WO2023092739A1 - 抗sar-cov-2全人源单克隆抗体6g18及其制法与应用 - Google Patents

抗sar-cov-2全人源单克隆抗体6g18及其制法与应用 Download PDF

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WO2023092739A1
WO2023092739A1 PCT/CN2021/138486 CN2021138486W WO2023092739A1 WO 2023092739 A1 WO2023092739 A1 WO 2023092739A1 CN 2021138486 W CN2021138486 W CN 2021138486W WO 2023092739 A1 WO2023092739 A1 WO 2023092739A1
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antibody
antigen
cov
sar
binding fragment
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万晓春
李俊鑫
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • C07K2317/50—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
    • 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]
    • 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

Definitions

  • the invention belongs to the field of immunology, and in particular relates to an anti-SAR-COV-2 (COVID-19) fully human monoclonal antibody and its preparation method and application.
  • HAMA anti-mouse antibody reaction
  • COVID-19 is an acute respiratory infectious disease caused by a SAR-COV-2 coronavirus, and there is still a lack of effective drugs and vaccines.
  • SAR-COV-2 coronavirus When the new coronavirus invades cells, it needs to rely on specific molecules expressed by the virus itself to bind to receptors on human cells in order to infect cells and further amplify.
  • the human antibody that neutralizes the virus is some specific antibody produced by human B lymphocytes, which can bind to the antigen on the surface of the virus, thereby preventing the virus from adhering to the target cell receptor, preventing the virus from invading cells, and can effectively prevent and treat SAR-COV-2 influenza.
  • the present invention provides an anti-SAR-COV-2 antibody or an antigen-binding fragment thereof, which specifically binds to the S protein of SAR-COV-2.
  • One aspect of the present invention provides an isolated anti-SAR-COV-2 antibody or antigen-binding fragment thereof; it has the complementarity determining region (HCDR) of the three heavy chain variable regions of any one of the following groups and three light chains can be Complementarity Determining Regions (LCDRs) of Variable Regions:
  • 6G18-HCDR1 GFTFSSYA SEQ ID No: 1;
  • 6G18-HCDR2 ISGSGGST SEQ ID No: 2;
  • 6G18-HCDR3 AKMVTPGYGDYSLYYFEY SEQ ID No: 3;
  • 6G18-LCDR1 SGSIASNY SEQ ID No: 4;
  • 6G18-LCDR:2 EDN SEQ ID No: 5;
  • 6G18-LCDR3 QSYDSSNLIWV SEQ ID No: 6;
  • Another aspect of the present invention provides an isolated anti-SAR-COV-2 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region and light chain variable region of antibody 6G18 are:
  • the antibody or antigen-binding fragment thereof is a humanized antibody, more preferably a fully humanized antibody.
  • the antibody is an IgG1 antibody.
  • the antibody or antigen-binding fragment thereof specifically binds to the surface S protein of SAR-COV-2.
  • the antibody is a monoclonal antibody or a polyclonal antibody, preferably a monoclonal antibody.
  • the antibody or antigen-binding fragment thereof specifically binds to the surface S protein of SAR-COV-2.
  • Another aspect of the present invention provides a nucleotide sequence encoding the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a vector comprising the aforementioned nucleotide sequence.
  • Another aspect of the present invention provides a host cell, comprising the aforementioned vector or vector group, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or suitable for the preparation of antibodies other cells or antigen-binding fragments thereof.
  • kits which comprises the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides a detection reagent, which comprises the aforementioned antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, and the reagent is a reagent for the following purposes: enzyme-linked immunosorbent assay (ELISA), western blot (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immuno-PCR.
  • ELISA enzyme-linked immunosorbent assay
  • FACS flow cytometry
  • IHC immunohistochemistry
  • the antibody or its antigen-binding fragment can be coupled alone or with a chemical bond, electrostatic adsorption or hydrophilic and hydrophobic adsorption, and the linking conjugate includes horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin (Biotin), fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose and other conjugates.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • Biotin biotin
  • FITC fluorescein isothiocyanate
  • Cy3, Cy5 magnetic beads and agarose and other conjugates.
  • the detection reagent can be used for non-diagnostic or therapeutic detection.
  • Another aspect of the present invention provides a pharmaceutical composition, which is the isolated antibody or antigen-binding fragment thereof as described above and pharmaceutically acceptable excipients.
  • the antibody or its antigen-binding fragment blocks or reduces the binding of the S protein of SAR-COV-2 to the cell surface receptor of the subject
  • the cell surface receptor is preferably a cell blood vessel Tensin-converting enzyme-associated carboxypeptidase (ACE2).
  • Another aspect of the present invention provides the use of an anti-SAR-COV-2 antibody or an antigen-binding fragment thereof in the preparation of a medicament for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection.
  • the drug is an oral or injection preparation.
  • Another aspect of the present invention provides a method for preventing, treating or alleviating at least one symptom or indication of SAR-COV-2 infection, the method comprising administering any of the aforementioned antibodies or antigen-binding fragments thereof or the aforementioned drugs
  • the composition is administered to a subject.
