WO2022170983A1 - 抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用 - Google Patents

抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用 Download PDF

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WO2022170983A1
WO2022170983A1 PCT/CN2022/074135 CN2022074135W WO2022170983A1 WO 2022170983 A1 WO2022170983 A1 WO 2022170983A1 CN 2022074135 W CN2022074135 W CN 2022074135W WO 2022170983 A1 WO2022170983 A1 WO 2022170983A1
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coronavirus
antibody
spike protein
dissociation
force
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French (fr)
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陈伟
胡炜
费攀宇
张同同
娄继忠
张勇
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Zhejiang University
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Zhejiang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • C07K16/102—Coronaviridae (F)
    • C07K16/104—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—RNA viruses
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • 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
    • G01N33/557—Immunoassay; Biospecific binding assay; Materials therefor using kinetic measurement, i.e. time rate of progress of an antigen-antibody interaction
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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 embodiments of the present invention relate to the field of cellular immunity, and in particular, to an anti-coronavirus antibody, a screening method thereof, a pharmaceutical composition containing the antibody, and applications.
  • the coronavirus mainly uses its spike protein (S protein) to bind to the receptor on the host cell, activate the fusion mechanism of the spike protein, and realize the virus invasion of the host.
  • S protein spike protein
  • the mature spike protein is mainly composed of two domains S1 and S2 through strong intramolecular non-covalent interactions.
  • the SARS-CoV-2 new coronavirus mainly uses the receptor binding domain (RBD) in its surface spike protein to recognize and bind to the angiotensin-converting enzyme 2 receptor (ACE2) of the host cell to activate virus invasion.
  • RBD receptor binding domain
  • ACE2 angiotensin-converting enzyme 2 receptor
  • the existing technology mainly focuses on the development of RBD-targeted neutralizing antibodies to directly block its interaction with ACE2, but single blocking of RBD/ACE2 interaction cannot resist the invasion of coronaviruses using other receptors.
  • the RBD region of the coronavirus mutates rapidly, and it is still doubtful whether antibodies against the RBD region can be effective against the mutated virus.
  • the inventors found in the study that the use of a single RBD-targeting antibody may also induce resistance mutations in the coronavirus.
  • Fc receptor-binding RBD blocking antibodies on the surface of immune cells may also activate the activation of the S2 membrane fusion machinery and instead help the virus to invade. Therefore, the development of antibodies targeting non-RBD epitopes and their screening methods are of great significance for the treatment of coronavirus-infected diseases.
  • the object of the present invention is to provide an anti-coronavirus antibody targeting non-RBD epitopes, to try to replace the existing RBD targeting antibody, to overcome the failure of single blocking RBD/ACE2 interaction to resist the invasion of coronavirus using other receptors Defects, or used in conjunction with existing RBD-targeted antibodies, reduce the chance of long-term use of a single RBD-targeted antibody to induce resistance mutations in coronaviruses.
  • the first aspect of the present invention provides an anti-coronavirus antibody, and the anti-coronavirus antibody can inhibit the dissociation of the S1 domain and the S2 domain of the coronavirus spike protein; and/or inhibit the coronavirus spike. Allosteric rearrangement of spike protein S2.
  • the dissociation force of the spike protein S1/S2 of the coronavirus treated with the anti-coronavirus antibody is greater than the dissociation force of the spike protein S1/S2 of the coronavirus not treated with the anti-coronavirus antibody away force.
  • the dissociation force of the spike protein S1/S2 of the coronavirus treated with the anti-coronavirus antibody is dissociated from the spike protein S1/S2 of the coronavirus not treated with the anti-coronavirus antibody
  • the ratio of the dissociated force is greater than or equal to 1.2, preferably, the dissociation force of the spike protein S1/S2 of the coronavirus treated with the anti-coronavirus antibody and the spike of the coronavirus not treated with the anti-coronavirus antibody
  • the ratio of protein S1/S2 dissociation force is greater than or equal to 1.2, or 1.3, or 1.4, or 1.5, etc., more preferably, the dissociation force is greater than or equal to 2.
  • the dissociation force of the spike protein S1/S2 of the coronavirus treated with the anti-coronavirus antibody is dissociated from the spike protein S1/S2 of the coronavirus not treated with the anti-coronavirus antibody
  • the ratio of the dissociation force is 1.2 ⁇ 200;
  • the force ratio of spike protein S1/S2 dissociation is 1.2-100.
  • the anti-coronavirus antibody specifically targets the spike protein S1 domain of the coronavirus and/or the spike protein S2 domain of the coronavirus.
  • the method for determining the activity of the antibody to inhibit the dissociation of the S1 and S2 domains of the coronavirus spike protein is single-molecule force spectroscopy.
  • the single-molecule force spectroscopy technology includes: magnetic tweezers, optical tweezers, atomic force microscopy, biofilm mechanics probe technology, glass fiber technology; more preferably, the single-molecule force spectroscopy technology is single-molecule magnetic tweezers technology .
  • the "force for dissociation of the spike protein S1/S2 of coronavirus” is determined by a single-molecule magnetic tweezers stretching experiment; preferably, the single-molecule magnetic tweezers stretching experiment includes repeating the steps in sequence: (1) use magnetic tweezers to apply force to the spike protein of coronavirus to dissociate it; and (2) reduce the force to 0 pN and let the protein return to its initial state; (3) record the magnitude of the force.
  • the acceleration of the increase of the force is preferably 0.8-1.2 pN/s; for example, 1 pN/s.
  • the acceleration of the force reduction is preferably -3 to -6 pN/s; for example, -5 pN/s.
  • the standing time is preferably 50-70s; for example, 60s.
