WO2024061061A1 - 中和新型冠状病毒的c-型单域抗体及应用 - Google Patents

中和新型冠状病毒的c-型单域抗体及应用 Download PDF

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WO2024061061A1
WO2024061061A1 PCT/CN2023/118405 CN2023118405W WO2024061061A1 WO 2024061061 A1 WO2024061061 A1 WO 2024061061A1 CN 2023118405 W CN2023118405 W CN 2023118405W WO 2024061061 A1 WO2024061061 A1 WO 2024061061A1
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seq
loop
antibody
type single
domain antibody
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French (fr)
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龚睿
吴必浩
张海伟
秦也
易雨晴
张哲�
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武汉班科生物技术有限公司
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biomedical engineering technology, and specifically to a C-type single-domain antibody for neutralizing the novel coronavirus and its application.
  • the new coronavirus mediates infection through the combination of the envelope glycoprotein (S protein) on the virus surface with the host receptor angiotensin-converting enzyme 2 (ACE2).
  • S protein is functionally divided into The S1 subunit containing the receptor binding domain (RBD) is responsible for binding to host cells and the S2 subunit is responsible for mediating the fusion of the virus and the host cell membrane.
  • the S protein is the main immunogen and antibody target of SARS-CoV-2.
  • this application provides a C-type single domain antibody that neutralizes the new coronavirus and its application.
  • the present invention uses the optimized CH2, that is, CH2-m01sm2 as a phage display library as the skeleton, and uses the SARS-CoV-2 envelope protein receptor binding domain RBD expressed in the eukaryotic system as the antigen. After multiple rounds of screening and affinity maturation, it is obtained A candidate clone BBT-VC002-1.
  • BBT-VC002-1 was expressed and purified prokaryotically in Escherichia coli, and its biological specificity, in vitro and in vivo neutralizing activity were evaluated, and a new C-type single domain antibody BBT that broadly neutralized SARS-CoV-2 was obtained.
  • -VC002-1 The optimized CH2, that is, CH2-m01sm2 as a phage display library as the skeleton, and uses the SARS-CoV-2 envelope protein receptor binding domain RBD expressed in the eukaryotic system as the antigen. After multiple rounds of screening and affinity maturation, it is obtained A candidate clone BBT-VC002-1.
  • BBT-VC002-1 has three loop regions, namely Loop BC, Loop DE and Loop FG, and their amino acid sequences are shown in SEQ ID No.1, SEQ ID No.6 and SEQ ID No.8, respectively.
  • the nucleotide sequences encoding Loop BC, Loop DE and Loop FG in BBT-VC002-1 are shown in SEQ ID No. 51, SEQ ID No. 52 and SEQ ID No. 53 respectively.
  • the amino acid sequence of BBT-VC002-1 is shown in SEQ ID No. 25, and the nucleotide sequence is shown in SEQ ID No. 54.
  • the above-mentioned single domain antibody against the SARS-CoV-2 envelope protein provided by the present invention is obtained by the following method: first, a phage display library with optimized CH2 as the skeleton is constructed, and then the SARS-CoV-2 expressed in mammalian cells is used.
  • the envelope protein receptor binding domain RBD is the antigen ( Figure 2), and candidate clones were obtained after four rounds of screening. Affinity maturation of candidate clones is performed through random mutation, resulting in a high-affinity, specific-binding clone.
  • the clone ( Figure 3) was expressed, purified, and identified to obtain an antibody BBT-VC002-1 that neutralizes SARS-CoV-2.
  • BBT-VC002-1 can inhibit SARS-CoV-2 from invading cells and is used for the prevention, treatment and diagnosis of SARS-CoV-2 infection.
  • the present invention constructs pre-fusion conformation wild type (WT), Delta and Omicron BA.1, BA.2, BA.4/5 mutant antigen spike trimer proteins, and determines the binding activity of BBT-VC002-1 through ELISA , the results show that BBT-VC002-1 can bind to the wild type (WT), Delta and Omicron BA.1, BA.2, BA.4/5 mutant strain antigen spike proteins ( Figure 4A), and bind to the wild type and previous mutant strains. Antigen RBD binding ( Figure 4B).
  • BBT-VC002-1 could inhibit the infection of Vero E6 cells by pseudoviruses such as wild type, Omicron mutant strains and their sublines ( Figure 5A and B), and previous popular mutant strains ( Figure 5A and B). 5C), can inhibit the infection of Vero E6 cells by SARS-CoV-2 wild-type, Delta, BA.1, BA.2, and BA.5 live viruses ( Figure 6), proving that the antibody has broad spectrum neutralization of SARS-CoV-2 role.
  • the present invention uses Syrian hamsters as an animal model of novel coronavirus infection to evaluate the in vivo efficacy of BBT-VC002-1.
  • Administration of the antibody by nasal drops or pulmonary atomization can significantly reduce the viral load in the lungs and respiratory tract of hamsters, demonstrating that the antibody has a good in vivo protective effect ( FIG. 7 ).
  • the amino acid sequence point mutants of the BBT-VC002-1 antibody of the present invention are named B5, B10, D6, D7, D10, E7, F2, F11, H1, H7, S-H4, R-H4, G7, A4 (Q50K, E90Q, R96S), A5, C9, D4, G10, and 3-C2 have binding and neutralizing activities similar to those of the BBT-VC002-1 antibody ( Figure 8).