  • the at least one symptom or indication is selected from the group consisting of pulmonary inflammation, alveolar damage, fever, cough, dyspnea, hypoxemia, acute respiratory distress syndrome, sepsis shock, coagulopathy, metabolic acidosis, nasal congestion, rhinorrhea, sore throat, diarrhea, organ failure, septic shock, and death.
  • the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered in combination with a second therapeutic agent.
  • the second therapeutic agent is selected from the group consisting of anti-inflammatory drugs (such as corticosteroids and non-steroidal anti-inflammatory drugs), antiviral drugs, different antibodies against the S protein of SAR-COV-2, for SAR-COV -2 Vaccines, antibiotics, dietary supplements such as antioxidants and any other palliative treatments for SAR-COV-2 infection, drugs that relieve the above symptoms or indications.
  • the pharmaceutical composition or the antibody or antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, intramuscularly or intracranially.
  • the anti-SAR-COV-2 antibody of the present invention can target and bind the S protein of the SAR-COV-2 virus, has high specificity, and can effectively block the S protein on the surface of the SAR-COV-2 virus and the cells of the subject Binding of surface receptors.
  • the gene of the fully human-derived antibody of the present invention is completely derived from human genes, without components of other species, and no toxic and side effects such as anti-mouse and anti-antibodies occur in the human body, and has a better biological phase. Capacitive, more suitable and more potential to become a macromolecular drug for the treatment of influenza virus.
  • the single B cell used in the present invention develops an anti-SAR-COV-2 virus antibody with simple operation It is fast, and the produced human antibodies have the advantages of high affinity and specificity.
  • Fig. 1 is the result figure of ELISA experiment of embodiment 1.
  • the term “antibody” refers to a molecule comprising at least one antigen-binding site that immunospecifically binds to a specific antigenic target of interest.
  • the term “antibody” includes, but is not limited to, full-length antibodies and/or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibodies composed of at least two Multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, and antibody mimetics that mimic the structure and/or function of antibodies formed from intact antibodies, or designated fragments or portions thereof, including single chain antibodies and their fragment.
  • Binding of an antibody to a target can result in various effects such as, but not limited to, such binding modulates, decreases, increases, antagonizes, agonizes, alleviates, slows, blocks, inhibits, eliminates and/or in vitro, in situ and/or in vivo Interfering with at least one target activity or binding, or receptor activity or binding.
  • antibodies of the present disclosure encompass antibody fragments capable of binding biomolecules (e.g., antigens or receptors) or portions thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragment (Fv), single-chain variable fragment (scFv) or disulfide-linked Fv (sdFv); diabody or bivalent diabody; linear antibody; single-chain antibody molecule; Multispecific antibodies formed from fragments.
  • biomolecules e.g., antigens or receptors
  • portions thereof including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragment (Fv), single-chain variable fragment (scFv) or disulfide-linked Fv (sdFv); diabody or bivalent diabody; linear antibody; single-chain antibody molecule; Multispecific antibodies formed from fragments.
  • Antibodies can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible small amounts of possible naturally occurring mutations. Monoclonal antibodies are highly specific, directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, in contrast to a polyclonal antibody preparation comprising different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contaminating other antibodies. The modifier "monoclonal" should not be interpreted as requiring that the antibody be produced by any particular method.
  • HCDR has the same meaning as heavy chain complementarity determining region and LCDR has the same meaning as light chain complementarity determining region.
  • monoclonal antibodies include “chimeric" antibodies, in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular class or subclass of antibodies, wherein all The remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and fragments of these antibodies exhibit the desired biological activity.
  • SAR-COV-2 also known as “novel coronavirus” refers to the newly emerged virus that causes novel coronavirus pneumonia (COVID-19).
  • the S protein refers to the Spike protein on the coronavirus.
  • SARS-CoV-2 recognizes ACE2 on the surface of cells in the human body through the Spike protein on the surface of the virus and infects the host cell. By blocking the S protein on the surface of the coronavirus SARS-CoV-2, it can effectively inhibit the virus from adhering to the target cell receptor and prevent the virus from invading cells.
  • humanized antibody as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term "antigen-binding fragment” or the like includes any naturally occurring, enzymatically available, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
  • the term "antigen-binding fragment” of an antibody has one or more fragments capable of binding to the S protein of SAR-COV-2.
  • the present invention provides an anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to SAR-COV-2, wherein the antibody's heavy and light chain CDR1, CDR2 and the amino acid sequences of the CDR3 region are as follows:
  • 6G18-HCDR1 GFTFSSYA SEQ ID No: 1;
  • 6G18-HCDR2 ISGSGGST SEQ ID No: 2;
  • 6G18-HCDR3 AKMVTPGYGDYSLYYFEY SEQ ID No: 3;
  • 6G18-LCDR1 SGSIASNY SEQ ID No: 4;
  • 6G18-LCDR:2 EDN SEQ ID No: 5;
  • 6G18-LCDR3 QSYDSSNLIWV SEQ ID No: 6;
  • amino acid sequence of the heavy chain variable region of the antibody 6G18 is shown in SEQ ID NO: 7, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acids to the sequence;
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 8, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acids to the sequence
  • the anti-SAR-COV-2 fully human monoclonal antibody of the present invention can target and bind the S protein of the SAR-COV-2 virus.