  • the dissociation force of the spike protein S1/S2 of the coronavirus treated with the anti-coronavirus antibody is greater than or equal to 15pN; preferably, the spike protein of the coronavirus treated with the anti-coronavirus antibody
  • the force for S1/S2 dissociation is 20 pN or more; eg, 24.3 pN.
  • the anti-coronavirus antibody-treated coronavirus spike protein S1/S2 has a dissociation force of 15-2000 pN; preferably, the anti-coronavirus antibody-treated coronavirus spike protein The dissociation force of S1/S2 is 20 to 1000 pN or more.
  • a second aspect of the present invention provides a method for screening anti-coronavirus antibodies, the method comprising the steps:
  • step (1) the step of determining the antibody to inhibit the dissociation activity of the S1 and S2 domains of the coronavirus spike protein comprises:
  • the step (1) also includes the steps before:
  • step (I) the screening for antibodies that simultaneously bind to the S1 and S2 domains of the coronavirus spike protein comprises the steps of:
  • the third aspect of the present invention provides an anti-coronavirus antibody obtained by screening by the above method.
  • the antibody is a monoclonal antibody, a polyclonal antibody or a nanobody.
  • the anti-coronavirus antibody is a humanized antibody.
  • a fourth aspect of the present invention provides a pharmaceutical composition comprising the anti-coronavirus antibody and a pharmaceutically acceptable carrier.
  • the fifth aspect of the present invention provides the application of the above-mentioned anti-coronavirus antibody or pharmaceutical composition in the preparation of a medicine for preventing and/or treating diseases caused by coronavirus.
  • the diseases caused by the coronavirus include Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and/or Novel Coronavirus Pneumonia (COVID-19).
  • MERS Middle East Respiratory Syndrome
  • SARS Severe Acute Respiratory Syndrome
  • COVID-19 Novel Coronavirus Pneumonia
  • the sixth aspect of the present invention provides a method for preventing and/or treating diseases caused by coronavirus, the method comprising administering to a patient a therapeutically effective amount of the above-mentioned antibody, or antigen-binding fragment thereof; A pharmaceutical composition comprising an amount of the above-mentioned antibody, or an antigen-binding fragment thereof.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the present invention has at least the following advantages:
  • the anti-coronavirus antibody provided by the invention has the potential to replace the existing RBD-targeted antibody because it targets non-RBD epitopes, overcomes the single blocking RBD/ACE2 interaction and cannot resist the use of other receptors by the coronavirus Defects in invasion, or used in conjunction with existing RBD-targeted antibodies, reduce the chance of long-term use of a single RBD-targeted antibody to induce resistance mutations in coronaviruses.
  • the method for screening anti-coronavirus antibodies provided by the present invention applies a new screening strategy for inhibiting the dissociation of coronavirus spike proteins S1 and S2 or inhibiting allosteric rearrangement of S2, which can be widely used in the invasion of coronaviruses Blocking.
  • Figure 1 is a comparison of the dissociation forces of the S1 and S2 domains of the spike protein under the action of antibodies;
  • Figure 2 is a comparison of the dissociation distances of the S1 and S2 domains of the spike protein under the action of antibodies;
  • Figure 3 is a schematic diagram of the variation of the dissociation rate of the S1 and S2 domains with force under the action of an antibody
  • Figure 4 is a schematic diagram of the efficiency of pseudovirus infection of ACE2-expressing 293T host cells under the action of antibodies.
  • coronavirus covers the four main subgroups of coronaviruses, namely Alpha, Beta, Gamma and Delta. More specifically, under this term are meant enveloped viruses with positive RNA genomes (ssRNA+) and nucleocapsids with helical symmetry; and large RNA viruses with genome sizes ranging from approximately 26 to 32 kilobases; and Other viruses with characteristic morphology of large spherical surface protrusions under electron microscope, such as: HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2 (2019-nCoV )Wait.
  • ssRNA+ positive RNA genomes
  • nucleocapsids with helical symmetry and large RNA viruses with genome sizes ranging from approximately 26 to 32 kilobases
  • Other viruses with characteristic morphology of large spherical surface protrusions under electron microscope such as
  • antibody encompasses whole antibodies as well as antibody fragments.
  • Antibody includes naturally occurring and artificially generated polyclonal antibodies, monoclonal antibodies and nanobodies, in particular, the term “antibody” refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen.
  • the antibody can be an intact antibody and any antigen-binding fragment thereof or a single chain thereof.
  • antibody includes a protein- or peptide-containing molecule containing at least a portion of an immunoglobulin molecule having the biological activity of binding to an antigen, eg, human antibody, chimeric antibody, humanized antibody, primate-derived Antibodies, veneered antibodies, single chain antibodies, and domain antibodies, etc.
  • an antigen eg, human antibody, chimeric antibody, humanized antibody, primate-derived Antibodies, veneered antibodies, single chain antibodies, and domain antibodies, etc.
  • epitope refers to a protein determinant capable of specifically binding an antibody.
  • Epitopes typically consist of chemically active surface groups of molecules such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • spike protein and “spike protein” are used interchangeably between “spike protein” and “S protein” and “s-protein” and “spike protein”. "Consists of a near-N-terminal S1 domain, a near-C-terminal S2 domain, a transmembrane domain, and an intracellular domain.
  • force of Spike S1/S2 dissociation refers to the force that dissociates between the S1 and S2 domains of the Spike protein.
  • S1 refers to the domain near the N-terminus of the coronavirus spike protein, and the terms “S1” and “S1 domain” are used interchangeably.
  • S2 refers to the C-terminal proximal domain of the coronavirus spike protein, and the terms “S2” and “S2 domain” are used interchangeably.
  • allosteric rearrangement refers to any structural change in a biomolecule, for example, non-limiting example: dissociation of the S1 and S2 domains of the coronavirus spike protein, exposing the S2 domain Recombination of the S2 structure caused by the fusogenic peptide on the membrane.