  • Figure 8 by replacing the three Loop regions of BBT-VC002-1 with the corresponding parts of other antibody fragments (SEQ ID No. 45, SEQ ID No. 46, SEQ ID No. 47), the binding activity and neutralization can still be obtained. Active combination (Figure 9).
  • BBT-VC002-1 is connected to polypeptides (ABD and 16L) or proteins (VH) to form fusion polypeptides or fusion proteins. Its antigen-binding activity remains unchanged ( Figure 10).
  • Figure 10 The sequence numbers of all the above mutants and antibodies are listed below. In Table 1 at the end of the article.
  • the present invention also discloses the use of the above-mentioned C-type single domain antibody for neutralizing the novel coronavirus in the preparation of preventive and therapeutic drugs, detection probes, fusion polypeptides, fusion proteins, and coupled antibodies for the novel coronavirus.
  • the C-type single domain antibody for neutralizing the novel coronavirus of the present invention can be prepared into nasal drops, nasal sprays, atomizers, and injection preparations related to the prevention and treatment of SARS-CoV-2 due to its small molecular weight and high binding activity.
  • C-type single domain antibody used in the present invention refers to a type of antibody with the CH2 domain of the antibody constant region as the backbone. Compared with full-length monoclonal antibodies, single-domain antibodies have better tissue penetration and can advantageously bind to epitopes with steric hindrance.
  • BBT-VC002-1 compared with the full-length monoclonal antibody (molecular weight ⁇ 150 kDa), BBT-VC002-1 has a smaller molecular weight (12 ⁇ 15 kDa), better tissue permeability, and is easier to identify the viral envelope.
  • the narrow conserved epitope caused by glycosylation masking or steric hindrance on the protein has better broad-spectrum neutralization ability against the new coronavirus, an RNA virus that is prone to mutation; finally, BBT-VC002-1 can not only neutralize the new coronavirus in mammalian cells It can also be expressed in prokaryotic expression systems, yeast expression systems, etc., with low production cost and short cycle.
  • FIG. 1 is a schematic diagram of the optimized CH2 structure.
  • the optimized CH2 introduces an additional pair of disulfide bonds (engineered disulfide bonds) on the basis of the pair of native disulfide bonds it contains.
  • the three Loop regions contained in CH2 are Loop BC, Loop DE and Loop FG, which can be used to introduce mutations to construct antibody libraries.
  • Figure 2 shows the expression and purification of Fc fusion SARS-CoV-2 envelope protein receptor binding domain (RBD).
  • the target protein was detected by polyacrylamide gel electrophoresis (SDS-PAGE).
  • Lane M is the molecular weight standard
  • lane RBD-Fc is the target protein with a size of approximately 55 kDa.
  • Figure 3 shows the purified C-type single domain antibody BBT-VC002-1.
  • A. The purified protein is detected by SDS-PAGE, and lane M is the molecular weight standard;
  • B Molecular size exclusion chromatography shows that it exists in a monomer state, and the purity can reach more than 97%.
  • Figure 4 shows the ELISA experiment to measure the binding of antibody BBT-VC002-1 to the S protein or RBD of wild-type and previously major popular mutant strains.
  • the EC 50 value is between 0.020 ⁇ g/mL - 0.040 ⁇ g/mL.
  • Figure 5 shows the pseudovirus neutralization experiment.
  • a and B BBT-VC002-1 neutralizes wild-type and Omicron mutant strains and their substrains at pseudovirus levels, with IC 50 values between 0.15 ⁇ g/mL and 1.40 ⁇ g/mL;
  • C Neutralizes previous pseudovirus levels “Variants of concern” (VOCs) have IC 50 values between 0.045 ⁇ g/mL and 0.19 ⁇ g/mL.
  • Figure 6 shows the live virus neutralization experiment.
  • BBT-VC002-1 neutralizes wild-type, Delta strain, BA.1 strain, BA.2 strain, and BA.5 strain live viruses, with IC 50 values of 0.44 ⁇ g/mL, 0.12 ⁇ g/mL, 0.90 ⁇ g/mL, respectively. 0.60 ⁇ g/mL, 0.24 ⁇ g/mL.
  • Figure 7 shows the in vivo protection experiment of BBT-VC002-1 against Omicron BA.1 infection in the Syrian hamster model.
  • A Virus titers in hamster lungs after prophylactic administration. Compared with the control group (PBS), intranasal administration of BBT-VC002-1 antibody at doses of 10 mg/kg, 2 mg/kg and 0.5 mg/kg can reduce virus titers by 3.00 log, 2.32 log and 1.07 log values respectively. .
  • B After treatment administration, the viral load in the lungs and bronchus of hamsters decreased compared with the control group (PBS).
  • the live virus titers in the 20 mg/kg group and 5 mg/kg group decreased by an average of 2.30 and 2.37 log compared with the control group.
  • the 20 mg/kg group and 5 mg/kg group decreased compared with the control group.
  • the live virus titer in the group decreased by 2.93 log and 1.77 log on average.
  • the number of RNA copies of hamster lungs and bronchial viruses in the treatment group was reduced compared with the control group.
  • the 20 mg/kg group and the 5 mg/kg group decreased by 2.04 and 2.00 logs respectively; in the bronchi, the 20 mg/kg group and the 5 mg/kg group decreased by 2.55 and 1.24 logs respectively; one-way analysis of variance was used Significant difference, ****p ⁇ 0.0001; ***p ⁇ 0.001; **p ⁇ 0.01; *p ⁇ 0.05; ns, no significance.
