WO2021244089A1 - 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用 - Google Patents

新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用 Download PDF

Info

Publication number
WO2021244089A1
WO2021244089A1 PCT/CN2021/079568 CN2021079568W WO2021244089A1 WO 2021244089 A1 WO2021244089 A1 WO 2021244089A1 CN 2021079568 W CN2021079568 W CN 2021079568W WO 2021244089 A1 WO2021244089 A1 WO 2021244089A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cov
sars
spike protein
cdr1
Prior art date
Application number
PCT/CN2021/079568
Other languages
English (en)
French (fr)
Inventor
张军方
Original Assignee
深圳市因诺赛生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市因诺赛生物科技有限公司 filed Critical 深圳市因诺赛生物科技有限公司
Priority to US18/000,515 priority Critical patent/US20230331822A1/en
Priority to JP2022574538A priority patent/JP2023527927A/ja
Priority to KR1020227046331A priority patent/KR20230018488A/ko
Priority to EP21817035.5A priority patent/EP4159757A4/en
Priority to AU2021285330A priority patent/AU2021285330A1/en
Publication of WO2021244089A1 publication Critical patent/WO2021244089A1/zh

Links

Classifications

    • 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
    • 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
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • 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

  • This solution relates to a new type of coronavirus (SARS-COV-2) spike protein binding molecule and its application, and belongs to the field of medical biotechnology.
  • SARS-COV-2 coronavirus
  • the first aspect of this solution provides a novel coronavirus (SARS-COV-2) spike protein binding molecule, which can specifically bind to the SARS-COV-2 spike protein and contains at least one immunoglobulin single variable domain, CDR1, CDR2 and CDR3 in the single variable domain of the immunoglobulin are selected from any one of the following combinations:
  • the immunoglobulin single variable domain is a single domain antibody.
  • the single domain antibody comprises an amino acid sequence having at least 80% sequence identity with any sequence in SEQ ID NO: 82-108.
  • the single domain antibody comprises an amino acid sequence having at least 90% sequence identity with any sequence in SEQ ID NO: 82-108.
  • the single domain antibody comprises an amino acid sequence having at least 99% sequence identity with any sequence in SEQ ID NO: 82-108.
  • the amino acid sequence of the single domain antibody contains one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 82-108.
  • the single domain antibody comprises any one of the amino acid sequences of SEQ ID NO: 82-108.
  • the SARS-COV-2 spike protein binding molecule also includes an immunoglobulin Fc region.
  • the inclusion of the immunoglobulin Fc region in the SARS-COV-2 spike protein binding molecule of this scheme can make the binding molecule form a dimer and further extend the half-life of the molecule in vivo.
  • the Fc region that can be used in this protocol can be from immunoglobulins of different subtypes, for example, IgG (IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.
  • the immunoglobulin Fc region is a human immunoglobulin Fc region.
  • the immunoglobulin Fc region is the Fc region of human IgG1.
  • the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO: 109.
  • At least one amino acid sequence in SEQ ID NO: 110-136 is included.
  • the stability and biological activity of the binding molecule after fusion with the above-mentioned Fc region are further improved, and the KD value of its binding to the SARS-COV-2 spike protein is further reduced.
  • the SARS-COV-2 spike protein binding molecule has at least one of the following characteristics:
  • the KD value combined with the SARS-COV-2 spike protein is less than 1 ⁇ 10-8M;
  • the second aspect of this solution provides a nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule.
  • the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis or from suitable Natural sources are separated and obtained.
  • the third aspect of this solution provides an expression vector containing the nucleic acid molecule and its expression control element.
  • the expression vector usually contains at least one nucleic acid molecule provided in this scheme, which is operably linked to one or more suitable expression control elements (promoter, enhancer, terminator, integration factor, selection marker, leader sequence, Reporter gene, etc.).
  • suitable expression control elements promoter, enhancer, terminator, integration factor, selection marker, leader sequence, Reporter gene, etc.
  • the fourth aspect of this solution provides a host cell containing and expressing the nucleic acid molecule.
  • the host cell is a cell for expressing heterologous protein, including bacterial cells, fungal cells or mammalian cells.
  • the fifth aspect of this solution provides a method for obtaining the SARS-COV-2 spike protein binding molecule, including:
  • step b Collect the SARS-COV-2 spike protein binding molecule expressed by the host cell from the culture of step a.
  • the SARS-COV-2 spike protein binding molecule provided in this solution can also be obtained by other methods known in the art to produce proteins, such as chemical synthesis.
  • the sixth aspect of this solution provides an immunoconjugate comprising the SARS-COV-2 spike protein binding molecule described in any one of the above conjugated to a therapeutic moiety.
  • the seventh aspect of this solution provides a pharmaceutical composition
  • a pharmaceutical composition comprising the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate described in any one of the above, and a pharmaceutically acceptable carrier .
  • the "pharmaceutically acceptable carrier” mentioned in this scheme includes any physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (such as by injection or infusion).
  • the active compound ie, binding molecule, immunoconjugate
  • the active compound can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound, as is well known to those skilled in the art .
  • composition described in this scheme may also include other adjuvants and excipients as required.
  • the eighth aspect of this scheme provides the application of the pharmaceutical composition in the preparation of drugs for the treatment or prevention of novel coronavirus pneumonia.
  • the ninth aspect of this solution provides a kit for detecting SARS-COV-2, which contains the SARS-COV-2 spike protein binding molecule described in any one of the above.
  • the tenth aspect of this solution provides a method for using the kit for detecting SARS-COV-2.
  • the SARS-COV-2 spike protein binding molecule described in any one of the above and the SARS-COV-2 spike Under the condition that a complex can be formed between the spike proteins, the test sample and the control sample are brought into contact with the SARS-COV-2 spike protein binding molecule described in any one of the above to detect the formation of the complex; through the test sample and the control The difference in the formation of complexes between samples determines the presence of SARS-COV-2 in the samples.
  • the SARS-COV-2 spike protein (SARS-COV-2-Spike protein) binding molecule provided in this program can specifically bind to the SARS-COV-2-Spike protein and effectively block the SARS-COV-2-Spike protein Binding with the ACE2 receptor of human cells, thereby blocking the infection process of SARS-COV-2 on cells, and inhibiting the infection and expansion of SARS-COV-2.
  • the SARS-COV-2-Spike protein binding molecule provided by this solution also has the characteristics of good specificity in binding to SARS-COV-2-Spike protein, high biological activity and stability, and no toxic side effects.
  • Figure 1 is an agarose gel electrophoresis diagram of the total RNA extracted in Example 1 of this scheme, where M: DNA marker 2000, lane 1: total RNA;
  • Figure 2 is an agarose gel electrophoresis diagram of the Step1-PCR amplification product of the nested PCR amplification of the single domain antibody gene in Example 1 of this scheme, where M: DNA marker 2000, lane 1: amplified product;
  • Figure 3 is an agarose gel electrophoresis diagram of the Step2-PCR amplification product of the nested PCR amplification of the single domain antibody gene in Example 1 of the present scheme.
  • Figure 4 is an agarose gel electrophoresis diagram of the target single-domain antibody gene and the SfiI and Not1 double digestion products of the vector pHEN1 in Example 1 of this scheme, in which the DNA marker 2000, lane 1: pHEN1; lane 2: pHEN1 digested with sfil/notI; lane 3: single domain antibody gene digested with sfil/notI;
  • Fig. 5 is an agarose gel electrophoresis diagram of the colony PCR amplification product used to measure the insertion rate of the library in Example 1 of this scheme, where M: DNA marker 2000; lanes 1-48: 48 colonies picked;
  • Fig. 6 is a graph showing changes in viral load of rhesus monkeys in the treatment group and the control group as the number of days changes in Example 2 of the present scheme.
  • antibody or “immunoglobulin” used interchangeably herein, whether referring to heavy chain antibodies or conventional 4-chain antibodies, are used as general terms to include full-length antibodies, individual The chain and all of its parts, domains or fragments (including but not limited to antigen binding domains or fragments).
  • sequence as used herein (for example, in “immunoglobulin sequence", “antibody sequence”, In terms of “single variable domain sequence”, “single domain antibody sequence” or “protein sequence”, etc., it should generally be understood to include not only related amino acid sequences, but also nucleic acid sequences or nucleotide sequences encoding said sequences, Unless this article requires a more limited explanation.
  • immunoglobulin variable domain refers to the term "framework region 1" or “FR1”, “framework region 2” or “FR2”, and “framework region 3”, respectively, referred to in the art and hereinafter, respectively.