  • RBD refers to the receptor binding domain of the S1 subunit on the coronavirus spike protein, responsible for binding to the cellular receptor protein ACE2.
  • humanized antibody refers to a class of engineered antibodies having CDRs derived from a non-human donor immunoglobulin whose remaining immunoglobulin portion is derived from a ( or more) human immunoglobulins.
  • Trizol method refers to a method for extracting RNA using Trizol reagent, which specifically includes the steps of culturing tissue cells; treating tissue cells with Trizol reagent to obtain supernatant; Process the tissue cells by centrifugation, discard the supernatant and dry.
  • OD600 refers to the absorbance of a solution at a wavelength of 600 nm.
  • composition means that the composition is of a grade and purity suitable for prophylactic or therapeutic administration to human subjects and is compatible with a pharmaceutically acceptable carrier, diluent, excipient At least one of the agents and/or additives is present together.
  • a pharmaceutically acceptable carrier As mentioned above, "a pharmaceutically acceptable carrier, excipient, adjuvant, excipient or diluent" as used herein is well known to those skilled in the art and readily available to the public.
  • a pharmaceutically acceptable carrier is one which is chemically inert to the active compound and which will not have deleterious side effects or toxicity under the conditions of use.
  • treatment refers to both therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent or slow (reduce) undesired physiological changes or disorders, such as the progression of an autoimmune disease.
  • beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable, including relief of symptoms, reduction in disease severity, stabilization of disease state (ie, no worsening), delay or slowdown in disease progression, Improvement or alleviation and alleviation (whether partial or total) of disease state.
  • Treatment also means prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder, as well as those susceptible to the condition or disorder, or those in need of prevention of the condition or disorder.
  • ectodomain spike protein About 1 mg of the full-length ectodomain spike protein was injected near the cervical lymph nodes of healthy alpaca every 2 weeks, for a total of 7 immunizations; about 60 mL of blood was collected from the neck vein of the alpaca during the last two immunizations; by centrifugation ( 25°C, 400g, centrifugation for 30 minutes) to separate lymphocytes.
  • the total RNA of lymphocytes was extracted by the Trizol method, and the corresponding cDNA library was established using a reverse transcription kit; then the antibody fragments were specifically amplified from the above cDNA library by PCR polymerase, and the antibody fragments were amplified by restriction endonuclease and T4 ligase.
  • the immunotubes coated with the full-length extracellular domain spike protein were selectively combined with the phage library obtained above (3% BSA, room temperature, 1 hour); after that, washed with PBS containing 0.01% Tween; The bound phage was eluted with 100 mM Trimethymime; the eluted phage was re-amplified and purified, and the concentration of the spike protein coated with the full-length extracellular domain was successively reduced, and the phage specific for the full-length extracellular domain was obtained after multiple rounds of screening. library. Finally, the gene sequences of the corresponding antibodies were obtained by ELISA identification and sequencing of the corresponding SS320 colonies.
  • Polystyrene microplates were coated with 2 ⁇ g/mL of S2 overnight at 4°C, and antibodies that specifically bound Spike S1 were screened for specific binding to Spike using the same method used to screen antibodies that specifically bind Spike S1.
  • Protein S2 antibody or first screen for antibodies that specifically bind to spike protein S2, and then screen for antibodies that specifically bind to spike protein S1, that is, antibodies that bind to both coronavirus spike proteins S1 and S2 at the same time. Finally, two antibodies were screened and denoted as Antibody 1 and Antibody 2.
  • the selected antibodies 1 and 2 were subjected to single-molecule magnetic tweezers stretching experiments.
  • the coverslips (dimensions of 30 ⁇ 24 mm and 22 ⁇ 22 mm, respectively) were soaked in 10% concentration of Decon90 solution, and washed with double distilled water after 30 minutes. After drying the cleaned long glass slides, put them into an oxygen plasma cleaner for 5 minutes, and soak them in 1% APTES methanol solution to aminate the surface of the glass slides. After 60 minutes, use methanol and double-distillation. The slides were washed three times with water. After drying the long glass slides, they and short glass slides were assembled into a laboratory chamber (Chamber) in a "sandwich" style. The Chamber was placed on a platform at 85°C, and the sealing film was gently pressed with tweezers to improve its sealing.
  • Extruded silica is stacked on both ends of the Chamber's slide to create "reservoirs" for adding and aspirating solutions.
  • PBS solution was added to wash the Chamber, and then a 0.5% concentration of glutaraldehyde in PBS was added to form the surface of the glass slide.
  • the Chamber was washed twice with PBS solution, and 100 ⁇ L of 50 ⁇ g/mL RBD monoclonal antibody solution was added to make the RBD antibody covalently linked to the surface of the glass slide.
  • 30 ⁇ L of amino-modified polystyrene beads on the surface (10 7 /mL) were added to fix the beads to the glass slide surface by covalent attachment.
  • the Chamber was washed twice with PBS solution and blocked with 1% BSA in PBS solution. After 8 hours or overnight, take out the spike protein from the -80°C refrigerator, dilute it to 1/10 4 to 1/10 6 times the storage concentration, and add it to the Chamber, so that the RBD area of the spike protein is covered by the surface of the glass slide. RBD antibody capture. After 30 minutes, the two sides of the Chamber were washed with PBS solution, and then 100uL of BSA-blocked magnetic beads (10 6 /mL) were added to the Chamber and incubated for 20 minutes, so that the C-terminus of the spike protein was linked to the streptavidin by biotin. protein (SA) on a magnetic sphere.