  • C. Indirect immunofluorescence method was used to detect the replication of SARS-CoV-2 in the lung tissue of the treatment group. Compared with the control group, the virus fluorescence signal was significantly reduced in the treatment group.
  • Figure 8 shows the binding activity (8A and B) and neutralizing activity (8C and D) of the antibody mutants obtained after point mutations in the Loop region and/or backbone region of BBT-VC002-1, EC 50 and IC 50 The value is equivalent to BBT-VC002-1.
  • Figure 9 shows the binding activity (9A) and neutralizing activity (9B) of the combination obtained by replacing the three Loop region sequences in BBT-VC002-1 with the Loop regions of the CH2 backbone of different types of antibodies, which is consistent with BBT-VC002 -1 is quite active.
  • FIG. 10 shows that after coupling BBT-VC002-1 to polypeptides or proteins, the activity is still maintained.
  • the RBD in the spike glycoprotein (S protein) of the new coronavirus SARS-CoV-2 was fused with the Fc fragment of human IgG1, and constructed into the mammalian cell expression vector pSecTag2 A to obtain the recombinant plasmid RecVec- RBD-Fc is expressed using the mammalian cell 293F expression system.
  • RBDs of mutant strains such as Beta (GenBank: QUN71037.1), Gamma (GenBank: QXF23725.1), Delta (GenBank: QUX81264.1), and BA.1 (GenBank: UGN73932.1) were constructed using the same method as above.
  • the culture supernatant was collected, and the expression of RBD-Fc was detected by Western Blot using Goat Anti-human-IgG Fc Antibody as an antibody.
  • the scale of cell culture and transfection was expanded to express the receptor binding domain protein in large quantities.
  • the culture supernatant was collected, and the target protein was purified with Protein A packing, and then the buffer was replaced by ultrafiltration using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and its molecular size and purity were verified by SDS-PAGE, as shown in Figure 2.
  • a phage library was constructed according to existing literature (Gong R, et al ., PLoS ONE, 2012), screened with antigens expressed in mammalian cells.
  • the purified antigen was coated in a 96-well plate and incubated at 4oC overnight.
  • the constructed phage display library was used for panning. Specific phages were captured by the antigen and washed with PBS+0.05% Tween-20 for 4 rounds. Screen to obtain candidate clones.
  • candidate clone as a template, random mutations were performed on it to construct a random mutation phage library. Through antigen sequential screening, a specific high-affinity clone was obtained, named BBT-VC002-1.
  • BBT-VC002-1 coding gene sequence was found.
  • the three loop regions of BBT-VC002-1 are: Loop BC, Loop DE and Loop FG.
  • Their respective coding gene sequences and amino acid sequences are listed in the table below.
  • BBT-VC002-1 was expressed and purified according to existing literature (Gong R, et al ., Methods Mol Biol., 2012).
  • the BBT-VC002-1 plasmid was extracted and transformed into E.coli HB2151.
  • Example 4 ELISA determination of the binding of BBT-VC002-1 and antigen receptor binding domain RBD
  • FIG. 4A shows the binding of BBT-VC002-1 to the viral S protein
  • Figure 4B shows the binding of BBT-VC002-1 to the RBD region of the viral S protein.
  • Example 5 Determination of pseudovirus neutralizing activity of BBT-VC002-1 on Vero E6 cells
  • 2-DMEM DMEM maintenance medium
  • Vero E6 cells were seeded at 1.5 ⁇ 10 4 cells/well in a 96-well cell culture plate, and the cells were cultured in a 37oC, 5% CO 2 incubator overnight before use.
  • the experiment was performed in duplicate for each antibody concentration.
  • the experiment set up 50 ⁇ L of antibody-free medium and 50 ⁇ L of pseudovirus as virus control wells.
  • Figure 5A and B show the effects of BBT-VC002-1 on wild strain Wuhan-Hu-1 and Omicron BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.4/5, BF .7. Neutralizing effect of BQ.1.1, XBB, , Neutralizing effect of Delta mutant strain.
  • Example 6 Determination of live virus neutralizing activity of BBT-VC002-1 on Vero E6 cells
  • Vero E6 cells were seeded into a 24-well cell culture plate at 1.5 ⁇ 10 cells per well, and the cells were cultured overnight in a 37oC and 5% CO2 incubator before use.
  • Example 7 In vivo protection experiment of BBT-VC002-1 in preventing and treating Omicron BA.1 infection in hamster model
  • the hamsters were sacrificed and their lung tissues were dissected and used for viral load detection.
  • live virus titer detection dilute the supernatant from the centrifuged lung tissue homogenate at 1:10, 1:100, and 1:1000, add 200 ⁇ L/well to Vero E6 cells in a pre-plated 24-well plate, and culture for 5 After 0.5% crystal violet staining, the number of plaques was counted and the virus titer was calculated (Figure 7A).
  • Treatment experiment 11 Syrian hamsters used in animal experiments were divided into 3 groups, 4 in the high-dose group, 4 in the low-dose group, and 3 in the PBS control group to evaluate the therapeutic effect of BBT-VC002-1.
  • Hamsters were anesthetized with tribromoethanol (Avertin) and infected by intranasal inoculation of 1 ⁇ 10 4 PFU of SARS-CoV-2 Omicron BA.1 per animal.