  • FR3 and “framework region 4" or “FR4" four "framework regions” composed of immunoglobulin domains, wherein the framework regions are referred to as “complementarity determining region 1" in the art and hereinafter, respectively
  • CDR1", “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3" or “CDR3” are separated by three “complementarity determining regions” or "CDRs”.
  • an immunoglobulin variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the immunoglobulin variable domains have antigen binding sites to give antibodies the specificity for the antigen.
  • Single domain antibody refers to a type of antibody that lacks the light chain of the antibody but only the variable region of the heavy chain. Because of its small molecular weight, it is also called Nanobody. Single domain antibodies specifically bind to epitopes without the need for other antigen-binding domains. Single domain antibodies are small, stable and efficient antigen recognition units formed by a single immunoglobulin domain.
  • the total number of amino acid residues in each CDR in a single domain antibody may be different.
  • the total number of amino acid residues in a single domain antibody will usually be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences can also be adapted for the purposes described in this scheme.
  • Single domain antibodies (which have been naturally "designed” to functionally bind to the antigen in the absence and interaction with the light chain variable domain) can be used as a single and relatively small function Sexual antigen binding structural unit, domain or polypeptide. This distinguishes the VH and VL domains of single-domain antibodies and conventional 4-chain antibodies. These VH and VL domains themselves are usually not suitable for practical applications as single antigen binding proteins or immunoglobulin single variable domains, but they need to be The form or another form is combined to provide a functional antigen binding unit (e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFv composed of VH domains covalently linked to VL domains).
  • a functional antigen binding unit e.g., in the form of conventional antibody fragments such as Fab fragments; or in the form of scFv composed of VH domains covalently linked to VL domains).
  • single domain antibodies or as part of a larger polypeptide provides many advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (such as Fab- or F(ab')2-fragments).
  • single domain antibodies require only a single domain to bind antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains are in proper spatial conformation and configuration.
  • single-domain antibodies can be expressed from a single gene without post-translational folding or modification; single-domain antibodies can be easily transformed into multivalent and multispecific formats; single-domain antibodies Highly soluble and no tendency to aggregate; single-domain antibodies are highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without using freezing equipment, thereby saving cost, time and environment; Single-domain antibodies are easy to prepare and relatively inexpensive, even on the scale required for production; single-domain antibodies are relatively small compared to conventional 4-chain antibodies and their antigen-binding fragments (about 15kDa or 1/10 the size of conventional IgG) ), therefore, compared with the conventional 4-chain antibody and its antigen-binding fragments, it shows higher tissue permeability and can be administered at a higher dose; single-domain antibodies can display the so-called cavity binding properties (especially due to the comparison with conventional VH domains) Its extended CDR3 loop), which can reach targets
  • the term “specificity” refers to the number of different types of antigens or epitopes that a specific antigen-binding molecule or antigen-binding protein (such as the immunoglobulin single variable domain of this protocol) can bind.
  • the specificity of the antigen-binding molecule can be determined based on its affinity and/or avidity.
  • the affinity expressed by the dissociation equilibrium constant (KD) of the antigen and the antigen-binding protein is a measure of the binding strength between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the difference between the epitope and the antigen-binding molecule
  • the stronger the binding strength between (or, the affinity can also be expressed as the association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner. Dear
  • the resultant force is a measure of the binding strength between an antigen-binding molecule (for example, an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide containing it) and a related antigen.
  • an antigen-binding molecule for example, an immunoglobulin, an antibody, an immunoglobulin single variable domain or a polypeptide containing it
  • Avidity is related to both: the affinity between the antigen-binding sites on its antigen-binding molecule, and the number of related binding sites present on the antigen-binding molecule.
  • SARS-COV-2 Spike Protein Binding Molecule (SARS-COV-2-Spike Protein Binding Molecule)" used in the invention means any molecule that can specifically bind to SARS-COV-2 Spike protein.
  • the SARS-COV-2 spike protein binding molecule may include a single domain antibody or a conjugate thereof as defined in this scheme against the SARS-COV-2 spike protein.
  • SARS-COV-2 spike protein binding molecules also encompass so-called "SMIP” (“Small Modular Immune Drugs”), or immunoglobulin superfamily antibodies (IgSF) or CDR grafted molecules.
  • SIP Mall Modular Immune Drugs
  • IgSF immunoglobulin superfamily antibodies
  • the "SARS-COV-2 spike protein binding molecule" of this scheme may include at least one immunoglobulin single variable domain such as a single domain antibody that binds to the SARS-COV-2 spike protein.
  • the "SARS-COV-2 spike protein binding molecule” of this scheme may comprise two immunoglobulin single variable domains such as single domain antibodies that bind to the SARS-COV-2 spike protein.
  • SARS-COV-2 spike protein binding molecules containing more than one immunoglobulin single variable domain are also called “formatted" SARS-COV-2 spike protein binding molecules.
  • the formatted SARS-COV-2 spike protein binding molecule can also include linkers and/or parts with effector functions, such as half-life extension parts, in addition to the immunoglobulin single variable domain that binds to the SARS-COV-2 spike protein (Such as an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (such as serum albumin) and/or a conjugated polymer (such as PEG) and/or an Fc region.
  • the "SARS-COV-2 spike protein binding molecule" of this scheme also covers bispecific antibodies, which contain immunoglobulin single variable domains that bind to different antigens.
  • the SARS-COV-2 spike protein binding molecule of this scheme will be preferably 10 -8 to 10 -12 moles/liter (M), more preferably 10 -9 to 10 -11 as measured in the Biacore or KinExA assay. mol / l, and even more preferably 10-10 to 10-12, and even more preferably 10 -11 to 10 -12 or less, a dissociation constant (KD). Any KD value greater than 10 -4 M is generally regarded as indicative of non-specific binding.
  • the specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, the surface plasmon resonance (SPR) assay described herein, and/or a competitive binding assay (e.g., enzyme immunoassay). Assay (EIA) and sandwich competitive assay.
  • SPR surface plasmon resonance
  • EIA enzyme immunoassay
  • sandwich competitive assay e.g., enzyme immunoassay.
  • Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as known and agreed in the art.
  • the conservative amino acid substitutions are well known in the art.
  • a conservative amino acid substitution is preferably one amino acid in the following groups (1)-(5) is replaced by another amino acid residue in the same group: (1) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) Polar negatively charged residues and (uncharged) amides: Asp, Asn, Glu and Gln; (3) Polar positively charged residues: His, Arg and Lys; (4) Larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (5) Aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala Or Pro replacement; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr Replace; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.
  • Sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is usually measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity. For the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • the polypeptide or nucleic acid molecule is regarded as "substantially separated".
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when it has been purified at least 2 times, especially at least 10 times, more particularly at least 100 times and up to 1000 times or more.
  • suitable techniques e.g. suitable chromatographic techniques, such as polyacrylamide gel electrophoresis
  • the "substantially separated" polypeptides or nucleic acid molecules are preferably substantially homogeneous.
  • the alpaca was immunized with the Spike-RBD protein of the new coronavirus, and immunized at 1, 2, 4, and 6 weeks respectively. A total of 4 immunizations were carried out, with a dose of 200 ug each time.
  • RNA reverse transcription system is as follows:
  • Oligo dT (2.5 ⁇ M) 1 ⁇ L dNTP(10 mM each) 1 ⁇ L Total RNA 2.428 ⁇ g H2O Up to 10 ⁇ L
  • Reverse transcription conditions are: 42°C, 30 min; 50°C, 15 min; 70°C, 15 min.
  • VHH Single domain antibody
  • the sequence of the amplification primer is: Primer For-1: 5 ⁇ -GTCCTGGCTGCTCTTCTACAAGG-3 ⁇ (SEQ ID NO: 138); Primer Rev-1: 5 ⁇ -GGTACGTGCTGTTGAACTGTTCC-3 ⁇ (SEQ ID NO: 139).
  • the DNA product gel recovery kit is used to recover 750 bp band, quantified by UV spectrophotometer;
  • the sequence of the amplification primer is: Primer For-2: 5 ⁇ - CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT -3 ⁇ (SEQ ID NO: 140); Primer Rev-2: 5 ⁇ - GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ ID NO: 141).
