype RBD，Alpha variant RBD，Beta variant RBD，Delta variant RBD,Lamdavariant RBD,Omicron variant RBD蛋白作为固定相，流动相为需要检测的IMCAS-123新冠中和抗体蛋白，之后通过BIA evaluation软件分析动力学参数并作图。
实验步骤：利用生物素-链霉亲和素的偶联作用，我们首先将Prototype RBD，Alpha variant RBD，Beta variant RBD，Delta variant RBD,Lamda variant RBD,Omicron variant RBD蛋白与生物素化试剂按比例室温放置30分钟，将蛋白进行生物素化标记，之后用浓缩管换液至PBS，去除多余的生物素化试剂。将生物素化的抗原蛋白SARS-CoV-2 Prototype RBD，SARS-CoV-2 Alpha variant RBD，SARS-CoV-2 Beta variant RBD，SARS-CoV-2 Delta variant RBD,SARS-CoV-2 Lamda variant RBD,SARS-CoV-2 Omicron variant RBD以10μg/ml的浓度固定在SA芯片(GE)上。
然后将浓度梯度为6.25nM，12.5nM，25nM，50nM和100nM的抗体IMCAS-123注入芯片，分析在恒温25℃进行，使用的缓冲液是0.05％PBST。芯片表面的再生使用的是10mM PH＝1.7的Glynice溶液，结合曲线如图所示，不同浓度的曲线组成图示的动力学曲线。结合动力学常数的计算是利用BIA evaluation software version 3.2(Biacore,Inc.)软件进行的。结果如图4所示，抗体IMCAS-123和SARS-CoV-2 Prototype RBD蛋白的亲和力常数为0.18nM，抗体IMCAS-123b和SARS-CoV-2 Alpha variant RBD蛋白的亲和力常数为0.15nM。抗体 IMCAS-123和SARS-CoV-2 Beta variant RBD蛋白的亲和力常数为0.32nM；抗体IMCAS-123和SARS-CoV-2 Delta variant RBD蛋白的亲和力常数为0.48nM，抗体IMCAS-123和SARS-CoV-2 Lamda variant RBD蛋白的亲和力常数为0.45nM，抗体IMCAS-123和SARS-CoV-2 Omicron variant RBD蛋白的亲和力常数为1.41nM，上述数据表明：抗体IMCAS-123与SARS-CoV-RBD有很强的亲和力。
实施例3、抗体IgG全抗构建和表达及纯化
1.抗体IgG全抗构建
为了获得人源抗体进行后续评价，设计了全抗IgG1构建。策略如下：
重链H：CMV promoter-EcoRI-信号肽(SP)-重链可变区(VH)-重链恒定区(CH)-Xhol；
轻链κ：CMV promoter-EcoRI-信号肽(SP)-轻链可变区(VK)-轻链恒定区(CLκ)-Xhol；
分别将轻重链可变区序列与相应的含由重链CH和轻链CLκ的恒定区的表达载体pCAGGS，通过同源重组连接，克隆至表达载体pCAGGS中，得到含有特定抗体轻、重链编码基因的重组质粒；其中，使用酶切位点ScaI和KpnI将轻重链可变区连入含有恒定区的载体中。
2.全抗的表达及纯化
用IMCAS-123轻、重链编码基因的质粒按照重链：轻链1：1.5比例共转染密度3*10^ 6 293F细胞。用150mM NaCl稀释质粒1ml细胞加1ug质粒，用150mM NaCl稀释1mg/ml PEI 1ml细胞加3ul，静置5min；上述二者混匀后静置20min，逐滴加入293F细胞。转染24h后按照1ml加0.035ml补料液，随后每48h加一次补料液。
转染5d后收上清，6500rpm离心30min去除细胞沉淀，与含有20mM磷酸钠(pH 7.4)等体积混合，经过0.22um滤膜过滤后，与protein A预装柱结合(5mL,GE Healthcare)。以10mM甘氨酸(pH 3.0)洗脱结合的蛋白。收集此蛋白浓缩后进行分子筛层析。目的峰通过SDS-PAGE确定，结果如图5所示。
实施例4、抗体IMCAS-123与SARS-CoV-2假病毒中和实验