  • SA biotin. protein
  • the single-molecule magnetic tweezers experiment was started after finding the spike protein with both ends connected to the surface of the magnetic ball and the glass slide under the microscope. Control the magnet to apply force to the protein at a force loading speed of 1pN/s, and then reduce the force to 0pN at a speed of -5pN when the force reaches the peak value; observe the S1/S2 dissociation of the spike protein during the increase of the force. After observing the dissociation of S1/S2, wait for 60 seconds to ensure that the protein returns to the original state, and repeat the above process. After determining the force-induced dissociation of the spike protein S1/S2, the magnitude of the force and the change in distance were recorded.
  • SARS2-GFP pseudovirus was obtained after 50 hours . Subsequently, 1 mL of the above SARS2-GFP pseudovirus solution was taken, and different concentrations of antibody 1 solutions were added to infect 2 ⁇ 10 5 ACE2-expressing 293T host cells in a 24-well plate.

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Abstract

本申请公开了一种抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用。本申请中，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力大于未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力。本申请提供的靶向非RBD表位的抑制S1/S2解离和或S2变构重排的抗冠状病毒抗体，以尝试替代现有的RBD靶向抗体，克服单一阻断RBD/ACE2互作无法抵抗冠状病毒利用其它受体入侵的缺陷，或与现有的RBD靶向抗体配合使用，降低长期使用单一的RBD靶向抗体诱发冠状病毒产生抵抗性突变的几率。

Description

抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用
相关申请交叉引用
本专利申请要求于2021年02月10日提交的、申请号为2021101830936、发明名称为“抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明实施例涉及细胞免疫领域，特别涉及一种抗冠状病毒抗体、其筛选方法、含有该抗体的药物组合物及应用。
背景技术
冠状病毒主要利用其刺突蛋白(S蛋白)结合宿主细胞上的受体，激活刺突蛋白的融合机制，实现病毒入侵宿主。成熟的刺突蛋白主要由S1和S2两个结构域通过分子内强非共价的相互作用组装而成。例如，SARS-CoV-2新冠病毒主要利用其表面刺突蛋白中的受体结合域(RBD)识别并结合宿主细胞的血管紧张素转化酶2受体(ACE2)，激活病毒入侵。
因此，现有技术主要聚焦于RBD靶向中和抗体的研发，用于直接阻断其与ACE2的相互作用，但单一阻断RBD/ACE2互作无法抵抗冠状病毒利用其他受体入侵。同时，冠状病毒RBD区域变异较快，针对RBD区域的抗体能否对变异的病毒起效仍存疑。另外，发明人在研究中发现，使用单一的RBD靶向抗体还可能会诱发冠状病毒产生抵抗性突变。此外，免疫细胞表面的Fc受体结合RBD阻断抗体还有可能激活促进S2膜融合机器的活化反而帮助病毒入侵。因此，开发靶向非RBD表位的抗体及其筛选方法对冠状病毒感染疾病的治疗意义重大。
发明内容
本发明的目的在于提供一种靶向非RBD表位的抗冠状病毒抗体，以尝试替代现有的RBD靶向抗体，克服单一阻断RBD/ACE2互作无法抵抗冠状 病毒利用其它受体入侵的缺陷，或与现有的RBD靶向抗体配合使用，降低长期使用单一的RBD靶向抗体诱发冠状病毒产生抵抗性突变的几率。
为解决上述问题，本发明第一方面提供了一种抗冠状病毒抗体，所述抗冠状病毒抗体可抑制冠状病毒刺突蛋白S1结构域与S2结构域的解离；和/或抑制冠状病毒刺突蛋白S2变构重排。
在一些优选的方案中，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力大于未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力。
在一些优选的方案中，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力与未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力之比大于等于1.2，优选地，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力与未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力之比大于等于1.2，或1.3，或1.4，或1.5等，更优选地该解离的力大于等于2。
在一些优选的方案中，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力与未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力之比为1.2～200；优选地，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力与未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力之比为1.2～100。