  • PCR products were subjected to agarose gel electrophoresis, and the agarose gel electrophoresis diagram was shown in Fig. 3, which was recovered by a DNA product gel recovery kit and quantified by an ultraviolet spectrophotometer.
  • 200 ⁇ L of the target gene (VHH) of about 500 bp was obtained at a concentration of 400ng/ ⁇ L.
  • the obtained target gene and vector pHEN1 were digested with SfiI and Not1, and the digested product was subjected to agarose gel electrophoresis. The result is shown in Figure 4.
  • the digested target gene and pHEN1 were ligated with T4 DNA ligase Later, it was transformed into E. coli electrocompetent cells TG1 to construct a single domain antibody gene library against SARS-COV-2-Spike protein, named J2-Lib.
  • the calculated library capacity is 1.395 ⁇ 10 8 cfu.
  • the agarose gel electrophoresis is shown in Figure 5. , Indicating that the insertion rate of the library is 100%, and the actual library capacity is 1.395 ⁇ 10 8 cfu.
  • the colony PCR reaction conditions were: 98°C for 10 s, 50°C for 30 s, 72°C for 1 min, a total of 30 cycles.
  • J2-Lib gene library live cells with 10-100 times the library capacity were inoculated and cultured. After the log phase was cultivated, the M13K07 phage was used for rescue. After the rescue culture, the phage was collected by centrifugation, and the phage was purified by PEG-NaCl.
  • the phage display library was obtained, named J2-PDL, and the titer was 3.4 ⁇ 10 13 cfu/mL. It can be directly used for subsequent affinity screening of specific phage.
  • the obtained antibody gene sequences were respectively constructed on the pcDNA3.4 vector, and HEK-293 cells were used to express the antibody, and the antibody in the supernatant of the culture medium was purified and collected with proteinA medium.
  • the purified antibodies were incubated with Spike-RBD-coated plates for ELISA measurement. Obtain antibodies that specifically bind to Spike-RBD protein.
  • the single-domain antibody sequences are as SEQ ID NO: 82-108, which respectively carry 27 sets of CDR1-3 sequences in SEQ ID NO: 1-81, as shown in Table 2:
  • the obtained gene coding sequences of 27 different CDR1-3 single domain antibodies were recombined into the expression vector PET32b (Novagen, product number: 69016-3), and the correct recombinant plasmids identified by sequencing were transformed into expression host bacteria.
  • BL1 (DE3) (Tiangen Biochemical Technology, product number: CB105-02), spread it on an LB plate containing 100 ⁇ g/mL ampicillin, overnight at 37°C. A single colony was selected for inoculation and cultured overnight. The next day, the overnight bacterial species were transferred and expanded. When the OD value reached 0.5-1 at 37°C, it was induced by adding 0.5mM IPTG and cultured overnight at 28°C. On the second day, the cells were collected by centrifugation, and the collected cells were crushed to obtain a crude antibody extract. Then, 27 single-domain antibody proteins were purified to achieve a purity of more than 90%.
  • the Spike-RBD protein and ACE2 protein were first expressed by HEK293 cells (pCDNA4, Invitrogen, Cat V86220). Then use Thermo's Biotinlytion kit to obtain biotinylated ACE2 protein.
  • Control group 1 2.250 Control group 2 0.041 COV2-114-1 0.213 COV2-187-2 0.181 COV2-360-3 0.132 COV2-143-4 0.157 COV2-13-5 0.195 COV2-356-6 0.192 COV2-115-7 0.216 COV2-22-8 0.412 COV2-116-9 0.388 COV2-122-10 0.293 COV2-171-11 0.358 COV2-136-12 0.196 COV2-237-13 0.147 COV2-353-14 0.223 COV2-121-15 0.361 COV2-324-16 0.431 COV2-120-17 0.382 COV2-153-18 0.153 COV2-39-19 0.248 COV2-124-20 0.328 COV2-18-21 0.384 COV2-119-22 0.258 COV2-151-23 0.152 COV2-214-24 0.423 COV2-331-25 0.169 COV2-32-26 0.188 COV2-336-27 0.197
  • the purified 27 single domain antibodies were added to the culture system separately.
  • the specific operation is: adding 10 4 /well VERO cells to a 96-well plate After 24 hours, the cells were washed twice with PBS, and 27 single domain antibodies were mixed with the virus and added to a 96-well plate.
  • the initial concentration of the antibody was 100 ⁇ g/mL, and then two-fold dilutions of 10 gradients, 5 replicate wells, 37 Incubate for 2 hours at a high temperature. On the 5th day, check whether the VERO cells have lesions. If the cells are not affected by the single-domain antibody treatment, it means that the single-domain antibody has the effect of neutralizing the virus and blocking the virus from infecting VERO cells.
  • test results showed that none of the VERO cells had any disease after treatment with the above 27 single domain antibodies, while most of the VERO cells that only added the virus and did not add the single domain antibody were diseased and died, indicating that all the 27 single domain antibodies obtained were able to block the disease.
  • the process by which the virus infects cells shows that it is an effective neutralizing antibody.
  • the amino acid sequence of the human immunoglobulin (IgG1) on the protein database Uniprot the amino acid sequence of the human IgG1-Fc region (SEQ ID NO: 109) was obtained.
  • the nucleic acid fragment encoding human IgG1-Fc (nucleic acid sequence such as SEQ ID NO: 137) was obtained from the total RNA of human PBMC, and then the SARS-COV-2-Spike protein single domain antibody and Fc were obtained by overlapping PCR
  • the nucleic acid fragment encoding the fusion protein was recombined into the vector pCDNA4 (Invitrogen, Cat V86220).
  • the successfully constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was transfected into HEK293 cells for expression.
  • the recombinant expression plasmid is diluted with Freestyle293 medium and added to the PEI (Polyethylenimine) solution required for transformation, the plasmid/PEI mixture is added to the HEK293 cell suspension, and placed in a shaker at 37°C, 10% CO 2 , and 100 rpm Medium culture; supplement with 50 ⁇ g/L IGF-1. 4h later supplement with EX293 medium, 2mM glutamine and 50 ⁇ g/L IGF-1, shake culture at 120rpm. Add 3.8mM VPA after 24h. After culturing for 5 days, the expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein.
  • sequence of the obtained 27 SARS-COV-2-Spike protein single domain antibody and Fc fusion protein is as SEQ ID NO: 110-136.
  • SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to SARS-COV-2-Spike protein was identified by SPR method.
  • the specific operation is: the binding kinetics of the 27 SARS-COV-2-Spike protein single domain antibodies and the Fc fusion protein against spike-RBD were measured by the surface plasmon resonance (SRP) method using the BIAcoreX100 instrument.
  • SRP surface plasmon resonance
  • the spike-RBD protein is directly coated on the CM5 biosensor chip to obtain approximately 1000 response units (RU).
  • SARS-COV-2-Spike protein single domain antibody and Fc fusion protein have a higher binding rate to SARS-COV-2-Spike protein, a lower dissociation rate, and an equilibrium dissociation constant KD less than 1.05 E -9, indicating that the fusion protein can bind to the SARS-COV-2-Spike protein more quickly and is difficult to dissociate. It further illustrates that the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein act as a blocking antibody. Has an excellent blocking effect.
  • ACE2 protein was obtained by expressing in HEK293 cells. Using Thermo Company's Biotinlytion kit, the biotinylated protein ACE2-Biotin was obtained.
  • the plate was coated with Spike-RBD protein 0.5 ⁇ g/well at 4°C overnight, and then each well was added with 27 SARS-COV-2-Spike protein single domain antibodies and Fc fusion protein 200ng and ACE2-Biotin 5ug, no fusion protein is added to the control group 1, and ACE2-Biotin is not added to the control group 2, and react for 2h at room temperature.
  • SA-HRP purchased from Sigma
  • Control group 1 2.319
  • Control group 2 0.021 COV2-114-1 0.135 COV2-187-2 0.163 COV2-360-3 0.175 COV2-143-4 0.129 COV2-13-5 0.195 COV2-356-6 0.137 COV2-115-7 0.253 COV2-22-8 0.321 COV2-116-9 0.297 COV2-122-10 0.203 COV2-171-11 0.192 COV2-136-12 0.196 COV2-237-13 0.107 COV2-353-14 0.256 COV2-121-15 0.410 COV2-324-16 0.302 COV2-120-17 0.188 COV2-153-18 0.124 COV2-39-19 0.199 COV2-124-20 0.278 COV2-18-21 0.264 COV2-119-22 0.156 COV2-151-23 0.201 COV2-214-24 0.303 COV2-331-25 0.142 COV2-32-26 0.167 COV2-336-27 0.129
  • SARS-COV-2-Spike protein single domain antibody and Fc fusion protein SARS-COV-2-Spike protein single domain antibody and Fc fusion protein (SARS-COV) provided by this program.
  • SARS-COV-2-Spike protein single domain antibody and Fc fusion protein 100 ⁇ g/Rhesus monkey
  • the other 6 (control group) were not treated with administration.
  • the viral load will be tested once a day. After testing, the average load of new coronavirus per milliliter of blood in the 6 rhesus monkeys in the treatment group was significantly lower than that in the control group, as shown in Figure 6.
  • the detection process of the load of the new coronavirus is: take the blood of rhesus monkeys (treatment group) and rhesus monkeys (control group) that are not treated with medication, and extract the nucleic acid of the virus in the blood for detection.
  • the detection process is as follows: Use the RNA extraction kit (Qiagen) to extract the RNA of SARS-COV-2 in accordance with the instructions, dissolve the obtained RNA in a 50 ⁇ L elution buffer and use it as a template for RT-PCR amplification.
  • the virus was amplified with primers RBD-qF1 (5 ⁇ -CAATGGTTTAACAGGCACAGG-3 ⁇ , SEQ ID NO: 142) and RBD-qR1 (5 ⁇ -CTCAAGTGTCTGTGGATCACG-3 ⁇ , SEQ ID NO: 143) S region gene.
  • RBD-qF1 5 ⁇ -CAATGGTTTAACAGGCACAGG-3 ⁇ , SEQ ID NO: 142
  • RBD-qR1 5 ⁇ -CTCAAGTGTCTGTGGATCACG-3 ⁇ , SEQ ID NO: 143
  • HiScriptR II One Step qRT-PCR SYBRRGreen Kit (Vazyme Biotech Co., Ltd) kit, operate according to the kit instructions, set the PCR amplification conditions as follows: 50°C 3min, 95°C 10s, 60°C 30s, 40 cycles, the PCR amplification instrument used is an ABI quantitative PCR instrument.