  • the antibody of the present invention is a fully human monoclonal antibody. Compared with the mouse antibody, the gene of the fully human antibody is completely derived from human genes, and there are no components of other species, and no anti-mouse anti-antibody occurs in the human body. Toxic and side effects, better biocompatibility, more suitable and more potential to become a macromolecular drug for the treatment of influenza virus.
  • the present invention provides a gene encoding the anti-SAR-COV-2 fully human monoclonal antibody of the present invention.
  • the gene comprises a nucleotide sequence encoding the amino acids shown above.
  • the nucleotide sequence is as follows (the following sequences are only exemplary, and those skilled in the art can design other nucleotide sequences that can be translated into the desired amino acid sequence according to the specific amino acid sequence):
  • the nucleotide sequence encoding the heavy chain variable region of antibody 6G18 is:
  • the nucleotide sequence encoding the light chain variable region of antibody 6G18 is:
  • the underlined region shows the CDR region.
  • the present invention provides a vector comprising the gene as described above.
  • the present invention provides a cell containing the gene as described above or the vector as described above.
  • the present invention provides a method for producing the anti-SAR-COV-2 fully human monoclonal antibody or a biologically active fragment derived from the monoclonal antibody that can specifically bind to SAR-COV-2, the method Including cultivating the genetically engineered cells containing the above-mentioned genes or the above-mentioned vectors encoding the heavy and light chains of the anti-SAR-COV-2 fully human monoclonal antibody or directly culturing the above-mentioned cells, collecting and purifying the anti-SAR-COV-2 fully human source monoclonal antibodies.
  • This technology is simple and quick to operate, and the human antibody produced has high affinity and specificity.
  • the improved technology of isolating the monoclonal antibody of the present invention with the function of neutralizing virus or killing tumor from memory B cells can be further adopted, It also greatly reduces the cumbersome operation and cost.
  • the present invention provides a pharmaceutical composition, which comprises the anti-SAR-COV-2 fully human monoclonal antibody described in the present invention or the antibody that can specifically bind to SAR-COV-2 derived from the monoclonal antibody. biologically active fragments.
  • the present invention provides the anti-SAR-COV-2 fully human monoclonal antibody or the biologically active fragment derived from the monoclonal antibody that can specifically bind to SAR-COV-2 or the pharmaceutical composition Application in the preparation of medicines for treating diseases caused by the SAR-COV-2 virus.
  • the present invention provides a kit for detecting the level of the SAR-COV-2 virus, which contains the anti-SAR-COV-2 fully human monoclonal antibody described in the present invention or a specific antibody derived from the monoclonal antibody. biologically active fragments that are sexually bound to SAR-COV-2; in some embodiments, the kit also contains a second antibody and an enzyme or fluorescent or radioactive label for detection, as well as a buffer; the second antibody For example, it is an anti-antibody against the monoclonal antibody of the present invention.
  • 3T3-CD40L feeder cells were established using lentivirus.
  • the lentiviral expression vector pLVX-CD40L was constructed, transfected into 293T cells, and the virus supernatant was collected on the fourth day of transfection.
  • NIH-3T3 cells were activated, cultured for 3 generations, infected with lentivirus, continued to be cultured and passed 3 times.
  • cryopreservation solution Place in a radiometer for 5000 rads radiation, resuspend the cells in the cryopreservation solution at a concentration of 3.5 ⁇ 10 7 cells per milliliter, aliquot 1 mL into cryovials, and freeze in liquid nitrogen (can be stored for 2 years).
  • the memory B cells were added to the mixed medium, and after mixing, they were limitedly diluted in a 384-well plate, 1 cell per well, with a volume of 50 ⁇ l, and placed in a 37°C, 5% CO 2 incubator for static culture. After 13 days, the supernatant was taken for ELISA to obtain human monoclonal antibodies.
  • the surface antigen S protein was purchased from Yiqiao Shenzhou Company, which is immunogenic, and the anti-S protein antibody can detect SAR-COV-2 influenza virus.
  • ELISA experiments were performed on the supernatant human monoclonal antibodies obtained above, specifically:
  • Example 1 The B cells obtained in Example 1 capable of secreting antibodies binding to the SAR-COV-2 virus were lysed, and the lysate was taken for reverse transcription of RNA to obtain the PCR template cDNA of the human antibody gene.
  • Design and synthesize primers for cloning antibody genes use cDNA as a template to clone the genes of heavy and light chains of antibodies, and send them to Jinweizhi Company for sequencing. specifically:
  • Reverse transcription system 150ng random primer (invitrogen, 48190-011), 0.5 ⁇ L 10mM dNTP (Invitrogen, 18427-088), 1 ⁇ L 0.1M DTT (Invitrogen, 18080-044), 0.5% v/v Igepal CA -630 (Sigma, I3021-50ML), 4U RNAsin (Promega), 6U Prime RNAse Inhibitor (Eppendorf) and 50 U III reverse transcriptase (Invitrogen, 18080-044), add DEPC water to 14 ⁇ L/well.