在另一优选例中，所述抗冠状病毒抗体特异性靶向冠状病毒的刺突蛋白S1结构域，和/或冠状病毒的刺突蛋白S2结构域。
在一些优选的方案中，所述测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离的活性的方法为单分子力谱技术。优选地，所述单分子力谱技术包括：磁镊、光镊、原子力显微镜技术、生物膜力学探针技术、玻璃纤维技术；更优选地，所述单分子力谱技术为单分子磁镊技术。
在一些优选的方案中，通过单分子磁镊拉伸实验测定“冠状病毒的刺突蛋白S1/S2解离的力”；优选地，所述单分子磁镊拉伸实验包括依次重复执行步骤：(1)使用磁镊对冠状病毒的刺突蛋白施加力使其解离；和(2)将力减小至0pN静置使蛋白质恢复初态；(3)记录力的大小。
步骤(1)中，所述力的增加的加速度优选为0.8～1.2pN/s；例如1pN/s。
步骤(2)中，所述力的减小的加速度优选为-3～-6pN/s；例如-5pN/s。
步骤(2)中，所述静置的时间优选为50～70s；例如60s。
在一些优选的方案中，所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力为大于等于15pN；优选地，所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力为大于等于20pN；例如24.3pN。
在一些优选的方案中，所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力为15～2000pN；优选地，所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力为大于等于20～1000pN。
本发明第二方面提供了一种筛选抗冠状病毒抗体的方法，所述方法包括步骤：
(1)测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离的活性；和/或
(2)测定抗体抑制冠状病毒刺突蛋白S2变构重排的活性。
在一些优选的方案中，步骤(1)中，所述测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离活性步骤包括：
(A)测定未经抗体处理的刺突蛋白S1/S2解离的力的大小，记为F 1；和
(B)测定抗体处理后的刺突蛋白S1/S2解离的力的大小，记为F 2；
(C)筛选F 2大于F 1时的抗体，即得所述抗冠状病毒抗体。
在一些优选的方案中，所述步骤(1)前还包括步骤：
(Ⅰ)筛选同时结合冠状病毒刺突蛋白S1结构域和S2结构域的抗体。
在一些优选的方案中，步骤(Ⅰ)中，所述筛选同时结合冠状病毒刺突蛋白S1结构域和S2结构域的抗体包括步骤：
(a)从抗体库中筛选特异性结合刺突蛋白S1结构域的抗体；
(b)从特异性结合刺突蛋白S1的抗体中筛选特异性结合S2结构域的抗体。
本发明第三方面提供了一种由上述方法筛选获得的抗冠状病毒抗体。
在一些优选的方案中，所述抗体为单克隆抗体、多克隆抗体或纳米抗体。
在一些优选的方案中，所述抗冠状病毒抗体为人源化抗体。
本发明第四方面提供了一种药物组合物，所述药物组合物包括所述的抗冠状病毒抗体以及药学可接受的载体。
本发明第五方面提供了上述抗冠状病毒抗体或药物组合物在制备预防和/或治疗冠状病毒所导致的疾病的药物中的应用。
在一些优选的方案中，所述冠状病毒所导致的疾病包括中东呼吸综合征(MERS)、严重急性呼吸综合征(SARS)和/或新型冠状病毒肺炎(COVID-19)。
本发明第六方面提供了预防和/或治疗冠状病毒所导致的疾病的方法，所述方法包括向患者施用治疗有效量的上述的抗体，或其抗原结合片段；或者向患者施用包含有治疗有效量的上述的抗体，或其抗原结合片段的药物组合物。
在不违背本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
相对现有技术而言，本发明至少具有下述优点：
(1)本发明提供的抗冠状病毒抗体，由于其靶向非RBD表位，因此具有潜力替代现有的RBD靶向抗体，克服单一阻断RBD/ACE2互作无法抵抗冠状病毒利用其它受体入侵的缺陷，或与现有的RBD靶向抗体配合使用，降低长期使用单一的RBD靶向抗体诱发冠状病毒产生抵抗性突变的几率。
(2)本发明提供的筛选抗冠状病毒抗体的方法，应用全新的抑制冠状病毒刺突蛋白S1与S2解离或抑制S2变构重排的筛选策略，该策略可以广泛应用于冠状病毒的入侵阻断中。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。
图1是根据刺突蛋白在抗体作用下S1与S2结构域解离力的大小的比较；
图2是刺突蛋白在抗体作用下S1与S2结构域解离的距离大小的比较；
图3是抗体作用下S1与S2结构域解离速率随力的变化示意图；
图4是抗体作用下假病毒感染ACE2表达的293T宿主细胞的效率示意 图。
具体实施方式
如本文所用，术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文所用，术语“冠状病毒”的范围涵盖冠状病毒的四个主要亚组，分别为Alpha，Beta，Gamma和Delta。更具体地，在该术语下是指具有正向RNA基因组(ssRNA+)和具有螺旋对称的核衣壳的包膜病毒；以及基因组大小范围约为26至32千个碱基的大型RNA病毒；以及在电子显微镜下具有大球形表面突起特征形态的其他病毒，例如：HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV和SARS-CoV-2(2019-nCoV)等。
如本文所用，术语“抗体”涵盖完整抗体以及抗体片段。“抗体”包括自然生成的和人工生成的多克隆抗体、单克隆抗体和纳米抗体，具体地，术语“抗体”是指特异性识别和结合抗原的多肽或多肽复合物。所述抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此，术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的含蛋白质或肽，例如：人抗体、嵌合抗体、人源化抗体、灵长类源化抗体、镶饰的抗体、单链抗体以及结构域抗体等。