  • One hour after the challenge the hamsters were anesthetized, and two high and low doses of 20 mg/kg and 5 mg/kg were atomized into the lungs, with a volume of 100 ⁇ L each, and an equal amount of PBS was given as a virus group control.
  • the antibody or PBS was given once a day for a total of 2 consecutive days.
  • the hamsters were sacrificed and their lung tissue and bronchi were dissected and used for viral load detection.
  • live virus titer detection dilute the supernatant after centrifugation of lung tissue and bronchial homogenate at 1:10, 1:100, and 1:1000, and add 200 ⁇ L/well to Vero E6 cells in a pre-plated 24-well plate. , after 5 days of culture, count the number of plaques after staining with 0.5% crystal violet and calculate the virus titer; for viral RNA copy number detection, extract 140 ⁇ L homogenate supernatant RNA according to the QIAamp® Viral RNA Mini manual. HiScript® II One Step qRT-PCR SYBR Green Kit (Vazyme) was used to design amplification primers based on the ORF1ab fragment for fluorescence quantitative PCR to detect RNA copy number ( Figure 7B).
  • Paraffin sections were made. The lung tissue samples were fixed with 8% paraformaldehyde for 7 days, embedded in paraffin, and cut into 3.5 ⁇ m sections. Paraffin sections were dewaxed and rehydrated, and the sections were immersed in heated EDTA (pH 8.0) buffer for antigen retrieval. The slides were permeabilized with PBS/0.02% Triton hour, after washing with PBS, the secondary antibody Cy3-conjugated goat anti-rabbit IgG (1:200) was added to continue incubation, and after washing with PBS, 1:200 DAPI was added for staining. The sections were fixed with neutral glue and images were collected (Figure 7C).
  • Example 8 Construction and activity of point mutants, combinations and fusions of BBT-VC002-1 antibody
  • hIgG1 CH2-com hIgG3 CH2-com
  • Monkey IgG1 CH2-com are comparable to the BBT-VC002-1 antibody in terms of binding activity and neutralizing activity.
  • the results suggest that the three loop areas of BBT-VC002-1 can be placed on different skeletons to function.
  • BBT-VC002-1 was coupled to peptides (16L and ABD) or proteins (VH), named BBT-VC002-1-16L, BBT-VC002-1-ABD, and BBT-VC002 respectively. -1-VH. Then carry out gene synthesis, protein expression, and activity detection (methods are the same as above). The test results are shown in Figure 10. BBT-VC002-1 still maintains activity after fusion with polypeptide or protein. The results show that BBT-VC002-1 can be used in the preparation of fusion peptides and fusion proteins for the prevention and treatment of new coronaviruses.

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Abstract

本发明公开了一种中和新型冠状病毒的C-型单域抗体及应用。本发明的C-型单域抗体能够抑制新型冠状病毒（SARS-CoV-2）入侵细胞，用于SARS-CoV-2感染的预防、治疗和诊断。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