  • KOD-Plus-Neo (TOYOBO, KOD401) kit to PCR amplify the heavy chain and light chain of the antibody gene, 40 ⁇ L system: 3.5 ⁇ L cDNA, 20 nM mixed primers, 4 ⁇ L buffer (buffer), 4 ⁇ L 2mM dNTPs , 2.4 ⁇ L MgSO 4 , 1 ⁇ L KOD.
  • Reaction program 94°C, 2min; 45 cycles: 98°C, 10s; 58°C, 30s; 68°C, 28s.
  • the nucleotide sequence of the heavy chain variable region of antibody 6G18 is the sequence shown in SEQ ID No: 9:
  • amino acid sequence of the heavy chain variable region of antibody 6G18 is as shown in SEQ ID No: 7:
  • the nucleotide sequence of the light chain variable region of antibody 6G18 is the sequence shown in SEQ ID No: 10,
  • amino acid sequence of the light chain variable region of antibody 6G18 is shown in SEQ ID No: 8
  • 6G18-HCDR1 GFTFSSYA SEQ ID No: 1;
  • 6G18-HCDR2 ISGSGGST SEQ ID No: 2;
  • 6G18-HCDR3 AKMVTPGYGDYSLYYFEY SEQ ID No: 3;
  • 6G18-LCDR1 SGSIASNY SEQ ID No: 4;
  • 6G18-LCDR:2 EDN SEQ ID No: 5;
  • 6G18-LCDR3 QSYDSSNLIWV SEQ ID No: 6;

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Abstract

提供抗SAR-COV-2抗体或其抗原结合片段及其应用，具体涉及，抗SAR-COV-2的抗体6G18重链和轻链互补决定区以及重链和轻链可变区序列。所述抗体为人源化抗体，其副作用低，亲和力和特异性高。

Description

抗SAR-COV-2全人源单克隆抗体6G18及其制法与应用 技术领域
本发明属于免疫学领域，具体地涉及抗SAR-COV-2(COVID-19)全人源单克隆抗体及其制法与应用。
背景技术
在2018年全球十大畅销药中，有8个是全人源或人源化单克隆抗体药物。排名第一的是艾伯维公司治疗关节炎的抗TNFa单克隆抗体Humira，这是一个全人源单克隆抗体，已经是连续6年销售额100亿以上的药王。从1986年第一个单克隆抗体药物上市开始，单抗药物经历了鼠源单抗药物(如Orth℃lone OKT3)、嵌合单抗药物(Rituximab)、人源化单抗药物(Herceptin)和全人源单抗药物(Humira)等阶段。由于人体出现抗鼠抗体反应(HAMA)，鼠源单抗药物、嵌合单抗药物已经逐渐被淘汰，目前占据市场的单克隆抗体药物全都是人源化单克隆抗体药物。
人源单克隆抗体在治疗炎症、癌症、流行性感冒特别是冠状病毒等方面具有高特异性的显著疗效。COVID-19是由一种SAR-COV-2冠状病毒引起的急性呼吸道传染病，至今仍然缺乏有效的药物和疫苗。新冠状病毒在入侵细胞时需要依赖病毒自身表达的特定分子与人细胞上的受体结合，才能感染细胞，并进一步扩增。中和病毒的人源抗体是人B淋巴细胞产生的某些特异抗体，能够与病毒表面的抗原结合，从而阻止该病毒黏附靶细胞受体，防止病毒侵入细胞，能够高效防治SAR-COV-2流行性感冒。
发明内容
为解决上述问题，本发明提供一种抗SAR-COV-2的抗体或其抗原结合片段，其特异性结合SAR-COV-2的S蛋白。
本发明一方面提供一种分离的抗SAR-COV-2的抗体或其抗原结合片段；其具有如下任一组的三个重链可变区的互补决定区(HCDR)和三个轻链可变区的互补决定区(LCDR)：
抗体6G18
6G18-HCDR1：GFTFSSYA SEQ ID No：1；
6G18-HCDR2：ISGSGGST SEQ ID No：2；
6G18-HCDR3：AKMVTPGYGDYSLYYFEY SEQ ID No：3；
6G18-LCDR1：SGSIASNY SEQ ID No：4；
6G18-LCDR:2：EDN SEQ ID No：5；以及
6G18-LCDR3：QSYDSSNLIWV SEQ ID No：6；
本发明另一个方面提供了一种分离的抗SAR-COV-2的抗体或其抗原结合片段，其中，抗体抗体6G18的重链可变区和轻链可变区为：
6G18重链可变区：
Figure PCTCN2021138486-appb-000001
6G18轻链可变区：
Figure PCTCN2021138486-appb-000002
在本发明的技术方案中，所述的抗体或其抗原结合片段为人源化抗体，更优选为全人源化抗体。
在本发明的技术方案中，所述的抗体为IgG1抗体。
在本发明的技术方案中，所述的抗体或其抗原结合片段特异性结合SAR-COV-2表面S蛋白。