如本文所用，术语“表位”是指能够特异性结合抗体的蛋白质决定簇。表位通常由分子如氨基酸或糖侧链的化学活性表面基团组成，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，术语“刺突蛋白”和“突刺蛋白”和“棘突蛋白”和“S蛋白”和“s-protein”和“spike protein”之间可以互换使用，所述“刺突蛋白”由近N-末端S1结构域、近C端S2结构域，跨膜结构域和细胞内结构域组成。
如文本所用，术语“刺突蛋白S1/S2解离的力”是指解离刺突蛋白S1结构域和S2结构域之间的力。
如本文所用，术语“S1”是指冠状病毒刺突蛋白近N-末端的结构域，术语“S1”和“S1结构域”可互换使用。
如本文所用，术语“S2”是指冠状病毒刺突蛋白近C端的结构域，术语“S2”和“S2结构域”可互换使用。
如本文所用，术语“变构重排”是指生物分子发生任一种结构的改变，非限制性的例如：冠状病刺突蛋白的S1结构域和S2结构域发生解离，暴露S2结构域上的膜融合肽引起的S2结构的重组。
如文本所用，术语“RBD”是指冠状病刺突蛋白上的S1亚基的受体结合域，负责与细胞受体蛋白ACE2的结合。
如本文所用，术语“人源化抗体”是指一类工程化抗体，其具有源自非人供体免疫球蛋白的CDR，该人源化抗体的剩余免疫球蛋白部分是来源自一种(或多种)人免疫球蛋白。
如本文所用，术语“Trizol法”是指一种使用Trizol试剂提取RNA的方法，具体包括步骤培养组织细胞；Trizol试剂处理组织细胞离心取上清；氯仿处理组织细胞离心取上清；异丙醇处理组织细胞离心弃上清并干燥。
如本文所用，术语“OD600”是指某种溶液在600nm波长处的吸光值。
如本文所用，术语“药物组合物”是指该组合物具有适于对人类受试者进行预防性或治疗性给药的等级和纯度，并且与药学上可接受的载体，稀释剂，赋形剂和/或添加剂中的至少一种一起存在。
如上所述，本文所用的“药学上可接受的载体，赋形剂，佐剂，赋形剂或稀释剂”是本领域技术人员公知的，易于公众获得。优选地，药学上可接受的载体是对活性化合物具有化学惰性的载体和在使用条件下没有有害副作用或毒性的载体。
如本文所用，术语“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防或减缓(减少)不期望发生的生理改变或紊乱，例如自身免疫性疾病的进程。有益的或期望的临床结果包括但不限于以下无论是可检测还是不可检测的结果，包括症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善或缓和以及减轻(无论是部分还是全部)。“治疗”还意指与不接受治疗时预期的生存期限相比所延长的生存期限。需要治疗的包括那些已经患有病症或紊乱的人，以及那些容易患有病症或紊乱的人，或者那些需要预防该病症或紊乱的人。
除非另有说明，本文所用的所有科学和技术术语具有本领域常用的含义。这里提供的定义是为了便于理解这里经常使用的某些术语，而不是为了限制本公开的范围。
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合实施例对本发明的各实施方式进行详细的阐述。然而，本领域的普通技术人员可以理解，在本发明各实施方式中，为了使读者更好地理解本申请而提出了许多技术细节。但是，即使没有这些技术细节和基于以下各实施方式的种种变化和修改，也可以实现本申请所要求保护的技术方案。
实施例1 抗冠状病毒抗体库构建
在健康羊驼颈部淋巴结附近每2周注射一次约1mg胞外域全长的刺突蛋白，共7次免疫；最后两次免疫期间进行羊驼颈部静脉取约60mL血液；通过离心的方式(25℃，400g，离心30分钟)分离得到淋巴细胞。
用Trizol法提取淋巴细胞总RNA，使用逆转录试剂盒建立相应的cDNA库；之后用PCR聚合酶从上述cDNA库中特异性地扩增抗体片段，并通过限制性内切酶与T4连接酶将其连接到噬菌体载体上；通过电转的方式将连接产物转化到SS320感受态细胞中，在抗性(四环素和氨苄)固体LB板上培养过夜；将含有抗性的细胞在抗性(四环素和氨苄)YT培养液扩增(37℃，220rpm)；当OD600≈0.5时，加入辅助噬菌体(辅助噬菌体数：细菌细胞数＝20:1)并继续培养30分钟；并用0.2mM的IPTG诱导30℃过夜培养；最后通过沉淀-离心的方式得到噬菌体库。
将胞外域全长的刺突蛋白包被好的免疫管与上述得到的噬菌体库进行选择性地结合(3％BSA，室温，1小时)；之后，用含有0.01％吐温的PBS清洗；用100mM Trimethymime洗脱结合的噬菌体；将洗脱的噬菌体再次扩增及纯化，并逐次减低包被胞外域全长的刺突蛋白浓度，经过多轮筛选得到胞外域全长的刺突蛋白特异的噬菌体库。最后通过ELISA鉴定并将相应的SS320菌落进行测序获得相应抗体的基因序列。
实施例2 抗冠状病毒抗体筛选
(1)从抗体库中筛选同时结合冠状病毒刺突蛋白S1结构域和S2结构域的抗体
用2μg/mL的S1 4℃过夜包被聚苯乙烯微孔板；用含0.2％Tween-20的PBS溶液清洗上述微孔板；之后用含2％BSA的PBST溶液37℃封闭1小时；微孔板经PBST清洗后用含有不同浓度的抗体在37℃条件下孵育1小时；PBST清洗后，用辣根过氧化物酶偶连的二抗以1:10000的稀释倍数37℃孵育1小时；清洗后，加入TMB显色液，室温孵育10分钟；加入等体积的硫酸溶液终止反应，并在波长450nm处测定吸光值，筛选波长450nm处有响应的抗体，即得特异性结合刺突蛋白S1的抗体。
用2μg/mL的S2 4℃过夜包被聚苯乙烯微孔板，采用与筛选特异性结合刺突蛋白S1的抗体相同的方法从特异性结合刺突蛋白S1的抗体中筛选特异性结合刺突蛋白S2的抗体；或者先筛选特异性结合刺突蛋白S2的抗体，再筛选特异性结合刺突蛋白S1的抗体，即得同时结合冠状病毒刺突蛋白S1与S2的抗体。最终筛选出两个抗体记为抗体1和抗体2。
(2)通过单分子磁镊拉伸实验筛选抑制冠状病毒刺突蛋白S1结构域与S2结构域的解离或抑制冠状病毒刺突蛋白S2变构重排的抗体。
将筛选出的抗体1和抗体2进行单分子磁镊拉伸实验。