中和新型冠状病毒的C-型单域抗体及应用 技术领域
本发明涉及生物医药工程技术领域，具体涉及一种中和新型冠状病毒的C-型单域抗体及应用。
 背景技术
新型冠状病毒（SARS-CoV-2）通过病毒表面的囊膜糖蛋白（spike glycoprotein，S蛋白）与宿主受体血管紧张素转换酶2（ACE2）结合介导感染，S蛋白在功能上分为负责与宿主细胞结合的含受体结合域（receptor binding domain，RBD）的S1亚基和负责介导病毒与宿主细胞膜融合的S2亚基。S蛋白是SARS-CoV-2主要免疫原和抗体靶点。随着多种突变株的出现，如奥密克戎（Omicron BA.1）以高传播速率在2022年初迅速成为主要流行株，其S蛋白携带的大量突变位点造成与受体亲和力增强并产生严重的免疫逃逸，极大地影响了现有的疫苗和抗体药物的有效性（Iketani S, et al., Nature, 2022）。奥密克戎在传播过程中又进化出一系列亚系如BA.2和BA.5，并迅速取代前者成为新的主要流行株，这些亚系能继续逃逸针对BA.1产生的免疫（Cao Y, et al., Nature, 2022），造成突破感染。所以亟需寻找靶向高度保守表位的广谱中和抗体应对现有或未来出现的突变株，研发单域抗体（纳米抗体）成为新的选择。
发明内容
针对现有技术中的问题，本申请提供了一种中和新型冠状病毒的C-型单域抗体及应用。
本发明从优化的CH2即CH2-m01sm2为骨架的噬菌体展示库中，以真核系统表达的SARS-CoV-2囊膜蛋白受体结合域RBD为抗原，经过多轮筛选和亲和力成熟后，获得一个候选克隆BBT-VC002-1。用大肠杆菌原核表达并纯化BBT-VC002-1，对其生物学特异性、体内外中和活性进行了评价，得到一种广谱中和SARS-CoV-2的新型C-型单域抗体BBT-VC002-1。
BBT-VC002-1具有三个Loop区，三个Loop区为Loop BC、Loop DE 和Loop FG，其氨基酸序列分别如SEQ ID No.1、SEQ ID No.6和SEQ ID No.8所示。
BBT-VC002-1中编码Loop BC、Loop DE 和Loop FG的核苷酸序列分别如SEQ ID No.51、SEQ ID No.52和SEQ ID No.53所示。
BBT-VC002-1的氨基酸序列如SEQ ID No.25所示，核苷酸序列如SEQ ID No.54所示。
本发明提供的上述针对SARS-CoV-2囊膜蛋白的单域抗体，是通过以下方法得到：首先构建以优化的CH2为骨架的噬菌体展示库，然后以哺乳动物细胞表达的SARS-CoV-2囊膜蛋白受体结合域RBD为抗原（图2），经过4轮筛选得到候选克隆。通过随机突变对候选克隆进行亲和力成熟，最终得到一个高亲和力、特异性结合的克隆。表达纯化该克隆（图3），进行鉴定，即得到一种中和SARS-CoV-2的抗体BBT-VC002-1。
BBT-VC002-1能够抑制SARS-CoV-2入侵细胞，用于SARS-CoV-2感染的预防、治疗和诊断。
本发明构建了融合前构象野生型（WT）、Delta及Omicron BA.1、BA.2、BA.4/5突变株抗原spike三聚体蛋白，通过ELISA测定了BBT-VC002-1的结合活性，结果表明BBT-VC002-1能够与野生型（WT）、Delta及Omicron BA.1、BA.2、BA.4/5突变株抗原spike蛋白结合（图4A），与野生型和以往突变株抗原RBD相结合（图4B）。通过体外中和实验测定了中和活性，结果表明BBT-VC002-1可抑制野生型、Omicron突变株及其亚系（图5A和B）、以往流行突变株等假病毒感染Vero E6细胞（图5C），能够抑制SARS-CoV-2野生型、Delta、BA.1、BA.2、BA.5活病毒感染Vero E6细胞（图6），证明该抗体具有广谱中和SARS-CoV-2的作用。
本发明以叙利亚仓鼠作为新冠病毒感染动物模型评价BBT-VC002-1的体内药效，滴鼻或雾化肺部给抗体后可显著降低仓鼠体内肺和呼吸道的病毒载量，证明该抗体具有良好体内保护效果（图7）。
本发明BBT-VC002-1抗体的氨基酸序列点突变体命名为B5、B10、D6、D7、D10、E7、F2、F11、H1、H7、S-H4、R-H4、G7、A4(Q50K、E90Q、R96S)、A5、C9、D4、G10、3-C2具有与BBT-VC002-1抗体类似的结合活性及中和活性（图8）。同时，将BBT-VC002-1的三个Loop区替换到其他抗体片段的相应部位（SEQ ID No.45、SEQ ID No.46、SEQ ID No.47），依然可以获得具有结合活性和中和活性的组合体（图9）。将BBT-VC002-1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10），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新型冠状病毒的C-型单域抗体在制备针对新型冠状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。本发明的中和新型冠状病毒的C-型单域抗体由于分子量小，结合活性高，可以制备成SARS-CoV-2防治相关的滴鼻剂、喷鼻剂、雾化药、注射制剂。
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：相对于全长单抗（分子量~150 kDa），BBT-VC002-1分子量较小（12~15 kDa），具有更好的组织渗透性，并且更易于识别病毒囊膜蛋白上糖基化遮蔽或空间位阻造成的狭小保守表位，针对新冠病毒这一易于突变的RNA病毒具有更好的广谱中和能力；最后，BBT-VC002-1不仅可以在哺乳动物细胞中表达，也可以在原核表达系统、酵母表达系统等中进行表达，其生产成本低、周期短。
 附图说明
图1为优化的CH2结构示意图。优化的CH2在本身含有的一对二硫键（native disulfide bond）基础上，多引入了一对二硫键（engineered disulfide bond）。CH2所含的三个Loop区分别是Loop BC，Loop DE和Loop FG，它们可以用来引入突变从而构建抗体库。
图2为Fc融合SARS-CoV-2囊膜蛋白受体结合域（RBD）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M为分子量标准，泳道RBD-Fc为目的蛋白，大小约为55 kDa。