在本发明的技术方案中，所述的抗体为单克隆抗体或多克隆抗体，优选为单克隆抗体。
在本发明的技术方案中，所述的抗体或其抗原结合片段特异性结合SAR-COV-2表面S蛋白。
本发明再一个方面提供了一种核苷酸序列，其编码如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种载体，包含前述的核苷酸序列。
本发明再一个方面提供了一种宿主细胞，包含前述载体或载体组，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
本发明再一个方面提供了一种试剂盒，所述试剂盒包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种检测试剂，所述检测试剂包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了上述抗体或其抗原结合片段作为检测试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测(ELISA)、免疫印迹(Western Blot)、流式细胞术(FACS)、免疫组织化学(IHC)检测或者免疫PCR。
在上述在免疫学检测中，抗体或其抗原结合片段可单独或与通过化学键偶联，静电吸附 或者亲疏水性吸附，而连接缀合物包括辣根过氧化物酶(HRP)，碱性磷酸酶(AP)，生物素(Biotin)，异硫氰酸荧光素(FITC)，Cy3、Cy5、磁珠和琼脂糖等缀合物连接。
在本发明的技术方案中，检测试剂可用于非诊断或治疗目的检测。
本发明再一个方面提供了一种药物组合物，其如前所述的分离的抗体或其抗原结合片段和药物上可接受辅料。
在本发明的技术方案中，其中所述抗体或其抗原结合片段阻断或降低SAR-COV-2的S蛋白与受试者的细胞表面受体结合，所述细胞表面受体优选为细胞血管紧张素转化酶相关性羧肽酶(ACE2)。
本发明再一个方面提供了一种抗SAR-COV-2的抗体或其抗原结合片段在制备预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的药物中的用途。
在本发明的技术方案中，所述的药物为口服或注射制剂。
本发明再一个方面提供了一种预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的方法，所述方法包括将前述任一项的抗体或其抗原结合片段或前述药物组合物施用于受试者。
在本发明的技术方案中，其中所述至少一种症状或适应症选自下组：肺部炎症、肺泡损伤、发热、咳嗽、呼吸困难、低血氧症、急性呼吸窘迫综合征、脓毒症休克、凝血功能障碍、代谢性酸中毒、鼻塞、流涕、咽痛、腹泻、器官衰竭、败血性休克和死亡。
在本发明的技术方案中，其中所述药物组合物或所述抗体或其抗原结合片段与第二治疗剂组合施用。其中所述第二治疗剂选自下组：抗炎症药物(如皮质类固醇和非甾体抗炎症药物)、抗病毒药物、针对SAR-COV-2的S蛋白的不同的抗体、对于SAR-COV-2的疫苗、抗生素、膳食补充剂如抗氧化剂和治疗SAR-COV-2感染的任何其他姑息疗法、缓解上述症状或适应症的药物。
在本发明的技术方案中，其中所述药物组合物或所述抗体或其抗原结合片段通过皮下、静脉内、皮内、腹膜内、口服、肌内或颅内施用。
有益效果
(1)本发明所述抗SAR-COV-2抗体可以靶向结合SAR-COV-2病毒的S蛋白，特异性高，能有效阻断SAR-COV-2病毒表面S蛋白与受试者细胞表面受体的结合。
(2)相比鼠源抗体，本发明全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
(3)相较于现有技术提供的噬菌体展示技术制备抗SAR-COV-2病毒人源单克隆抗体的方法，本发明采用的单个B细胞开发抗SAR-COV-2病毒的抗体具有操作简单快捷，生产的人源抗体具有高亲和力和特异性等优点。
附图说明
图1为实施例1ELISA实验结果图。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
如本文所用，术语“抗体”是指包含至少一个抗原结合位点的分子，所述抗原结合位点免疫特异性地结合至特定的目标抗原靶标。因此，术语“抗体”包括但不限于全长抗体和/或其变体，其片段，肽体及其变体，单克隆抗体(包括全长单克隆抗体)、多克隆抗体、由至少两个完整的抗体形成的多特异性抗体(例如双特异性抗体)、人抗体、人源化抗体和模拟抗体的结构和/或功能的抗体模拟物或其指定片段或部分，包括单链抗体及其片段。抗体与靶标的结合可引起多种作用，例如但不限于这种结合在体外、原位和/或体内调节、减少、增加、拮抗、激动、减轻、减缓、阻断、抑制、消除和/或干扰至少一种靶标活性或结合，或受体活性或结合。因此，本公开的抗体涵盖能够结合生物分子(例如抗原或受体)或其部分的抗体片段，包括但不限于Fab、Fab'和F(ab')2、pFc'、Fd、单结构域抗体(sdAb)、可变片段(Fv)、单链可变片段(scFv)或二硫化物连接的Fv(sdFv)；双功能抗体或二价双功能抗体；线性抗体；单链抗体分子；由抗体片段形成的多特异性抗体。抗体可以是任何类型(例如IgG、IgE、IgM、IgD、IgA和IgY)，类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。