将盖玻片(尺寸分别是30×24mm和22×22mm)浸泡在10％浓度的Decon90溶液中，30分钟后用双蒸水清洗盖玻片。将清洗完成的长玻片烘干后放入氧气等离子清洗机清洗5分钟，并将其浸泡在1％浓度的APTES甲醇溶液中对玻片表面进行氨基化修饰，60分钟后用甲醇和双蒸水先后分别清洗三次玻片。将长玻片烘干后，将其和短玻片按照“三明治”样式组装成实验小室(Chamber)，将Chamber放在85℃平台上，用镊子轻轻按压封口膜处提高其封闭性。挤压硅胶堆积在Chamber的玻片两头制造“蓄水池”，用于加入和吸出溶液。待硅胶凝固后，先加入PBS溶液清洗Chamber，随后加入0.5％浓度的戊二醛PBS溶液，使玻片表面醛基化。30分钟后，用PBS溶液清洗Chamber两次，加入100μL 50μg/mL的RBD单克隆抗体溶液，使RBD抗体共价连接到玻片表面上。10分钟后，加入30μL表面带有氨基修饰的聚苯乙烯小球(10 7个/mL)，使小球通过共价连接固定至玻片表面。4小时后， 用PBS溶液清洗Chamber两次，再用1％BSA的PBS溶液封闭Chamber。8小时或过夜后，从-80℃冰箱中取出刺突蛋白，将其稀释至存储浓度的1/10 4～1/10 6倍后加入Chamber中，使刺突蛋白的RBD区域被玻片表面的RBD抗体捕获。30分钟后，用PBS溶液清洗两边Chamber，接着将100uL BSA封闭的磁球(10 6个/mL)加入Chamber中孵育20分钟，使刺突蛋白的C端通过生物素连接到带链霉亲和素蛋白(SA)的磁球上。
在显微镜下找到两端分别连接在磁球和玻片表面的刺突蛋白后开始做单分子磁镊实验。控制磁铁以1pN/s的力加载速度对蛋白质施加力，当力达到峰值后以-5pN的速度将力减小至0pN；观察刺突蛋白在力增加的过程中的S1/S2解离情况。观察到S1/S2解离后，等待时间60秒以保证蛋白质回到最初状态，如此反复执行上述过程。在确定刺突蛋白S1/S2发生力致解离后，记录其力的大小和距离变化的大小。
在Chamber中轻柔地加入一定浓度的实施例2筛选的抗体1溶液，将力维持在较低水平(～1pN)，等待20分钟，使待测抗体结合到刺突蛋白相应的抗原表位上。控制磁镊对刺突蛋白施加逐渐增加的力，统计此时刺突蛋白出现S1/S2解离时的力和距离变化的大小，并与没有加入待测抗体的数据进行比较。结果见图2。
同样的，在Chamber中轻柔地加入一定浓度的实施例2筛选的抗体2溶液，将力维持在较低水平(～1pN)，等待20分钟，使待测抗体结合到刺突蛋白相应的抗原表位上。控制磁镊对刺突蛋白施加逐渐增加的力，统计此时刺突蛋白出现S1/S2解离时的力和距离变化的大小，并与没有加入待测抗体的数据进行比较。结果见图2。抗体1使刺突蛋白出现S1/S2解离时的力约为24pN。
根据上述数据，根据Bell模型，分别得到的抗体1和抗体2对S1结构域与S2结构域解离速率影响情况，见图3。
实施例3 抗冠状病毒抗体人源化改造
利用在线工具(例如IG Blast、IMGT/V-QUEST、AbYsis等)将得到的纳米抗体与人源抗体或成熟的人源化抗体的氨基酸序列划分为的骨架区 (FR1到FR4区)和互补决定区(CDR1～CDR3区)；以人源抗体或成熟的人源化抗体为模板，保留骨架区(FR1到FR4区)，将其CDR1～CDR3区移植为目标纳米抗体的CDR1～CDR3，即CDR区移植法对纳米抗体进行人源化改造。将改造得到的人源化纳米抗体进行同源建模，对产生空间结构影响的CDR区进行适当地改造，并对其进行能量最小化优化，提升人源化纳米抗体结构的稳定性。
合成人源化抗体重链与轻链基因并在AbVec2.0-IGHG1载体上构建抗体表达的质粒；通过PEI脂质体瞬时转染的方式将其在Expi-293F细胞上分泌表达。取适量的Protein A/G Beads用平衡液进行离心清洗(500g，3分钟；重复两次)；将上述Protein A/G Beads中加入Expi-293F细胞分泌的抗体溶液，混合孵育约30分钟；离心并用洗杂液清洗抗体富集的Protein A/G Beads(500g，3分钟；重复两次)；之后，加入5倍Protein A/G Beads体积的洗脱液，均匀混匀10分钟，离心(500g，3分钟)获得抗体上清液；之后，立即加入十分之一体积的中和液讲pH值调至7.4；之后通过离子交换柱及分子筛得到高纯度的抗体。
实施例4 抗冠状病毒抗体中和效果鉴定
用PEI脂质体共转染新冠病毒刺突蛋白质粒(pCAG-SARS2-SΔC19)及辅助质粒(PLP1、PLP2、pCDH-CMV-CopGFP)到HEK 293T细胞中，50小时后获得SARS2-GFP假病毒。随后，取1mL上述SARS2-GFP假病毒溶液，并加入不同浓度的抗体1溶液，在24孔板中感染2×10 5个表达ACE2的293T宿主细胞。12小时后更换新鲜DMEM完全培养基；细胞再培养36小时后用FACS检测GFP阳性的293T的比例，即指示新冠假病毒感染ACE2宿主细胞的效率。相比于对照组抗体2的中和效果，抗体1的加入显著地降低了新冠假病毒入侵的效率；并且随着其浓度的提高，新冠假病毒感染ACE2表达的293T宿主细胞的效率从15％降低到10％；结果见图4。
上述结果表明，抗体1使S1/S2解离时的力明显增加，约2倍左右，从而显著地降低了新冠假病毒入侵的效率；而抗体2使S1/S2解离时的力增加不明显，其降低新冠假病毒入侵的效率效果不及抗体1。显而易见地，采用 本发明所述的抗冠状病毒抗体筛选的方法，通过对刺突蛋白S1/S2解离时的力的测定，筛选出的抗体可显著降低冠状病毒入侵的效率。
本领域的普通技术人员可以理解，上述各实施方式是实现本发明的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。

Claims (12)

  1. 一种抗冠状病毒抗体，其特征在于，所述抗冠状病毒抗体可抑制冠状病毒刺突蛋白S1结构域与S2结构域的解离；和/或
    抑制冠状病毒刺突蛋白S2变构重排。
  2. 根据权利要求1所述的抗冠状病毒抗体，其特征在于，所述抗体抑制冠状病毒刺突蛋白S1结构域与S2结构域的解离指经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力大于未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力。
  3. 根据权利要求2所述的抗冠状病毒抗体，其特征在于，经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力与未经所述抗冠状病毒抗体处理的冠状病毒的刺突蛋白S1/S2解离的力之比大于等于1.2。
  4. 一种筛选抗冠状病毒抗体的方法，其特征在于，所述方法包括步骤：
    (1)测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离的活性；和/或
    (2)测定抗体抑制冠状病毒刺突蛋白S2变构重排的活性；
    其中，所述测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离的活性包括步骤：
    (A)测定未经抗体处理的刺突蛋白S1/S2解离的力的大小，记为F 1；和
    (B)测定抗体处理后的刺突蛋白S1/S2解离的力的大小，记为F 2；和
    (C)筛选F 2大于F 1时的抗体，即得所述抗冠状病毒抗体。