1.准备部分：
样品：
IMCAS-123全抗
假病毒(WT，Alpha，Beta，Delta，Omicron)
耗材：枪头(无菌)，圆底96孔板，10cm细胞培养皿,平底12孔板，平底96孔板，流式固定液，流式管
试剂：DMEM+10％FBS(044)
假病毒包装：
pCAGGS-SARS-CoV-2-S各突变体质粒各30μg,转染293T细胞10cm2盘(细胞量80％-90％)，4h后换液DMEM(10％FBS)，转染后24h，加入VSV-ΔG-GFP假病毒5ml，2h后换液，加入DMEM 10％FBS，含VSVG抗体1:1000(10mg/ml由I1Hybridoma
Figure PCTCN2022085186-appb-000001
CRL2700 TM细胞表达，终浓度10ug/ml)，加入假病毒后20h收上清，3000rpm,10min离心，过0.45滤膜。分装冻-80。未转染S蛋白的细胞，后续同样加入VSV-ΔG-GFP假病毒和抗体的组作为假病毒包装对照。
假病毒颗粒定量：
假病毒用0.5U/μlBaseMuncher endonuclease(Abcam,ab270049)处理1.5小时。
提取RNA，用L蛋白的引物进行QPCR。并根据结果进行一致化处理。
假病毒滴度测定：
将vero细胞铺96孔板，24小时长到90％；
将假病毒用DMEM(10％FBS 044)3倍梯度稀释(2*，6*)，100ul/孔加入96孔板，每个样做三孔平行(每个假病毒每种细胞6个孔，共8种假毒)15h之后CQ1拍照读数，计算滴度。
2.中和实验：
将vero细胞铺96孔板，24小时长到90％；实验当天从-80℃取出灭活血清，冰上融化(血清需提前56℃30min灭活)。取DMEM培养基(10％FBS 044)倒入一个10cm皿，用于稀释血清。稀释抗体(初始200ug/ml)(3个重复，2倍稀释，8个梯度)，稀释假病毒(到1000TU/50ul/assay)。
将稀释好的假病毒倒入10cm皿，加入96孔板(与培养基体积1:1，即1个重复孔60ul培养基+60ul假病毒)，吹打混匀1次。将96孔板放入37℃孵育1h(如果超过2块，可两两摞起来，保证受热均匀)；孵育时间至40-50min，取出培养箱中事先准备好的vero细胞，将抽泵功力调至50％，吸尽vero细胞上清，加入血清与病毒的混合液100ul。
37℃孵育15h后，用CQ1显微镜读数法检测绿色荧光并拍照、计数。
实验结果如图6所示，其中半抑制率数据如下表：
  WT Alpha Beta Gamma Delta BA.1 BA.2 BA.3
半抑制率 0.007867 0.03605 0.02559 0.02952 0.6633 0.04397 0.03324 0.1195
由结果可见，IMCAS-123对WHO提出的全部VOCs病毒株的假病毒均非常好的中和作用，是目前已知唯一对三种Omicron突变型均可以中和的抗体。
实施例5、Octet检测IMCAS-123与其它已确认结合靶位抗体的竞争性实验
利用生物分子相互作用分析仪Octet red96检测IMCAS-123与SARS-CoV-2 Prototype RBD的结合是否和hACE2以及其他抗体存在竞争关系。使用生物素-链霉亲和素偶联法(SA芯片)捕获Prototype RBD(10ug/ml)，使其响应值在一个合适的值。然后将需检测抗体稀释到浓度为400nM，每个孔的体积为200μl。先通过浓度为400nM的IMCAS-123抗体，使其与SARS-CoV-2 Prototype RBD的结合达到饱和状态，400nM的另外一个抗体在400nM IMCAS-123抗体存在的条件下通过尖端，同样的方法，先将另一个抗体通过尖端，使其与抗原的结合达到饱和状态，再将含有同样浓度抗体和IMCAS-123的混合溶液通过尖端，进行反向验证，利用Octet RED96系统(FortéBio)的生物膜干涉仪(BLI)进行实时关联和解离，所有实验均在室温环境下进行，最后使用Octet数据分析软件对数据进行处理，并得出结合曲线。结果如图7所示，表明在与全部8种已知位点的代表抗体竞争中：IMCAS-123表位与ACE2、S309、REGN10933、REGN10987、C104均发生竞争，判定IMCAS-123是一类跨越RBD2、RBD4、RBD5三个已知表位同时可以抑制ACE2结合的抗体，这也是首次发现此类结合特点抗体。