Abstract

提供了一种新型冠状病毒(SARS-COV-2)刺突蛋白结合分子及其应用。该结合分子能特异性结合SARS-COV-2的刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，能有效阻断SARS-COV-2-Spike蛋白与人体细胞ACE2受体的结合，进而阻断SARS-COV-2对细胞的感染过程，抑制SARS-COV-2的传染和扩增。

Description

新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用
本方案要求于2020年06月02日提交的中国专利申请CN202010491549.0的优先权。在先申请的公开内容通过整体引用并入本方案。
技术领域
本方案涉及新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用，属于医药生物技术领域。
 背景技术
新型冠状病毒肺炎（COVID-19）在全球累计感染超过400多万人，且感染人数依然在快速增加，对COVID-19目前临床上缺乏特异有效的治疗手段。虽然我国疫情已经得到全面控制，但国外疫情却暴发出来，并且还在迅速增长。此外，越来越多的研究显示，新型冠状病毒（SARS-COV-2）感染可能存在慢性携带状态；部分出院复阳病人也提示病毒可能会长期存在人体。目前尚不清楚长期携带存在的机制、时间等关键因素，未来防止SARS-COV-2卷土重来至关重要。受COVID-19的影响，我国及世界各国由此而产生的经济损失、社会负担及其它负面影响难以计量。
目前针对COVID-19尚无特效药物，亟需快速研制有效的药物。国内外众多研发机构都在针对COVID-19的治疗策略研究上分秒必争。虽然已发掘的瑞德西韦、法匹拉韦等广谱小分子抗病毒药物对COVID-19具有一定疗效，但由于针对SARS-COV-2并无特异性，治疗效果受限，难以成为COVID-19的特效药。
 技术问题
针对现有抗病毒药物对新型冠状病毒新型冠状病毒无特异性，治疗效果受限，难以成为SARS-COV-2的特效药的问题，本方案提供一种新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用。
 技术解决方案
本方案的第一方面提供一种新型冠状病毒（SARS-COV-2）刺突蛋白结合分子，能特异性结合SARS-COV-2刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组：
1）SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；
2）SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；
3）SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；
4）SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；
5）SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；
6）SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；
7）SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；
8）SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；
9）SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；
10）SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；
11）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；
12）SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；
13）SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；
14）SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；
15）SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；
16）SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；
17）SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；
18）SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；
19）SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；
20）SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；
21）SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3；
22）SEQ ID NO:64所示的CDR1，SEQ ID NO:65所示的CDR2和SEQ ID NO:66所示的CDR3；
23）SEQ ID NO:67所示的CDR1，SEQ ID NO:68所示的CDR2和SEQ ID NO:69所示的CDR3；
24）SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2和SEQ ID NO:72所示的CDR3；
25）SEQ ID NO:73所示的CDR1，SEQ ID NO:74所示的CDR2和SEQ ID NO:75所示的CDR3；
26）SEQ ID NO:76所示的CDR1，SEQ ID NO:77所示的CDR2和SEQ ID NO:78所示的CDR3；
27）SEQ ID NO:79所示的CDR1，SEQ ID NO:80所示的CDR2和SEQ ID NO:81所示的CDR3。
作为本方案的一个实施例，所述免疫球蛋白单一可变域为单域抗体。
作为本方案的一个实施例，所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少80％的序列相同性的氨基酸序列。
作为本方案的一个实施例，所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少90％的序列相同性的氨基酸序列。
作为本方案的一个实施例，所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少99％的序列相同性的氨基酸序列。
作为本方案的一个实施例，所述单域抗体的氨基酸序列与SEQ ID NO: 82-108中任一项比包含一或多个氨基酸取代，优选保守氨基酸取代。
作为本方案的一个实施例，所述单域抗体包含 SEQ ID NO:82-108中任意一种氨基酸序列。
作为本方案的一个实施例，所述的SARS-COV-2刺突蛋白结合分子还包含免疫球蛋白Fc区。
在本方案的SARS-COV-2刺突蛋白结合分子中包含免疫球蛋白Fc区可以使所述结合分子形成二聚体，同时进一步延长所述分子的体内半衰期。可用于本方案的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。
作为本方案的一个实施例，所述免疫球蛋白Fc区是人免疫球蛋白Fc区。
作为本方案的一个实施例，所述免疫球蛋白Fc区是人IgG1的Fc区。
作为本方案的一个实施例，所述免疫球蛋白Fc区的氨基酸序列为SEQ ID NO:109。
作为本方案的一个实施例，包含SEQ ID NO:110-136中的至少一种氨基酸序列。
与上述Fc区融合后的结合分子，稳定性和生物活性进一步提高，并进一步降低了其与SARS-COV-2刺突蛋白结合的KD值。
作为本方案的一个实施例，所述SARS-COV-2刺突蛋白结合分子具有下述特征中的至少一种：
a、与SARS-COV-2刺突蛋白结合的KD值小于1×10-8M；
b、阻断SARS-COV-2与人体细胞受体ACE2的结合；
c、抑制SARS-COV-2的传染和扩增。
本方案的第二方面提供了编码所述SARS-COV-2刺突蛋白结合分子的核酸分子，所述核酸分子为RNA、DNA或cDNA，其可以通过人工合成的方式获得，也可从适合的天然来源加以分离获得。
本方案的第三方面提供了包含所述核酸分子及其表达调控原件的表达载体。该表达载体通常包含至少一种本方案提供的核酸分子，其可操作地连接至一个或多个适合的表达调控元件（启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因等）。针对在特定宿主细胞中的表达对所述元件及其序列进行选择为本领域技术人员的常识。
本方案的第四方面提供了包含所述核酸分子并进行表达的宿主细胞。所述的宿主细胞为用于表达异源蛋白的细胞，包括细菌细胞、真菌细胞或哺乳动物细胞。
本方案的第五方面提供了获得所述的SARS-COV-2刺突蛋白结合分子的方法，包括：
a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养上述宿主细胞；
b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。
将特定的核酸分子重组到表达载体上并通过转化或转染方法进入宿主细胞中表达、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。同时蛋白结合分子的分离及纯化技术为本领域技术人员所公知。
本方案提供的SARS-COV-2刺突蛋白结合分子也可以通过本领域已知的其它产生蛋白质的方法获得，例如化学合成。
本方案的第六方面提供了一种免疫缀合物，包含与治疗性部分缀合的上述任一项所述的SARS-COV-2刺突蛋白结合分子。
本方案的第七方面提供了一种药物组合物，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子和/或所述的免疫缀合物，以及药学上可接受的载体。
本方案所述的“药学上可接受的载体”包括生理学相容的任何溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即结合分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用，为本领域技术人员公知。
本方案所述的药物组合物根据需要还可以包括其它佐剂和辅料等。
本方案的第八方面提供了所述药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。
本方案的第九方面提供了一种用于检测SARS-COV-2的试剂盒，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子。
本方案的第十方面提供了所述用于检测SARS-COV-2的试剂盒的使用方法，在上述任一项所述的SARS-COV-2刺突蛋白结合分子与SARS-COV-2刺突蛋白之间能够形成复合物的条件下，使检测样品和对照样品接触上述任一项所述的SARS-COV-2刺突蛋白结合分子，检测复合物的形成；通过所述检测样品与对照样品之间复合物形成的差异判断样品中SARS-COV-2的存在。
 有益效果
本方案提供的SARS-COV-2刺突蛋白（SARS-COV-2-Spike蛋白）结合分子能够特异性的结合SARS-COV-2-Spike蛋白，并有效阻断SARS-COV-2-Spike蛋白与人体细胞ACE2受体的结合，进而阻断SARS-COV-2对细胞的感染过程，抑制SARS-COV-2的传染和扩增。且本方案提供的SARS-COV-2-Spike蛋白结合分子还具有与SARS-COV-2-Spike蛋白结合的特异性好，生物活性和稳定性高以及无毒副作用的特点。
 附图说明
图1是本方案实施例1中提取的总RNA的琼脂糖凝胶电泳图，其中，M：DNA marker 2000，泳道1：总RNA；
图2是本方案实施例1中巢式PCR扩增单域抗体基因的Step1-PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000，泳道1：扩增产物；
图3是本方案实施例1中巢式PCR扩增单域抗体基因的Step2-PCR扩增产物的琼脂糖凝胶电泳图，其中，DNA marker 2000，泳道1和2：扩增产物；
图4是本方案实施例1中目的单域抗体基因和载体pHEN1的SfiI和Not1双酶切产物的琼脂糖凝胶电泳图，其中，DNA marker 2000，泳道1：pHEN1；泳道2：sfil/notI酶切后的pHEN1；泳道3：sfil/notI酶切后的单域抗体基因；
图5是本方案实施例1中测算文库插入率的菌落PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000；泳道1-48：挑取的48个菌落；
图6是本方案实施例2中随天数变化治疗组与对照组恒河猴的病毒载量变化图。
本方案的实施方式
定义
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。
除非另有说明，否则可互换使用的术语“抗体”或“免疫球蛋白”在本文中无论是指重链抗体还是指常规4链抗体，均用作一般术语以包括全长抗体、其单个的链以及其所有部分、结构域或片段(包括但不限于抗原结合结构域或片段)。此外，本文所用的术语“序列”(例如在“免疫球蛋白序列”、 “抗体序列”、 “单一可变结构域序列”、 “单域抗体序列”或“蛋白序列”等的术语中)一般应理解为既包括相关氨基酸序列，又包括编码所述序列的核酸序列或核苷酸序列，除非本文需要更限定的解释。
如本文所用的术语“免疫球蛋白可变结构域”是指基本上由本领域及下文中分别称为“框架区1”或“FR1”、 “框架区2”或“FR2”、 “框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域及下文中分别称为“互补决定区1”或“CDR1”、 “互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。
传统IgG抗体分子一般由轻链和重链组成，轻链包含1个可变区(VL)和1个恒定区(CL)，重链包含1个可变区(VH)和3个恒定区(CH1，CH2，CH3)。单域抗体(Single domain antibody，sdAb)，是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。单域抗体特异性结合表位而无需其他抗原结合结构域。单域抗体为由单一免疫球蛋白结构域形成的小型稳定及高效的抗原识别单元。
如本领域中对于单域抗体中的各CDR中的氨基酸残基的总数可能不同。
单域抗体中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本方案所述的目的。
单域抗体及含有其的多肽的其他结构特性及功能性质可总结如下：
单域抗体(其已经天然“设计”以在不存在轻链可变结构域且不与轻链可变结构域相互作用的情况下与抗原功能性结合)可用作单一且相对较小的功能性抗原结合结构单元、结构域或多肽。此区分单域抗体与常规4链抗体的VH及VL结构域，这些VH及VL结构域自身通常不适于作为单一抗原结合蛋白或免疫球蛋白单一可变结构域进行实际应用，但需要以某种形式或另一形式组合以提供功能性抗原结合单元(如以诸如Fab片段等常规抗体片段的形式；或以由与VL结构域共价连接的VH结构域组成的scFv的形式)。
由于上述这些独特性质，使用单域抗体或作为较大多肽的一部分提供许多优于使用常规VH及VL结构域、scFv或常规抗体片段(例如Fab-或F(ab’)2-片段)的显著优势，例如，单域抗体仅需要单一结构域以高亲和力及高选择性结合抗原，从而使得既不需要存在两个单独结构域，也不需要确保该两个结构域以适当空间构象及构型存在(例如scFv一般需要使用经特别设计的接头)；单域抗体可自单一基因表达且不需要翻译后折叠或修饰；单域抗体可容易地改造成多价及多特异性格式；单域抗体高度可溶且无聚集趋势；单域抗体对热、pH、蛋白酶及其他变性剂或条件高度稳定，且因此可在制备、储存或运输中不使用冷冻设备，从而达成节约成本、时间及环境；单域抗体易于制备且相对廉价，甚至在生产所需的规模上亦如此；单域抗体与常规4链抗体及其抗原结合片段相比相对较小(大约15kDa或大小为常规IgG的1/10)，因此相比于常规4链抗体及其抗原结合片段，显示较高的组织渗透性且可以较高剂量给药；单域抗体可显示所谓腔结合性质(尤其由于与常规VH结构域相比其延长的CDR3环)，从而可到达常规4链抗体及其抗原结合片段不可到达的靶及表位。