图3为纯化的C-型单域抗体BBT-VC002-1。A. 纯化后的蛋白经SDS-PAGE检测，泳道M为分子量标准；B. 分子大小排阻色谱显示其以单体状态存在，纯度能达到97%以上。
图4为ELISA实验测定抗体BBT-VC002-1与野生型和以往主要流行突变株的S蛋白或者RBD的结合，EC 50值为0.020 μg/mL - 0.040 μg/mL之间。
图5为假病毒中和实验。A和B. 在假病毒水平BBT-VC002-1中和野生型和Omicron突变株及其亚系，IC 50值在0.15 μg/mL-1.40 μg/mL之间；C. 假病毒水平中和以往“高关注变异株”(variants of concern，VOCs)，IC 50值在0.045 μg/mL-0.19 μg/mL之间。
图6为活病毒中和实验。BBT-VC002-1中和野生型、Delta株、BA.1株、BA.2株、BA.5株活病毒，IC 50值分别为0.44 μg/mL、0.12 μg/mL、0.90 μg/mL、0.60 μg/mL、0.24 μg/mL。
图7为BBT-VC002-1在叙利亚仓鼠模型中对Omicron BA.1感染体内保护实验。A. 预防给药后仓鼠肺部病毒滴度。相比对照组（PBS），按10 mg/kg、2 mg/kg和0.5 mg/kg剂量进行BBT-VC002-1滴鼻给抗体可使病毒滴度分别降低3.00 log、2.32 log、1.07 log值。B.治疗给药后，仓鼠肺部和支气管病毒载量相较对照组（PBS）减少情况。在肺部，20 mg/kg组和5 mg/kg组相较于对照组活病毒滴度平均下降了2.30和2.37 log，在支气管，20 mg/kg组和5 mg/kg组相较于对照组活病毒滴度平均下降了2.93 log和1.77 log。仓鼠肺部和支气管病毒RNA拷贝数治疗组相较对照组减少情况。在肺部，20 mg/kg组和5 mg/kg组分别下降2.04 和2.00个log；在支气管，20 mg/kg组和5 mg/kg组分别下降2.55和1.24个log；使用单因素方差分析显著性差异，****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no significance。C. 治疗组间接免疫荧光法检测肺组织中SARS-CoV-2复制情况，治疗组相较于对照组病毒荧光信号显著减少。
图8为在BBT-VC002-1的Loop区和/或骨架区进行点突变后得到的抗体突变体的结合活性（8A和B）和中和活性（8C和D），EC 50和值IC 50值与BBT-VC002-1相当。
图9为将BBT-VC002-1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（9A）和中和活性（9B），其与BBT-VC002-1活性相当。
图10为将BBT-VC002-1与多肽或者蛋白偶联后，活性依然保持。
 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：表达纯化SARS-CoV-2囊膜蛋白受体结合域（RBD）
将新型冠状病毒SARS-CoV-2的spike glycoprotein（S蛋白)（GenBank: QHR63250.2）中的RBD与人IgG1的Fc片段融合，构建到哺乳动物细胞表达载体pSecTag2 A上，得到重组质粒RecVec-RBD-Fc，采用哺乳动物细胞293F表达体系表达。Beta（GenBank: QUN71037.1）、Gamma（GenBank: QXF23725.1）、Delta（GenBank: QUX81264.1）、BA.1（GenBank: UGN73932.1）等突变株的RBD用以上述同样方法构建。转染前1天将293F细胞（控制细胞密度为5×10 5个/mL）40 mL接种于125 mL悬浮细胞培养瓶。将40 μg重组质粒RecVec-RBD-Fc稀释于4 mL培养基中轻轻混匀，再将120μg PEI（polyethylenimine）稀释于培养液中，轻轻混匀。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，125转/分钟，37ºC、8% CO 2悬浮培养。
培养120 h后收集培养基上清，用Goat Anti-human-IgG Fc Antibody作为抗体通过蛋白免疫印迹（Western Blot）检测RBD-Fc的表达。在检测到RBD-Fc表达后，扩大细胞培养与转染规模，大量表达受体结合域蛋白。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10 kDa的超滤离心管超滤置换缓冲液，经SDS-PAGE验证其分子大小和纯度，如图2所示。
实施例2：噬菌体展示库的构建及抗体的筛选
以优化的CH2为骨架，按照已有的文献构建噬菌体库（Gong R, et al.,  PLoS ONE, 2012），以哺乳动物细胞表达的抗原进行了筛选。将纯化后的抗原包被于96孔板中4ºC孵育过夜后，用已经构建的噬菌体展示文库进行淘选，特异性的噬菌体被抗原所捕获，用PBS+0.05% Tween-20清洗，经过4轮筛选，得到候选克隆。以候选克隆为模板，对其进行随机突变，构建随机突变噬菌体文库，通过抗原序贯筛选，得到一个特异性的高亲和力克隆，命名为BBT-VC002-1。
测序可知，BBT-VC002-1编码基因序列。BBT-VC002-1的三个Loop区为：Loop BC、Loop DE和Loop FG，其各自的编码基因序列和氨基酸序列在后有表格列出。
实施例3：BBT-VC002-1的表达纯化
按照已有文献（Gong R, et al., Methods Mol Biol., 2012）对BBT-VC002-1进行表达和纯化。提取BBT-VC002-1质粒，转化入 E.coli HB2151中。再接种菌种于含100 μg/ml 氨苄青霉素的SB培养基（1 L培养基中含30 g胰蛋白胨、20 g酵母提取物和10 g MOPS，pH值用NaOH调至7.0）中，待OD 600达到0.7~1.0时加入IPTG至终浓度为200 μg/ml，于37ºC、220 rpm的条件下进行诱导表达14~16 h。4ºC、6000 rpm、15分钟离心收集菌体，弃培养基，沉淀重悬于PBS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（图3A）和分子排阻色谱（图3B）验证其纯度。随后用截留分子量为3 kDa的超滤离心管超滤浓缩。所得到的BBT-VC002-1的C-末端含6×His标签和FLAG标签。