实施例6、IMCAS-123的HCDR3区定点突变
根据IMCAS123结合位点特点通过引物阵列，针对HCDR3区开展定点突变，通过实施例4(候选抗体SS320原核细胞小规模表达与鉴定方式)变为scFv蛋白表达，通过实施例5(Octet检测IMCAS-123与其它已确认结合靶位抗体的竞争性实验)方式验证突变后结果特性，结果如图8所示。结果显示I102S、I102M、M104L三种突变，表现出抗体结合速度提升的特点。对应突变后HCDR3序列分别为AKDHL STMVQPEYFHHW、AKDHL MTMVQPEYFHHW、AKDHLIT LVQPEYFHHW。进一步研究筛选到L101F、L101F+H111Y两种突变(对应突变后HCDR3序列分别为AKDHFITMVQPEYFHHW、AKDHFITMVQPEYFYHW)，结果表现出抗体对奥密克戎突变株四种亚型假病毒中和能力的是更为显著的提升，分别见图9和图10。

Claims (18)

  1. 针对SARS-CoV-2的原型株、Alpha、Beta、Delta、Omicron株均有效的广谱新型冠状病毒的人源抗体或其抗原结合片段。
  2. 如权利要求1所述的人源抗体或其抗原结合片段，其特征在于，其包括重链可变区和轻链可变区，重链可变区包括的CDR3的氨基酸序列是SEQ ID NO：15、19、20、21、27、28所示；其轻链可变区包括的CDR3的氨基酸序列是：SEQ ID NO：18。
  3. 如权利要求2所述的人源抗体或其抗原结合片段，其特征在于，所述重链可变区包括的CDR1至2的氨基酸序列分别是：SEQ ID NO：13和14所示；所述轻链可变区包括的CDR1至2的氨基酸序列分别是：SEQ ID NO：16、17所示。
  4. 如权利要求3所述的人源抗体或其抗原结合片段，其特征在于，重链可变区氨基酸序列如SEQ ID NO：22、23、24、25所示，轻链可变区氨基酸序列如SEQ ID NO：26所示。
  5. 如权利要求4所述的人源抗体或其抗原结合片段，其特征在于，重链的全长氨基酸序列如SEQIDNO：10所示，或者存在I102S、I102M、M104L、L101F、或L101F且H111Y突变；轻链的全长氨基酸序列如SEQ ID NO：12所示。
  6. 如权利要求1至5任一项所述的人源抗体或其抗原结合片段，其特征在于，所述人源抗体为单链抗体。
  7. 如权利要求6所述的人源抗体或其抗原结合片段，其特征在于，其氨基酸序列如SEQ ID NO：8所示，或者存在I216S、I216M、M218L、L101F、或L101F且H111Y突变。
  8. 如权利要求1至5任一项所述的人源抗体或其抗原结合片段，其特征在于，其为全抗IG1抗体。
  9. 如权利要求1至8任一项所述的人源抗体或其抗原结合片段的编码核酸。
  10. 如权利要求9所述的编码核酸，其特征在于，重链的全长核苷酸序列如SEQ ID NO：9所示，轻链的全长核苷酸序列如SEQ ID NO：11所示。
  11. 如权利要求9所述的编码核酸，其特征在于，核苷酸序列如SEQIDNO：7所示。
  12. 含如权利要求9-11任一项所述的编码核酸的表达载体或重组细胞。
  13. 如权利要求12所述的表达载体或重组细胞，其特征在于，所述重链和轻链编码构建在同一个或不同的表达载体上。
  14. 含有权利要求1-8任一项所述的人源抗体或其抗原结合片段作为有效成分的预防或治疗SARS-CoV-2引起的疾病的药物组合物。
  15. 如权利要求14所述的药物组合物，其特征在于，还包括药学上可接受助剂。
  16. 权利要求1-8任一项所述的人源抗体或其抗原结合片段的用途，其特征在于，在制备治疗 SARS-CoV-2引起的疾病的预防或治疗的药物中应用。
  17. 如权利要求16所述的用途，其特征在于，所述SARS-CoV-2选自原型株、Alpha、Beta、Delta、Omicron株中一种或多种。
  18. 一种新型冠状病毒的人源抗体，其特征在于，基于IMGT网站分类原则由重链VH3-23，IGHJ1：轻链VK1-12，IGK4的序列组合。
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