获得结合特定抗原或表位的单域抗体的方法，先前已公开于以下文献中：R.van derLinden et al.,Journal of Immunological Methods,240(2000)185–195；Li et al.,JBiol Chem .,287(2012)13713–13721；Deffar et al .,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009和WO94/04678。
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本方案的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲
合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。
发明所用术语“SARS-COV-2刺突蛋白结合分子（SARS-COV-2-Spike蛋白结合分子）”意指任何能够特异性结合SARS-COV-2刺突蛋白的分子。SARS-COV-2刺突蛋白结合分子可以包括针对SARS-COV-2刺突蛋白的如本方案定义的单域抗体或其缀合物。SARS-COV-2刺突蛋白结合分子还涵盖所谓的“SMIP”( “小模块免疫药物”)，或者免疫球蛋白超家族抗体(IgSF)或CDR移植分子。
本方案的“SARS-COV-2刺突蛋白结合分子”可以包含至少一个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。在一些实施方案中，本方案的“SARS-COV-2刺突蛋白结合分子”可以包含两个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。含有一个以上的免疫球蛋白单一可变结构域的SARS-COV-2刺突蛋白结合分子亦称为“格式化的”SARS-COV-2刺突蛋白结合分子。格式化的SARS-COV-2刺突蛋白结合分子除结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。本方案的“SARS-COV-2刺突蛋白结合分子”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。
通常，本方案的SARS-COV-2刺突蛋白结合分子将以如于Biacore或KinExA测定中测量的优选10 -8至10 -12摩尔/升(M)、更优选10 -9至10 -11摩尔/升、甚至更优选10 -10至10 -12 、甚至更优选10 -11至10 -12或更低的解离常数(KD)。任何大于10 -4 M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、和/或竞争性结合测定(例如酶免疫测定(EIA)及夹心式竞争性测定。
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(1)-(5)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(1)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(2)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(3)极性带正电残基：His、Arg及Lys；(4)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(5)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,NewYork,1991。
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。
实施例1
筛选针对SARS-COV-2-Spike蛋白的单域抗体
1.1文库的构建
1.1.1免疫
用新型冠状病毒的Spike-RBD蛋白免疫羊驼，并分别在第1、2、4、6周免疫，共免疫4次，每次免疫剂量为200ug。
1.1.2提取总RNA
取第6周免疫后的羊驼外周血50ml，分离淋巴细胞，用Trizol提取淋巴细胞的总RNA，采用紫外分光光度计检测提取的RNA 的结果为：OD260/280＝1.99，OD260/230＝1.43，说明提取的RNA没有明显降解，纯度较好；总 RNA浓度为809.3 ng/μL。用提取的总 RNA进行琼脂糖凝胶电泳，结果如图1所示，可以看到28S和18S两条条带。
1.1.3 RNA反转录
RNA反转录体系如下：
[根据细则91更正 12.04.2021]　
Step 1：（表1）
Oligo dT（2.5 μM） 1 μL
dNTP(10 mM each) 1 μL
总 RNA 2.428 μg
H2O Up to 10 μL
混匀后，65℃ 保温5 min，迅速冰浴；
[根据细则91更正 12.04.2021]　
Step 2 ：（表2）
Step1 反应液 10 μL
5×PrimeScript  Buffer 4 μL
RNase inhibitor（40 U/μL） 0.5 μL
PrimeScript  RTase（200 U/μL） 0.5 μL
RNase free H2O Up to 20 μL
混匀后，进行反转录得到cDNA，反转录条件为：42℃，30 min；50℃，15 min ；70℃，15 min。
1.1.4 单域抗体（VHH）基因扩增
采用巢式PCR扩增VHH基因，方法如下：
[根据细则91更正 12.04.2021]　
Step1 ：（表3）
10×Ex Taq Buffer（Mg2+ plus） 5 μL
dNTP mixture(each 2.5 mM) 4 μL
Primer For-1（10 mM each）             1.5 μL
Primer Rev-1（10 mM each） 1.5 μL
cDNA 1 μL
Ex Taq酶 0.25 μL
H2O Up to 50 μL
混匀后，进行PCR反应，反应条件：98℃ 10 s，50℃ 30 s，72℃ 1min，共20个循环。扩增引物的序列为：Primer For-1: 5´-GTCCTGGCTGCTCTTCTACAAGG-3´（SEQ ID NO:138）；Primer Rev-1: 5´-GGTACGTGCTGTTGAACTGTTCC-3´（SEQ ID NO:139）。
   PCR产物经DNA纯化试剂盒纯化浓缩后，进行琼脂糖凝胶电泳，得到的琼脂糖凝胶电泳图如图2所示，采用DNA产物凝胶回收试剂盒回收750 bp条带，紫外分光光度计定量；
[根据细则91更正 12.04.2021]　
Step 2：（表4）
10×Ex Taq Buffer（Mg2+ plus） 5 μL
dNTP mixture(2.5 mM each) 4 μL
Primer For-2（10 mM each） 2 μL
Primer Rev-2（10 mM each） 2 μL
Step 1 DNA模板 1 μL
Ex Taq 0.25 μL
H2O Up to 50 μL
混匀后，进行PCR反应，反应条件：98℃ 10 s，55℃ 30 s，72℃ 30s，共20个循环。扩增引物的序列为：Primer For-2: 5´- CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT -3´（SEQ ID NO:140）；Primer Rev-2: 5´- GATGTGCAGCTGCAGGAGTCTGGRGGAGG -3´（SEQ ID NO:141）。
得到的PCR产物进行琼脂糖凝胶电泳，琼脂糖凝胶电泳图如图3所示，采用DNA产物凝胶回收试剂盒回收，紫外分光光度计定量。最终得到约500 bp的目的基因（VHH）200μL，浓度为400ng/μL。
1.1.5文库转化
将得到的目的基因和载体pHEN1采用SfiI和Not1进行双酶切，将酶切产物进行琼脂糖凝胶电泳，结果如图4所示，将酶切后的目的基因和pHEN1采用T4 DNA连接酶连接后，转化至大肠杆菌电转感受态细胞TG1中，构建针对SARS-COV-2-Spike蛋白的单域抗体基因文库，命名为J2-Lib。共转化15次，混合后均匀涂布于6块Ф150 mm的培养皿（含氨苄青霉素的LB固体培养基）中。
分别取0.1μL，0.01 μL，0.001 μL和0.0001 μL混合后的转化液均匀涂布于Ф90mm的培养皿（含氨苄青霉素的LB固体培养基），用于文库库容量的计算（以菌落数为30-300的平板为准计数），如表1所示，计算库容量为1.395×10 8 cfu。
[根据细则91更正 12.04.2021]　
表5
菌液体积（μL） 0.1 0.01 0.001 0.0001
菌落数 满板 93 12 1
在上述用于计算库容量的培养皿中随机挑选48个菌落，进行菌落PCR，并将PCR产物进行琼脂糖凝胶电泳，测算文库的目的基因插入率，琼脂糖凝胶电泳如图5所示，说明文库插入率为100%，文库实际库容量为1.395×10 8 cfu。
[根据细则91更正 12.04.2021]　
菌落PCR体系如下：（表6）
10×Ex Taq Buffer（Mg2+ plus） 5 μL
dNTP mixture(each 2.5 mM) 4 μL
Primer For-1（10 mM each）             1.5 μL
Primer Rev-1（10 mM each） 1.5 μL
菌落混悬液 1 μL
Ex Taq酶 0.25 μL
H2O Up to 50 μL
菌落PCR反应条件为：98℃ 10 s，50℃ 30 s，72℃ 1min，共30个循环。
1.1.6文库救援
从上述J2-Lib基因文库中取10-100倍库容量的活细胞进行接种培养，培养至对数期后采用M13K07噬菌体进行救援，救援培养后，离心收集噬菌体，采用PEG-NaCl纯化噬菌体，即得噬菌体展示文库，命名为J2-PDL，滴度为3.4×10 13 cfu/mL。可直接用于后续特异性噬菌体的亲和筛选。
1.2针对SARS-COV-2-Spike蛋白单域抗体的筛选
用Spike-RBD蛋白（刺突蛋白受体结合区蛋白）3μg/孔包被平板，4℃放置过夜；用1wt%脱脂奶粉室温封闭2h，加入100μl噬菌体(8×10 11tfu，来自1.1.6所构建的噬菌体展示文库J2-PDL)，在室温下作用1h。之后用PBST(PBS中含有0.05vt%吐温20)洗脱5遍，以洗掉不结合的噬菌体；用三乙基胺(100mM)将与Spike-RBD蛋白特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3-4轮。由此，阳性的克隆被富集，达到了利用噬菌体展示文库筛取抗体库中Spike-RBD蛋白特异抗体的目的。并将获得的阳性的噬菌体进行测序，获得抗体基因序列。
将获得的抗体基因序列分别构建在pcDNA3.4载体上，用HEK-293细胞表达抗体，用proteinA介质纯化收集培养基上清中的抗体。纯化后的抗体与包被Spike-RBD的板孵育进行ELISA测定。获得可特异性结合Spike-RBD蛋白的抗体。
根据序列比对软件Vector NTI分析获得的抗体序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。共获得27个不同的单域抗体株，单域抗体序列如SEQ ID NO:82-108，分别携带SEQ ID NO:1-81中的27组CDR1-3序列，具体如表2所示：
[根据细则91更正 12.04.2021]　
表7
单域抗体 CDR1 CDR2 CDR3
COV2-114-1 SEQ ID NO:1 SEQ ID NO:2 SEQ ID NO:3
COV2-187-2 SEQ ID NO:4 SEQ ID NO:5 SEQ ID NO:6
COV2-360-3 SEQ ID NO:7 SEQ ID NO:8 SEQ ID NO:9
COV2-143-4 SEQ ID NO:10 SEQ ID NO:11 SEQ ID NO:12
COV2-13-5 SEQ ID NO:13 SEQ ID NO:14 SEQ ID NO:15
COV2-356-6 SEQ ID NO:16 SEQ ID NO:17 SEQ ID NO:18
COV2-115-7 SEQ ID NO:19 SEQ ID NO:20 SEQ ID NO:21
COV2-22-8 SEQ ID NO:22 SEQ ID NO:23 SEQ ID NO:24
COV2-116-9 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27
COV2-122-10 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30
COV2-171-11 SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33
COV2-136-12 SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36
COV2-237-13 SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39
COV2-353-14 SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42
COV2-121-15 SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45
COV2-324-16 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48
COV2-120-17 SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:51
COV2-153-18 SEQ ID NO:52 SEQ ID NO:53 SEQ ID NO:54
COV2-39-19 SEQ ID NO:55 SEQ ID NO:56 SEQ ID NO:57
COV2-124-20 SEQ ID NO:58 SEQ ID NO:59 SEQ ID NO:60
COV2-18-21 SEQ ID NO:61 SEQ ID NO:62 SEQ ID NO:63
COV2-119-22 SEQ ID NO:64 SEQ ID NO:65 SEQ ID NO:66
COV2-151-23 SEQ ID NO:67 SEQ ID NO:68 SEQ ID NO:69
COV2-214-24 SEQ ID NO:70 SEQ ID NO:71 SEQ ID NO:72
COV2-331-25 SEQ ID NO:73 SEQ ID NO:74 SEQ ID NO:75
COV2-32-26 SEQ ID NO:76 SEQ ID NO:77 SEQ ID NO:78
COV2-336-27 SEQ ID NO:79 SEQ ID NO:80 SEQ ID NO:81
。
1.3针对SARS-COV-2-Spike蛋白的单域抗体的评价鉴定
1.3.1单域抗体在宿主菌大肠杆菌中表达、纯化
将获得的27个不同CDR1-3的单域抗体的基因编码序列分别重组至表达载体PET32b(Novagen，产品号：69016-3)中，并将测序鉴定正确的重组质粒分别转化到表达型宿主菌BL1(DE3)(天根生化科技，产品号：CB105-02)中，将其涂布在含有100μg/mL的氨苄青霉素的LB平板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.5-1时，加入0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收集菌体，并将收集的菌体破碎获得抗体粗提液。然后纯化27株单域抗体蛋白，使其纯度达到90%以上。
1.3.2竞争ELISA考察SARS-COV-2-Spike蛋白单域抗体对Spike-RBD蛋白与受体ACE2结合的阻断效果