实施例4：ELISA测定BBT-VC002-1和抗原受体结合域RBD的结合
将病毒Spike三聚体蛋白（WT、Delta、Omicron BA.1、BA.2、BA.4/5）或者受体结合域RBD（WT、Alpha (N501Y)、Beta (K417N/E484K/N501Y)、Gamma (K417N/E484K/N501Y)、Delta (L452R/T478K)、Delta plus (K417N/L452R/T478K)、Kappa (L452R/E484Q)、Lota (S477N)、Eta (E484K)、Epsilon (L452R)、Mu (R346K/E484K/N501Y)、Lambda (L452Q/F490S)）以4 μg/mL包被在ELISA板上，4°C孵育过夜后用PBS + 3% milk于37ºC封闭1.5小时，用PBST (PBS + 0.05% Tween 20) 洗3次。加入梯度稀释的抗体，37ºC孵育1.5小时后用PBST 洗6次，再加辣根过氧化物酶（HRP）标记的鼠抗FLAG单克隆抗体于37ºC孵育1小时后，用PBST洗6次，再加入ABTS底物后通过酶标仪在405 nm波长下进行读值检测。图4A展示了BBT-VC002-1与病毒S蛋白的结合；图4B展示了BBT-VC002-1与病毒S蛋白的RBD区域的结合。
实施例5：BBT-VC002-1在Vero E6细胞上假病毒中和活性的测定
假病毒包装：编码SARS-CoV-2 野生型和各突变株S蛋白的cDNA由公司合成或点突变PCR获得，克隆到pCAGGS载体上得到重组表达质粒；瞬时转染293T细胞，转染24小时后弃掉原培养基，加入MOI=2的VSV△G，在37ºC感染2小时后PBS洗三次，加入含2%FBS的DMEM维持培养基（2-DMEM）使细胞生产假病毒。第二天收上清0.45 μM滤膜抽滤，10倍梯度稀释感染Vero E6细胞测定滴度，根据滴度结果按照2×工作滴度（约2000个荧光点/100 μL）分装后-80ºC保存备用。
假病毒中和实验：将Vero E6细胞1.5×10 4 cell/孔接种于96孔细胞培养板中，细胞在37ºC、5% CO 2培养箱中培养过夜后备用。将终浓度15 μg/mL起始、3倍梯度稀释的抗体稀释液50 μL与50 μL假病毒按照1:1混匀，共100 μL。实验为每个抗体浓度双复孔检测。实验设置50 μL无抗体培养基与50 μL假病毒为病毒对照孔。将上述混合液在37ºC培养箱中孵育1小时后，去除Vero E6细胞上清，加到细胞上。将细胞培养板放入37ºC、5% CO 2培养箱中培养24小时。使用Operatta高内涵采集荧光图像和配套Harmony分析系统得到每孔荧光点数。随着抗体浓度的升高，呈现绿色荧光点逐渐减少，表明被新型冠状病毒的假病毒感染的细胞数目减少，即表示BBT-VC002-1可以中和新型冠状病毒的假病毒。将抗体浓度对应的荧光点数作抑制曲线图并进行四参数非线性拟合，计算出IC 50值。图5A和B展示了BBT-VC002-1对野生株Wuhan-Hu-1及Omicron BA.1、BA.1.1、BA.2、BA.2.12.1、BA.2.75、BA.4/5、BF.7、BQ.1.1、XBB、XBB.1.5、XBB.1.16、CH.1.1、CJ.1、EG.5突变株的中和作用；图5C展示了BBT-VC002-1对Alpha、Beta、Gamma、Delta突变株的中和作用。
实施例6：BBT-VC002-1在Vero E6细胞上活病毒中和活性的测定
将Vero E6细胞按每孔1.5×10 5个接种于24孔细胞培养板中，细胞在37ºC和5% CO 2培养箱中培养过夜后备用。将终浓度30 μg/mL起始、3倍梯度稀释的抗体与含有150 PFU的SARS-CoV-2 WT、Delta、Omicron BA.1、BA.2、BA.5活病毒等体积混匀，混合液37ºC孵育1小时。去除Vero E6细胞上清，加入抗体-病毒混合液37ºC感染1h后弃掉，加入0.5 mL含2% FBS和0.9%羧甲基纤维素的DMEM继续在37ºC 5%CO 2培养3-5天。8%的多聚甲醛固定后，洗掉覆盖物并用0.5%结晶紫染色后统计每孔空斑数量。实验为每个抗体浓度双复孔检测。实验设置只加维持培养基的孔为细胞对照孔，加维持培养基及病毒稀释液的孔为病毒对照孔。将抗体浓度对应的空斑数作抑制曲线图并进行四参数非线性拟合，计算出IC 50值（图6）。
实施例7：BBT-VC002-1在仓鼠模型中预防和治疗Omicron BA.1感染体内保护实验
预防实验：用于动物实验叙利亚仓鼠分为4组，每组4只，用以评估BBT-VC002-1的预防效果。用异氟烷麻醉仓鼠，按照10 mg/kg、2mg/kg和0.5 mg/kg剂量滴鼻给抗体，体积为100 μL每只，给等量PBS作为病毒组对照。给抗体1小时后，麻醉仓鼠，以1×10 4 PFU每只的SARS-CoV-2 Omicron BA.1鼻内接种感染。在第4天将仓鼠处死后解剖取其肺组织用于病毒载量检测。活病毒滴度检测，将肺组织匀浆离心后的上清1:10、1:100、1:1000稀释，在预先铺好的24孔板Vero E6细胞中按200 μL/孔加入，培养5天后0.5%结晶紫染色后统计空斑数并计算病毒滴度（图7A）。
治疗实验：用于动物实验的11只叙利亚仓鼠被分为3组，高剂量组4只、低剂量组4只，PBS对照组3只，用以评估BBT-VC002-1的治疗效果。用三溴乙醇（Avertin）麻醉仓鼠，1×10 4 PFU每只的SARS-CoV-2 Omicron BA.1鼻内接种感染。攻毒1小时后，麻醉仓鼠，20 mg/kg和5 mg/kg高低两组剂量肺部雾化给药，体积为100 μL每只，给等量PBS作为病毒组对照。而后每天给一次抗体或PBS，总共连续给2天，在第4天将仓鼠处死后解剖取其肺组织和支气管用于病毒载量检测。对于活病毒滴度检测，将肺组织和支气管匀浆离心后的上清1:10、1:100、1:1000稀释，在预先铺好的24孔板Vero E6细胞中按200 μL/孔加入，培养5天后0.5%结晶紫染色后统计空斑数并计算病毒滴度；对于病毒RNA拷贝数检测，按照QIAamp® Viral RNA Mini手册提取140 μL匀浆上清RNA。使用HiScript® II One Step qRT-PCR SYBR Green 试剂盒（Vazyme），以ORF1ab片段设计扩增引物进行荧光定量PCR检测RNA拷贝数（图7B）。