  5. 根据权利要求4所述的筛选抗冠状病毒抗体的方法，其特征在于，所述测定抗体抑制冠状病毒刺突蛋白S1结构域和S2结构域解离的活性的方法为单分子力谱技术。
  6. 根据权利要求4或5任一项所述的方法，其特征在于，所述步骤(1)前还包括步骤：
    (Ⅰ)筛选同时结合冠状病毒刺突蛋白S1结构域和S2结构域的抗体。
  7. 一种抗冠状病毒抗体，其特征在于，所述抗冠状病毒抗体由权利要求4～6中任一种方法筛选获得。
  8. 根据权利要求1或7任一项所述的抗冠状病毒抗体，其特征在于，所述抗冠状病毒抗体为人源化抗体。
  9. 一种药物组合物，其特征在于，所述药物组合物包括权利要求7或8任一项所述的抗冠状病毒抗体以及药学可接受的载体。
  10. 根据权利要求1～3中任一项所述的抗冠状病毒抗体或者7或8任一项所述的抗冠状病毒抗体在制备预防和/或治疗冠状病毒所导致的疾病的药物中的应用。
  11. 根据权利要求9所述的抗冠状病毒抗体在制备预防和/或治疗冠状病毒所导致的疾病的药物中的应用。
  12. 根据权利要求10或11任一项所述的应用，其特征在于，所述冠状病毒所导致的疾病包括中东呼吸综合征(MERS)、严重急性呼吸综合征(SARS)和/或新型冠状病毒肺炎(COVID-19)。
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111574623A (zh) * 2020-05-25 2020-08-25 西安咸辅生物科技有限责任公司 一种新冠病毒s1+s2抗独特型卵黄抗体疫苗的制备方法
CN111606980A (zh) * 2020-05-27 2020-09-01 中国医学科学院基础医学研究所 Sars-cov冠状病毒s2蛋白多肽及其应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7629443B2 (en) * 2004-06-02 2009-12-08 New York Blood Center, Inc. Neutralizing monoclonal antibodies against severe acute respiratory syndrome-associated coronavirus
CN111269911B (zh) * 2020-01-23 2023-05-02 南开大学 包括CpG位点的发夹结构及测量CpG毗邻序列影响蛋白解离时间常数的单分子力学方法
CN112094327A (zh) * 2020-09-25 2020-12-18 军事科学院军事医学研究院军事兽医研究所 基于新型冠状病毒rbd-sd1蛋白的截短体及其应用
CN112194711A (zh) * 2020-10-15 2021-01-08 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） 一种新型冠状病毒s蛋白的b细胞线性抗原表位、抗体、鉴定方法及应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111574623A (zh) * 2020-05-25 2020-08-25 西安咸辅生物科技有限责任公司 一种新冠病毒s1+s2抗独特型卵黄抗体疫苗的制备方法
CN111606980A (zh) * 2020-05-27 2020-09-01 中国医学科学院基础医学研究所 Sars-cov冠状病毒s2蛋白多肽及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAI LIANPAN: "Viral targets for vaccines against COVID-19", NATURE REVIEWS IMMUNOLOGY, pages 73 - 82, XP055784887, Retrieved from the Internet <URL:https://www.nature.com/articles/s41577-020-00480-0.pdf> [retrieved on 20210311] *
HATEM A. ELSHABRAWY, MELISSA COUGHLIN, SUSAN BAKER, BELLUR PRABHAKAR, KRZYSZTOF PYRC: "Human Monoclonal Antibodies against Highly Conserved HR1 and HR2 Domains of the SARS-CoV Spike Protein Are More Broadly Neutralizing", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 7, no. 11, 21 November 2012 (2012-11-21), pages e50366, XP055695602, DOI: 10.1371/journal.pone.0050366 *
LU YAN, WANG YAJUN, ZHANG ZHEN, HUANG JINGLIANG, YAO MEICUN, HUANG GUOBIN, GE YUANYUAN, ZHANG PEICHUN, HUANG HUAXIN, WANG YONG, LI: "Generation of Chicken IgY against SARS-COV-2 Spike Protein and Epitope Mapping", JOURNAL OF IMMUNOLOGY RESEARCH, HINDAWI PUBLISHING CORPORATION, US, vol. 2020, 16 October 2020 (2020-10-16), US , pages 1 - 8, XP055872677, ISSN: 2314-8861, DOI: 10.1155/2020/9465398 *
O WILLEMSEN: "Biomolecular Interactions Measured by Atomic Force Microscopy", BIOPHYSICAL JOURNAL, BIOPHYSICAL SOCIETY, vol. 79, no. 6, 1 December 2000 (2000-12-01), pages 3267 - 3281, XP055012915, ISSN: 00063495, DOI: 10.1016/S0006-3495(00)76559-3 *

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