先通过HEK293细胞(pCDNA4，Invitrogen，Cat V86220)表达获得 Spike-RBD蛋白与ACE2蛋白。再利用Thermo公司的Biotinlytion试剂盒，得到生物素化的ACE2蛋白。
用Spike-RBD蛋白0.5μg/孔，4℃过夜包被平板，之后每孔加入100ng的1.3.1纯化所得的单域抗体以及5μg生物素化的ACE2蛋白，并设置对照组，对照组1的孔中不加入单域抗体，对照组2的孔中不加入生物素化的ACE2蛋白，室温下反应2h。之后加入SA-HRP(购自Sigma公司)，室温反应1小时后加入显色液，450nm波长读取吸收值。当样品OD值比对照OD值<0.8时，则认为单域抗体有阻断效果。
结果如表3所示，出现27个不同的单域抗体株表现出对Spike蛋白/ACE2蛋白相互作用的阻断效应。
[根据细则91更正 12.04.2021]　
表8
样品 OD
对照组1 2.250
对照组2 0.041
COV2-114-1 0.213
COV2-187-2 0.181
COV2-360-3 0.132
COV2-143-4 0.157
COV2-13-5 0.195
COV2-356-6 0.192
COV2-115-7 0.216
COV2-22-8 0.412
COV2-116-9 0.388
COV2-122-10 0.293
COV2-171-11 0.358
COV2-136-12 0.196
COV2-237-13 0.147
COV2-353-14 0.223
COV2-121-15 0.361
COV2-324-16 0.431
COV2-120-17 0.382
COV2-153-18 0.153
COV2-39-19 0.248
COV2-124-20 0.328
COV2-18-21 0.384
COV2-119-22 0.258
COV2-151-23 0.152
COV2-214-24 0.423
COV2-331-25 0.169
COV2-32-26 0.188
COV2-336-27 0.197
在生物安全级别为P3的实验室，通过用病毒感染VERO细胞模型，将纯化得到的27株单域抗体分别加入到培养体系中，具体操作为：将10 4/孔VERO细胞加到96孔板，24小时后，用PBS洗细胞2次，将27株单域抗体分别与病毒混合加入96孔板，抗体初始浓度为100μg/mL，再分别2倍稀释10个梯度，5个复孔，37度孵育2小时，第5天检测VERO细胞是否发生病变。如果单域抗体处理后细胞不发生病变说明单域抗体具有中和病毒阻断病毒感染VERO细胞的效果。
检测结果显示，经上述27株单域抗体处理后VERO细胞均未发生病变，而只加入病毒未加入单域抗体的VERO细胞大部分病变和死亡，说明所获得的27株单域抗体全部能够阻断病毒感染细胞的过程，显示其为有效的中和抗体。
实施例2
1.1制备SARS-COV-2-Spike蛋白单域抗体的Fc融合蛋白
根据蛋白数据库Uniprot上人免疫球蛋白 (IgG1)的恒定区氨基酸序列，得到人IgG1-Fc区氨基酸序列(SEQ ID NO:109)。通过逆转录PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段（核酸序列如SEQ ID NO:137），再通过overlapping PCR得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的编码核酸片段，并重组至载体pCDNA4(Invitrogen，Cat V86220)。
将构建成功的含有SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的核酸片段的pCDNA4质粒转染HEK293细胞进行表达。具体是将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10%的CO 2，100rpm的摇床中培养；补加50μg/L IGF-1。4h后补加EX293培养基、2mM谷氨酰胺和50μg/L IGF-1，120rpm摇培。24h后加3.8mM VPA。培养5天后，收集表达培养上清液，通过Protein A亲和层析法，纯化得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白。
获得的27个SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的序列如SEQ ID NO:110-136。
1.2鉴定SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的功能
通过SPR法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合能力。具体操作是：将获得的27株SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白针对spike-RBD的结合动力学通过表面等离振子共振(SRP) 方法，使用BIAcoreX100 仪器测量，将spike-RBD蛋白直接包被于CM5 生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白用HBS-EP+1× 缓冲液(GE，cat#BR-1006-69) 三倍连续稀释(1.62nm 至1000nm)，在25℃进样120s，解离时间为30min，加入10mM 甘氨酸-HCl(pH2.0) 再生120s。使用简单一对一Languir 结合模型(BIAcore Evaluation Software version 3.2) 计算出融合蛋白与SARS-COV-2-Spike蛋白的结合速率(kon) 、解离速率(koff)和平衡解离常数(kD) （以比率koff/kon 计算）。计算结果如表4所示。
[根据细则91更正 12.04.2021]　
表9
抗体编号 结合速率(kon) 解离速率(koff) 平衡解离常数(kD;koff/kon)
COV2-114-1-Fc 1.74E+06 1.52E-05 0.87E-12
COV2-187-2-Fc 1.52E+05 3.26E-05 2.14E-11
COV2-360-3-Fc 2.12E+06 3.45E-04 1.63E-11
COV2-143-4-Fc 3.12E+06 6.25E-05 2.00E-12
COV2-13-5-Fc 1.45E+06 1.23E-05 0.85E-12
COV2-356-6-Fc 5.23E+05 5.68E-04 1.09E-10
COV2-115-7-Fc 1.25E+05 1.68E-05 1.34E-11
COV2-22-8-Fc 1.89E+05 5.96E-04 3.15E-10
COV2-116-9-Fc 2.56E+06 6.84E-05 2.67E-12
COV2-122-10-Fc 3.51E+06 9.23E-05 2.63E-12
COV2-171-11-Fc 4.23E+06 4.26E-05 1.01E-12
COV2-136-12-Fc 5.36E+04 5.63E-04 1.05E-9
COV2-237-13-Fc 5.47E+06 5.78E-05 1.06E-12
COV2-353-14-Fc 9.23E+06 6.34E-05 0.69E-12
COV2-121-15-Fc 1.29E+06 2.68E-05 2.08E-12
COV2-324-16-Fc 4.85E+06 3.71E-05 0.76E-12
COV2-120-17-Fc 6.52E+07 3.64E-04 0.56E-12
COV2-153-18-Fc 2.56E+06 3.15E-05 1.23E-12
COV2-39-19-Fc 1.89E+05 2.46E-05 1.30E-11
COV2-124-20-Fc 3.62E+06 3.51E-05 0.97E-12
COV2-18-21-Fc 5.23E+06 3.99E-05 0.76E-12
COV2-119-22-Fc 1.86E+06 4.22E-05 2.27E-12
COV2-151-23-Fc 2.58E+06 3.22E-05 1.25E-12
COV2-214-24-Fc 6.32E+06 1.29E-05 0.20E-12
COV2-331-25-Fc 4.23E+06 3.56E-05 0.84E-12
COV2-32-26-Fc 4.26E+06 3.14E-05 0.74E-12
COV2-336-27-Fc 1.56E+06 6.31E-05 4.04E-12
由表4可知，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合速率较高的，解离速率较低，平衡解离常数KD小于1.05 E-9，说明融合蛋白能更快速的结合SARS-COV-2-Spike蛋白并很难解离下来，进一步说明SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白作为一个阻断型抗体，具有极佳的阻断效果。
1.3通过竞争ELISA法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白/ACE2的相互作用的阻断能力
利用HEK293细胞表达获得ACE2蛋白。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白ACE2-Biotin。
用Spike-RBD蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入获得的27株SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白200ng和 ACE2-Biotin 5ug，对照组1中不加入融合蛋白，对照组2中不加入ACE2-Biotin，室温下反应2h。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时，洗涤之后加入显色液，450nm波长读取吸收值。结果如表5所示：
[根据细则91更正 12.04.2021]　
表10
样品 OD
对照组1 2.319
对照组2 0.021
COV2-114-1 0.135
COV2-187-2 0.163
COV2-360-3 0.175
COV2-143-4 0.129
COV2-13-5 0.195
COV2-356-6 0.137
COV2-115-7 0.253
COV2-22-8 0.321
COV2-116-9 0.297
COV2-122-10 0.203
COV2-171-11 0.192
COV2-136-12 0.196
COV2-237-13 0.107
COV2-353-14 0.256
COV2-121-15 0.410
COV2-324-16 0.302
COV2-120-17 0.188
COV2-153-18 0.124
COV2-39-19 0.199
COV2-124-20 0.278
COV2-18-21 0.264
COV2-119-22 0.156
COV2-151-23 0.201
COV2-214-24 0.303
COV2-331-25 0.142
COV2-32-26 0.167
COV2-336-27 0.129
结果显示，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白能有效阻断Spike蛋白与ACE2之间的相互作用。
1.4分析SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白结合的特异性
利用人HEK293细胞通过瞬时转染，获得带有目前已知的7种冠状病毒（SARS-COV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV）Spike蛋白全长基因的质粒(pCDNA4 ,Invitrogen，Cat V86220)，并于膜上瞬时表达Spike蛋白。该质粒使得Spike蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上Spike蛋白的表达水平。将构建好的细胞重悬于0.5%的PBS-BSA Buffer中，加入SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白，同时设置阴性对照，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl的0.5％PBS-BSABuffer中，流式细胞仪进行检测。结果显示，27株SARS-COV-2-Spike蛋白单域抗体-Fc融合蛋白只特异性结合SARS-COV-2-Spike蛋白，而不与其他冠状病毒的Spike蛋白结合。
1.5 SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白阻断SARS-COV-2感染细胞
将提前给药（SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白100μg/恒河猴）的15只健康恒河猴和15只未提前给药的健康恒河猴与感染SARS-COV-2的2只恒河猴在同一环境中喂养10天。10天后观察发现提前给药的15只健康恒河猴均未出现异常，病毒核酸检测均为阴性；而15只未提前给药的健康恒河猴有12只出现感染症状，经病毒核酸检测均呈阳性。
上述试验结果说明本方案提供的SARS-COV-2-Spike蛋白单域抗体能够完全阻断新冠病毒感染，具有优良的预防作用。
将上述感染SARS-COV-2病毒并出现症状的12只恒河猴中的6只（治疗组）利用本方案提供的SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白（SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白100μg/恒河猴）进行给药治疗，另外6只（对照组）不进行给药治疗。在治疗后的6天内，每天进行一次病毒载量的检测。经检测，治疗组的6只恒河猴每毫升血液中新冠病毒的平均载量相比对照组显著降低，如图6所示。其中新冠病毒的载量的检测过程为：分别采取给药治疗的恒河猴（治疗组）和未进行给药治疗的恒河猴（对照组）的血液，提取血液中的病毒的核酸进行检测，检测过程为：采用RNA提取试剂盒（Qiagen）按照说明书操作提取SARS-COV-2的RNA，将获得的RNA溶解在50μL洗脱buffer并作为模板进行RT-PCR扩增。以RT-PCR产物为模板，用引物RBD-qF1（5´-CAATGGTTTAACAGGCACAGG-3´，SEQ ID NO:142）和RBD-qR1（5´-CTCAAGTGTCTGTGGATCACG-3´，SEQ ID NO:143）扩增病毒S区基因。采用HiScriptR II One Step qRT-PCR SYBRRGreen Kit (Vazyme Biotech Co.,Ltd)试剂盒，根据试剂盒说明书进行操作，设置PCR扩增条件为；50℃ 3min，95℃ 10s，60℃ 30s，40个循环，所用PCR扩增仪为ABI定量PCR仪。
上述体内实验结果表明，本方案SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白在对感染SARS-COV-2的恒河猴体内表现出显著的抑制SARS-COV-2感染细胞并扩增的效果。
1.6 SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白稳定性研究
使用500mM碳酸氢铵作为碱性破坏试剂，37℃处理48h。选择1%双氧水作为氧化剂，室温处理10h。
使用竞争ELISA法检测处理前后的上述27种SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白的生物学活性变化。48h碱处理后的竞争ELISA活性相对碱处理0小时平均为107%；10h氧化后的竞争ELISA活性相对氧化0h平均为111%。
说明本方案提供的27种SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白具有较高的稳定性。
以上所述仅为本方案的较佳实施例而已，并不用以限制本方案，凡在本方案的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本方案的保护范围之内。