如本文所用，术语“单克隆抗体”是指得自一群基本上同种的抗体的抗体，即除了可能存在少量可能天然发生的突变之外，构成群体的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。此外，与包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物相反，每个单克隆抗体针对抗原上的单一决定簇。除了其特异性之外，单克隆抗体的优 点还在于其可以未污染其它抗体而合成。修饰语“单克隆”不应解释为需要通过任何特定方法产生抗体。
如本文所用，术语HCDR与重链互补决定区含义相同，LCDR与轻链互补决定区含义相同。
如本文所用，单克隆抗体包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定抗类别或亚类的抗体中的相应序列相同或同源，而所述链剩余的与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，且这些抗体的片段，表现出所需的生物活性。
如本文所用，术语“SAR-COV-2”也称作“新型冠状病毒”，指新发生的引起新型冠状病毒肺炎(COVID-19)的病毒。
如本文所用，S蛋白指冠状病毒上的刺突蛋白(Spike protein)，SARS-CoV-2通过病毒表面的刺突蛋白识别人体内细胞表面的ACE2并侵染宿主细胞。通过阻断冠状病毒SARS-CoV-2表面的S蛋白可以有效抑制病毒黏附靶细胞受体，防止病毒侵入细胞。
如本文使用的术语"人源化抗体"包括具有源自人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人人源化抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如由随机或体外位点特异性诱变或通过体内体细胞突变引入的突变)。
如本文使用的术语抗"抗原结合片段"等包括任何天然存在的、酶学可获得的、合成的或基因工程的特异性结合抗原以形成复合物的多肽或糖蛋白。如本文使用的术语抗体的"抗原结合片段"与SAR-COV-2的S蛋白具有结合能力的一个或多个片段。
一方面，本发明提供抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段，其中，抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
抗体6G18
6G18-HCDR1：GFTFSSYA SEQ ID No：1；
6G18-HCDR2：ISGSGGST SEQ ID No：2；
6G18-HCDR3：AKMVTPGYGDYSLYYFEY SEQ ID No：3；
6G18-LCDR1：SGSIASNY SEQ ID No：4；
6G18-LCDR:2：EDN SEQ ID No：5；以及
6G18-LCDR3：QSYDSSNLIWV SEQ ID No：6；
在一些实施方式中，该抗体6G18的重链可变区氨基酸序列如SEQ ID NO:7所示，或该 序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；
Figure PCTCN2021138486-appb-000003
且
该抗体的轻链可变区氨基酸序列如SEQ ID NO:8所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列
Figure PCTCN2021138486-appb-000004
经ELISA实验验证，本发明所述抗SAR-COV-2全人源单克隆抗体可以靶向结合SAR-COV-2病毒的S蛋白。本发明所述抗体为全人源性单克隆抗体，相比鼠源抗体，全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
另一方面，本发明提供编码本发明所述的抗SAR-COV-2全人源单克隆抗体的基因。在一些实施方式中，所述基因包含编码具有上述所示的氨基酸的核苷酸序列。
在一些具体实施方式中，该核苷酸序列如下所示(以下序列仅为示例性，本领域技术人员根据具体的氨基酸序列，可以设计其他可以翻译为所需氨基酸序列的核苷酸序列)：
编码抗体6G18重链可变区的核苷酸序列为：
Figure PCTCN2021138486-appb-000005
编码抗体6G18轻链可变区的核苷酸序列为：
Figure PCTCN2021138486-appb-000006
本发明抗体的重链可变区和轻链可变区序列中，其中下划线部分区域中的显示的是CDR区。
另一方面，本发明提供含如上所述基因的载体。
再一方面，本发明提供含如上所述基因或如上所述载体的细胞。
再一方面，本发明提供一种产生所述抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段的方法，该方法包括培养含编码抗SAR-COV-2全人源单克隆抗体的重轻链的上述基因或上述载体的基因工程细胞或直接培养上述细胞，收集，纯化得所述抗SAR-COV-2全人源单克隆抗体。
现有技术中存在采用噬菌体展示技术制备抗SAR-COV-2病毒人源单克隆抗体的方法，尽管该方法具有生产成本低、不经过免疫和细胞融合等繁琐工作的优点，但是其缺点也比较明显，从非免疫抗体库中获得的抗体往往亲和力不足、受外源基因转化率的限制、抗体库的库容量不足以涵盖动物的抗体多样性等。本发明从病人的血液中分离分泌功能抗体的B细胞，然后提取RNA和合成cDNA，从中克隆分泌目的抗体的基因，最后重组和表达全人源单克隆抗体。该技术操作简单快捷，生产的人源抗体具有高亲和力和特异性，此外，可进一步采用改进的从记忆B细胞中分离具有中和病毒功能或杀伤肿瘤功能的本发明所述单克隆抗体技术，更是大大减少了繁琐操作和成本。