间接免疫荧光法检测肺组织中SARS-CoV-2。制作石蜡切片，肺组织样本用8%多聚甲醛固定7天后石蜡包埋，切成3.5 μm的切片。石蜡切片脱蜡再水化，将切片浸泡在加热的EDTA（pH 8.0）缓冲液中用于抗原修复。载玻片用PBS/0.02% Triton X-100 渗透15分钟，在室温用5% BSA封闭1小时后与一抗（兔抗SARS-CoV-2 NP蛋白多克隆抗体，1:800）37ºC孵育1小时，PBS洗后加入二抗Cy3偶联羊抗兔IgG（1:200）继续孵育，PBS洗后加入1:200 DAPI染色。切片用中性胶固定后用采集图像（图7C）。
实施例8：BBT-VC002-1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-VC002-1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（B5(P20S, H32R, N48K, S103A)、B10(I34V, L71P, I105V)、D6(T51A, E90V, A116T)、D7(D28N, T51K)、D10(E90T)、E7(Q50K, E90T, R96S)、F2(E90K)、F11(P10S, K11N, T13A, V70I)、H1(Q50K, S88T, E90T, R96S)、H7(Q50K, E90A, R96S)、S-H4(Q50K, R96S)、R-H4(K38R、Q50K、K117N、E90T、R96N)、G7(N48K、Q50K、E90S、R96S、I105V)、A4(Q50K、E90Q、R96S)、A5(Q50K、E90S、R96S)、C9(Q50K、S88T、E90H、R96S)、D4(Q50K、E90H、R96S)、G10(Q50K、H72R、K84R、H32F、A62T、E90T、R96S)、3-C2(Q50K、D66S、K117N、E90T、R96G)），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和中和实验检测，检测方法分别参照实施例4和实施例5。检测结果如图8所示，这些点突变蛋白与SARS-CoV-2的RBD的结合活性在0.008μg/mL-0.015μg/mL之间，对Omicron BA.1假病毒的中和活性在0.15μg/mL-0.40μg/mL，与BBT-VC002-1抗体相当。表明在BBT-VC002-1蛋白的Loop区和/或骨架区进行一些点突变获得的突变体并不影响抗体的活性。
组合体的构建与活性：将BBT-VC002-1中的三个Loop区序列即Loop BC、Loop DE和Loop FG替换到不同类型抗体CH2骨架的Loop区上：人类的IgG1 CH2区、IgG3 CH2区、猴的IgG1 CH2区。获得的组合体分别命名为hIgG1 CH2-com、hIgG3 CH2-com、Monkey IgG1 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9所示，hIgG1 CH2-com、hIgG3 CH2-com、Monkey IgG1 CH2-com在结合活性和中和活性方面与BBT-VC002-1抗体相当。结果提示，BBT-VC002-1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-VC002-1与多肽（16L和ABD）或者蛋白（VH）偶联，分别命名为BBT-VC002-1-16L、BBT-VC002-1-ABD、BBT-VC002-1-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10所示，融合多肽或者蛋白后的BBT-VC002-1依然保持活性。结果表明，BBT-VC002-1可在制备针对新型冠状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. 一种中和新型冠状病毒的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 BC、Loop DE 和Loop FG，
    Loop BC的氨基酸序列选自SEQ ID No.1-5中任一项；
    Loop DE的氨基酸序列选自SEQ ID No.6或SEQ ID No.7；
    Loop FG的氨基酸序列选自SEQ ID No.8-24中任一项。
  2. 根据权利要求1所述中和新型冠状病毒的C-型单域抗体，其特征在于： Loop BC、Loop DE 和Loop FG氨基酸序列分别如SEQ ID No.1、SEQ ID No.6和SEQ ID No.8所示。
  3. 根据权利要求1所述中和新型冠状病毒的的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.25-47中任一项。
  4. 权利要求1~3任一项所述的中和新型冠状病毒的C-型单域抗体在制备针对SARS-CoV-2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任一项所述的中和新型冠状病毒的C-型单域抗体在制备SARS-CoV-2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SARS-CoV-2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.48所示。
  7. 一种SARS-CoV-2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.49所示。
  8. 一种SARS-CoV-2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.50所示。
  9. 根据权利要求2所述中和新型冠状病毒的C-型单域抗体，其特征在于：编码所述Loop BC、Loop DE 和Loop FG的核苷酸序列分别如SEQ ID No.51、SEQ ID No.52和SEQ ID No.53所示。
  10. 根据权利要求3所述中和新型冠状病毒的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.54所示。
PCT/CN2023/118405 2022-09-20 2023-09-12 中和新型冠状病毒的c-型单域抗体及应用 WO2024061061A1 (zh)

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