Claims (22)

  1. 一种SARS-COV-2刺突蛋白结合分子，其特征在于：能特异性结合SARS-COV-2刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组：
    1）SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；
    2）SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；
    3）SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；
    4）SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；
    5）SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；
    6）SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；
    7）SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；
    8）SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；
    9）SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；
    10）SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；
    11）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；
    12）SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；
    13）SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；
    14）SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；
    15）SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；
    16）SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；
    17）SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；
    18）SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；
    19）SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；
    20）SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；
    21）SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3；
    22）SEQ ID NO:64所示的CDR1，SEQ ID NO:65所示的CDR2和SEQ ID NO:66所示的CDR3；
    23）SEQ ID NO:67所示的CDR1，SEQ ID NO:68所示的CDR2和SEQ ID NO:69所示的CDR3；
    24）SEQ ID NO:70所示的CDR1，SEQ ID NO:71所示的CDR2和SEQ ID NO:72所示的CDR3；
    25）SEQ ID NO:73所示的CDR1，SEQ ID NO:74所示的CDR2和SEQ ID NO:75所示的CDR3；
    26）SEQ ID NO:76所示的CDR1，SEQ ID NO:77所示的CDR2和SEQ ID NO:78所示的CDR3；
    27）SEQ ID NO:79所示的CDR1，SEQ ID NO:80所示的CDR2和SEQ ID NO:81所示的CDR3。
  2. 如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白单一可变域为单域抗体。
  3. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少80％的序列相同性的氨基酸序列。
  4. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少90％的序列相同性的氨基酸序列。
  5. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO: 82-108中任一序列具有至少99％的序列相同性的氨基酸序列。
  6. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含 SEQ ID NO:82-108 中的任意一种氨基酸序列。
  7. 如权利要求1-6任一项所述的SARS-COV-2刺突蛋白结合分子，其特征在于：还包含免疫球蛋白Fc区。
  8. 如权利要求7所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人免疫球蛋白Fc区。
  9. 如权利要求8所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人IgG1的Fc区。
  10. 如权利要求9所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区的氨基酸序列为SEQ ID NO:109。
  11. 如权利要求10所述的SARS-COV-2刺突蛋白结合分子，其特征在于：包含SEQ ID NO:110-136中的至少一种氨基酸序列。
  12. 如权利要求1-6、8-11中任一项所述的SARS-COV-2刺突蛋白结合分子，其特征在于：具有下述特征中的至少一种：
    a、与SARS-COV-2刺突蛋白结合的KD值小于1×10 -8M；
    b、阻断SARS-COV-2与人体细胞受体ACE2的结合；
    c、抑制SARS-COV-2的传染和扩增。
  13. 如权利要求7所述的SARS-COV-2刺突蛋白结合分子，其特征在于：具有下述特征中的至少一种：
    a、与SARS-COV-2刺突蛋白结合的KD值小于1×10 -8M；
    b、阻断SARS-COV-2刺突蛋白与人体细胞受体ACE2的结合；
    c、抑制SARS-COV-2传染和扩增。
  14. 编码权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子的核酸分子。
  15. 包含权利要求14所述的核酸分子及其表达调控原件的表达载体。
  16. 包含权利要求14所述的核酸分子并进行表达的宿主细胞。
  17. 获得权利要求1-13中任一项所述的SARS-COV-2刺突蛋白结合分子的方法，其特征在于，包括：
    a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养权利要求16所述的宿主细胞；
    b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。
  18. 一种免疫缀合物，其特征在于，包含与治疗性部分缀合的权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子。
  19. 一种药物组合物，其特征在于，包含权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子和/或权利要求18所述的免疫缀合物，以及药学上可接受的载体。
  20. 权利要求19所述的药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。
  21. 一种用于检测SARS-COV-2的试剂盒，其特征在于，包含权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子。
  22. 权利要求21所述的试剂盒的使用方法，其特征在于，在权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子与SARS-COV-2刺突蛋白之间能够形成复合物的条件下，使检测样品和对照样品接触权利要求1-13任一项所述的SARS-COV-2刺突蛋白结合分子，检测复合物的形成；通过所述检测样品与对照样品之间复合物形成的差异判断样品中SARS-COV-2的存在。
PCT/CN2021/079568 2020-06-02 2021-03-08 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用 WO2021244089A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US18/000,515 US20230331822A1 (en) 2020-06-02 2021-03-08 SARS-COV-2 spike protein binding molecule and application thereof
JP2022574538A JP2023527927A (ja) 2020-06-02 2021-03-08 新型コロナウイルス（ｓａｒｓ－ｃｏｖ－２）スパイクタンパク質結合分子及びその使用
KR1020227046331A KR20230018488A (ko) 2020-06-02 2021-03-08 신종 코로나바이러스 스파이크 단백질 결합 분자 및 이의 응용
EP21817035.5A EP4159757A4 (en) 2020-06-02 2021-03-08 SARS-COV-2 SPICE PROTEIN BINDING MOLECULE AND ITS USE
AU2021285330A AU2021285330A1 (en) 2020-06-02 2021-03-08 SARS-CoV-2 spike protein binding molecule and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010491549.0 2020-06-02
CN202010491549.0A CN111647077B (zh) 2020-06-02 2020-06-02 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用