另一方面，本发明提供一种药物组合物，其包含本发明所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段。
另一方面，本发明提供所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段或所述的药物组合物在制备用于治疗由SAR-COV-2病毒引起的疾病的药物中的应用。
另一方面，本发明提供一种检测SAR-COV-2病毒水平的试剂盒，其含有本发明所述的抗SAR-COV-2全人源单克隆抗体或来源于该单克隆抗体的能够特异性结合SAR-COV-2的生物活性片段；在一些实施方式中，所述的试剂盒还含有第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液；所述第二抗体例如为抗本发明所述单克隆抗体的抗抗体。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)构建稳定表达CD40L的NTH-3T3细胞系(3T3-CD40L)
利用慢病毒建立3T3-CD40L饲养细胞。构建慢病毒表达载体pLVX-CD40L，转染293T细胞，转染第四天收集病毒上清液。活化NIH-3T3细胞，培养3代后用慢病毒感染，继续培养并传代3次。利用流式细胞仪进行分选FITC荧光强度在MFI附近的细胞，重新加入至培 养瓶中，37℃，5％CO 2培养箱中培养和检测，其是将表达CD40L的3T3细胞和空载体pLVX(带有ZxGreen)转染的3T3细胞分别用带有APC的抗CD40L染色，然后上流式细胞仪分析。结果发现，所有3T3-CD40L饲养细胞都表达CD40L。当细胞长到80％～90％时，消化收集细胞，浓度为每毫升1×10 7细胞。置于辐射仪中进行5000rads辐射，冻存液重悬细胞，浓度为每毫升3.5×10 7细胞，分装1mL在冷冻小管，液氮冻存(可以保存2年)。
(2)记忆B细胞的分选和活化
用淋巴分离液分离和冻存曾经感染SAR-COV-2病毒的康复病人的PBMC，每管10～50×10 6细胞，冻存在液氮罐中。配制PBMC流式染色液，其成分如下表1所示
表1 PBMC流式染色液
抗体 体积(μL)
CD19-PE-Cy7 0.5
IgM-PE 1.0
IgA-APC 2.5
IgD-FITC 2.5
PBS-1％(wt/vol)BSA 43.5
解冻PBMC，加入上述PBMC流式染色液并在流式细胞仪上分选，分选出CD19 +IgM –IgA –IgD –的记忆B细胞，细胞纯度需在90％以上，若低于90％，重复分选过程。配制激活B细胞的混合培养基，如下表2所示：
表2
组分 体积
完全IMDM培养基 336mL
IL-2(10,000U mL -1) 3.5mL
IL-21(100μg mL -1) 175μL
步骤(1)中得到的3T3-CD40L 10mL
将记忆B细胞加入到混合培养基中，混匀后有限稀释在384孔板，每孔1个细胞，体积为50μl，置于37℃，5％CO 2培养箱中静置培养。13天后，取上清液进行ELISA，获得人源单克隆抗体。
(3)人源单克隆抗体结合SAR-COV-2病毒的表面抗原S蛋白实验
表面抗原S蛋白购自义翘神州公司，具有免疫原性，抗S蛋白抗体可以SAR-COV-2流感病毒。对上述获得的上清液人源单克隆抗体进行ELISA实验，具体地：
(1)将100ng/100μL的SAR-COV-2病毒的HA蛋白包被在96孔酶标板中，每孔100μl；
(2)放置4℃冰箱过夜；
(3)用PBST溶液洗涤三遍，每孔加5％的脱脂奶粉溶液200μL，37 ℃孵育1小时；
(4)用PBST溶液洗涤三遍，加100μl没有感染病毒的正常人血清(阴性对照)或上清液，各三个重复；
(5)37℃孵育1小时后用PBST溶液洗涤三遍；
(6)以1:5000稀释带HRP的抗人IgG抗体(abcam)，加入酶标版中，每孔100μL；
(7)37℃孵育1小时后用PBST溶液洗涤三遍；
(8)每孔加100μL TMB底物溶液(Thermo Scientific)，37℃5分钟；
(9)每孔加终止溶液2M硫酸100μL，立刻在酶标仪中450nm波长检测吸光值。其结果如图1所示，ELISA实验表明本发明获得的人源单克隆抗体可以靶向结合SAR-COV-2病毒的S蛋白。
实施例2人源化单克隆抗体基因的克隆
将实施例1获得的能够分泌结合SAR-COV-2病毒的抗体的B细胞进行裂解，取裂解液进行RNA的反转录，获得人源抗体基因的PCR模板cDNA。设计和合成克隆抗体基因的引物，以cDNA为模板克隆抗体的重链和轻链的基因，并且送金唯智公司测序。具体地：
(1)将裂解后的B细胞液转移至96孔板(Eppendorf，030133366)。
(2)反转录体系：150ng随机引物(invitrogen,48190-011),0.5μL 10mM dNTP(Invitrogen,18427-088),1μL 0.1M DTT(Invitrogen,18080-044),0.5％v/v Igepal CA-630(Sigma,I3021-50ML),4U RNAsin(Promega),6U Prime RNAse Inhibitor(Eppendorf)and 50 U
Figure PCTCN2021138486-appb-000007
III reverse transcriptase(Invitrogen,18080-044)，补DEPC水至14μL/孔。