Publications (1)

Publication Number Publication Date
WO2021244089A1 true WO2021244089A1 (zh) 2021-12-09

Family

ID=72344706

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/079568 WO2021244089A1 (zh) 2020-06-02 2021-03-08 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用

Country Status (7)

Country Link
US (1) US20230331822A1 (zh)
EP (1) EP4159757A4 (zh)
JP (1) JP2023527927A (zh)
KR (1) KR20230018488A (zh)
CN (1) CN111647077B (zh)
AU (1) AU2021285330A1 (zh)
WO (1) WO2021244089A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024052764A1 (en) * 2022-09-06 2024-03-14 Nantcell, Inc. Peptide therapeutics against sars-cov-2 spike protein

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10787501B1 (en) 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
DK3972987T3 (da) 2020-04-10 2023-07-31 Akston Biosciences Corp Antigenspecifik immunterapi til COVID-19-fusionsproteiner og fremgangsmåder til anvendelse deraf
CN111647077B (zh) * 2020-06-02 2021-02-09 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
MX2022016215A (es) * 2020-06-15 2023-05-30 Neuome Peptides Pte Ltd Dispositivo de ensayo de flujo lateral para deteccion de analitos y metodo de deteccion de los mismos.
US20230399385A1 (en) * 2020-09-14 2023-12-14 Nanjing Vazyme Biotech Co., Ltd. Neutralizing antibodies against sars-cov-2
EP3970798A1 (en) * 2020-09-18 2022-03-23 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Sars-cov-2-nanobodies
CN112062840A (zh) 2020-09-22 2020-12-11 石河子大学 一种基于新型冠状病毒s蛋白的纳米抗体及其应用
CN112513076B (zh) * 2020-09-23 2022-03-18 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
WO2022061720A1 (zh) * 2020-09-25 2022-03-31 中国科学技术大学 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
IL300776A (en) * 2020-09-25 2023-04-01 DNARx Systems and methods for expressing biomolecules on the subject
WO2022075921A1 (en) * 2020-10-08 2022-04-14 Hummingbird Bioscience Holdings Limited Sars-cov-2 spike protein antigen-binding molecules
CN112409488B (zh) * 2020-10-23 2022-07-01 中国科学院上海药物研究所 针对多种冠状病毒的单克隆抗体及应用
KR20220054080A (ko) * 2020-10-23 2022-05-02 주식회사 와이바이오로직스 SARS-CoV-2 스파이크 단백질에 특이적으로 결합하는 항체 및 이의 용도
EP3992205A1 (en) * 2020-11-03 2022-05-04 Rheinische Friedrich-Wilhelms-Universität Bonn Sars coronavirus-2 spike protein binding compounds
CN112316130B (zh) * 2020-11-05 2023-11-28 武汉科技大学 一种SARS-CoV2粘膜免疫疫苗及其应用
WO2022095968A1 (en) * 2020-11-06 2022-05-12 Shanghaitech University ANTIBODIES AGAINST SARS-CoV-2 SPIKE PROTEIN
CN112625136B (zh) * 2020-11-18 2022-02-11 三优生物医药(上海)有限公司 针对冠状病毒具有中和活性的双特异性抗体及其用途
TW202241942A (zh) * 2020-12-21 2022-11-01 日商中外製藥股份有限公司 Ｓａｒｓ－ｃｏｖ－２結合分子及其用途
CN112782401A (zh) * 2021-02-08 2021-05-11 聊城大学 一种体外快速检测新型冠状病毒的方法及应用
CN112898437B (zh) * 2021-03-24 2024-02-02 思格(苏州)生物科技有限公司 一种新冠病毒抗原及其制备方法与应用
CN115160434B (zh) * 2022-05-26 2023-05-02 广东菲鹏制药股份有限公司 人源化单域抗体及其应用和药物
CN114773464B (zh) * 2022-06-20 2022-08-26 北京市疾病预防控制中心 一种针对新冠病毒omicron株S蛋白的单域抗体VHH-2及编码序列和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110974950B (zh) * 2020-03-05 2020-08-07 广州恩宝生物医药科技有限公司 一种用于预防SARS-CoV-2感染的腺病毒载体疫苗
CN111153991A (zh) * 2020-02-26 2020-05-15 北京博奥森生物技术有限公司 一种人SARS-CoV-2单克隆抗体及其制备方法和应用
CN111088283B (zh) * 2020-03-20 2020-06-23 苏州奥特铭医药科技有限公司 mVSV病毒载体及其病毒载体疫苗、一种基于mVSV介导的新冠肺炎疫苗

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (en) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulins devoid of light chains
CN111647077A (zh) * 2020-06-02 2020-09-11 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
DANIEL WRAPP, DE VLIEGER DORIEN, CORBETT KIZZMEKIA S., TORRES GRETEL M., WANG NIANSHUANG, VAN BREEDAM WANDER, ROOSE KENNY, VAN SCH: "Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies", CELL, vol. 181, no. 5, 28 May 2020 (2020-05-28), Amsterdam NL , pages 1004 - 1015.e15, XP055764639, ISSN: 0092-8674, DOI: 10.1016/j.cell.2020.04.031 *
DEFFAR ET AL., AFRICAN JOURNAL OF BIOTECHNOLOGY, vol. 8, no. 12, 17 June 2009 (2009-06-17), pages 2645 - 2652
HO MITCHELL: "Perspectives on the development of neutralizing antibodies against SARS-CoV-2", ANTIBODY THERAPEUTICS, vol. 3, no. 2, 30 April 2020 (2020-04-30), pages 109 - 114, XP055857346, DOI: 10.1093/abt/tbaa009 *
LI ET AL., JBIOL CHEM., vol. 287, 2012, pages 13713 - 13721
R. VAN DER LINDEN ET AL., JOURNAL OF IMMUNOLOGICAL METHODS, vol. 240, 2000, pages 185 - 195
See also references of EP4159757A4
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024052764A1 (en) * 2022-09-06 2024-03-14 Nantcell, Inc. Peptide therapeutics against sars-cov-2 spike protein

Also Published As

Publication number Publication date
EP4159757A4 (en) 2023-11-08
JP2023527927A (ja) 2023-06-30
CN111647077B (zh) 2021-02-09
KR20230018488A (ko) 2023-02-07
CN111647077A (zh) 2020-09-11
EP4159757A1 (en) 2023-04-05
AU2021285330A1 (en) 2023-01-19
US20230331822A1 (en) 2023-10-19

Similar Documents

Publication Publication Date Title
WO2021244089A1 (zh) 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
US10253104B2 (en) Bispecific antibodies against CD3ϵ and BCMA
WO2017049452A1 (zh) 抗人cd137的完全人抗体及其应用
WO2017201766A1 (zh) 抗人pd-1人源化单克隆抗体及其应用
CN113045647B (zh) 新冠病毒SARS-CoV-2的中和性抗体及其应用
WO2022061594A1 (zh) 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
EP4144758A1 (en) Single variable domain antibody targeting human programmed death ligand 1 (pd-l1) and derivative thereof
CN113150129A (zh) 抗新冠病毒SARS-CoV-2表面S2蛋白的单链抗体及其应用
CN111378048A (zh) 针对中东呼吸综合征冠状病毒的抗体-多肽双特异性免疫治疗剂
CN111349163A (zh) 针对cd123的单克隆抗体
CA3209675A1 (en) Anti-tslp nanobodies and their applications
CN113754770B (zh) 一种特异性结合人ctla4的抗体及包含其的药物和试剂盒
US20240117012A1 (en) Anti-sars-cov-2 antibody
TWI815136B (zh) 一種雙特異性抗體及其用途
WO2022037528A1 (zh) 结合bcma的单可变结构域及抗原结合分子
TW201102086A (en) Antibodies against human CCN1 and uses thereof
WO2024061021A1 (zh) 检测抗CD19 Car表达水平的单克隆抗体及其在激活CD19 CAR-T细胞中的应用
WO2022037527A1 (zh) 结合bcma的单可变结构域及抗原结合分子
CN113248610B (zh) 白细胞介素2结合分子、其衍生物、试剂盒及其生产方法和用途
WO2022188829A1 (zh) 新型冠状病毒抗体及其用途
WO2024094076A1 (zh) 一种gipr结合蛋白及其应用
CN110407942B (zh) 针对kn044的单域抗体
WO2024017326A1 (zh) 抗gprc5d纳米抗体及其应用
CN117843776A (zh) 抗体分子、核酸、制药用途及炎性疾病治疗方法
CN117924509A (zh) 靶向整合素α4β7和TNF-α的双特异性抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21817035

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022574538

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20227046331

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021817035

Country of ref document: EP

Effective date: 20230102

ENP Entry into the national phase

Ref document number: 2021285330

Country of ref document: AU

Date of ref document: 20210308

Kind code of ref document: A