(3)反转录反应程序：42℃，10min；25℃，10min；50℃，60min；94℃，5min。
(4)cDNA保存在-20℃。
(5)引物的设计和合成:
(6)用KOD-Plus-Neo(TOYOBO，KOD401)试剂盒PCR分别扩增抗体基因的重链和轻链，40μL体系：3.5μL cDNA，20nM混合引物，4μL缓冲液(buffer)，4μL 2mM dNTPs，2.4μL MgSO 4，1μL KOD。
(7)反应程序：94℃，2min；45个循环：98℃，10s；58℃，30s；68℃，28s。
(8)对扩增产物进行琼脂糖凝胶。
(9)抗体基因重链可变区PCR产物测序结果如下，其中：
抗体6G18的重链可变区核苷酸序列为如SEQ ID No:9所示序列：
Figure PCTCN2021138486-appb-000008
抗体6G18重链可变区氨基酸序列为如SEQ ID No:7所示序列:
Figure PCTCN2021138486-appb-000009
抗体6G18轻链可变区核苷酸序列为如SEQ ID No:10所示序列，
Figure PCTCN2021138486-appb-000010
抗体6G18轻链可变区的氨基酸序列如SEQ ID No:8所示序列
Figure PCTCN2021138486-appb-000011
对应地，各抗体的CDR区序列如下所示：
抗体6G18
6G18-HCDR1：GFTFSSYA SEQ ID No：1；
6G18-HCDR2：ISGSGGST SEQ ID No：2；
6G18-HCDR3：AKMVTPGYGDYSLYYFEY SEQ ID No：3；
6G18-LCDR1：SGSIASNY SEQ ID No：4；
6G18-LCDR:2：EDN SEQ ID No：5；以及
6G18-LCDR3：QSYDSSNLIWV SEQ ID No：6；
上述结果显示上清液中含有能够结合SAR-COV-2病毒的抗体。
最后说明的是：以上实施例仅用于说明本发明的实施过程和特点，而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应当理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，均应涵盖在本发明的保护范围当中。

Claims (10)

  1. 一种分离的抗SAR-COV-2的抗体或其抗原结合片段，其具有如下任一组的三个重链互补决定区和三个轻链互补决定区：
    6G18-HCDR1：GFTFSSYA SEQ ID No：1；
    6G18-HCDR2：ISGSGGST SEQ ID No：2；
    6G18-HCDR3：AKMVTPGYGDYSLYYFEY SEQ ID No：3；
    6G18-LCDR1：SGSIASNY SEQ ID No：4；
    6G18-LCDR:2：EDN SEQ ID No：5；以及
    6G18-LCDR3：QSYDSSNLIWV SEQ ID No：6。
  2. 根据权利要求1所述的分离的抗SAR-COV-2的抗体或其抗原结合片段，抗体6G18的重链可变区和轻链可变区的序列分别为；
    6G18重链可变区：
    EVQLLESGGDLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGSGGSTYYADSVKGRFTISRDNSKNTLNLQMSSLRAEDTAVYYCAKMVTPGYGDYSLYYFEYWGQGTLVTVSS SEQ ID No：7，
    6G18轻链可变区：
    NFMLTQPHSVSESPGKTVTISCTRTSGSIASNYVQWYQQRPGSAPTTVMFEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNLIWVFGGGTKLTVT SEQ ID No：8；
    优选地，所述的抗体或其抗原结合片段为人源化的抗体或其抗原结合片段。
  3. 一种核苷酸序列，其特征在于：其编码如权利要求1-2任一项所述的抗体或其抗原结合片段。
  4. 一种载体，其特征在于：包含权利要求3所述的核苷酸序列。
  5. 一种宿主细胞，其特征在于：包含权利要求4所述载体。
  6. 一种试剂盒，所述试剂盒包含如权利要求1-2任一项所述的抗体或其抗原结合片段。
  7. 一种检测试剂，所述检测试剂包含如权利要求1-2任一项所述的抗体或其抗原结合片段。
  8. 如权利要求1-2任一项所述的抗体或其抗原结合片段作为检测试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
  9. 一种药物组合物，其如权利要求1-2任一项所述的分离的抗体或其抗原结合片段和药物上可接受辅料。
  10. 权利要求1-2任一项所述的抗SAR-COV-2的抗体或其抗原结合片段，或权利要求9所述的药物组合物在制备预防、治疗或缓解SAR-COV-2感染的至少一种症状或适应症的药物中的用途；
    优选地，所述至少一种症状或适应症选自下组：新型冠状病毒肺炎、肺部炎症、肺泡损伤、发热、咳嗽、呼吸困难、低血氧症、急性呼吸窘迫综合征、脓毒症休克、凝血功能障碍、代谢性酸中毒、鼻塞、流涕、咽痛、腹泻、器官衰竭、败血性休克和死亡。
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