WO2022143815A1 - 抗新冠病毒受体结合区域表位中和抗体及其应用 - Google Patents

抗新冠病毒受体结合区域表位中和抗体及其应用 Download PDF

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WO2022143815A1
WO2022143815A1 PCT/CN2021/142693 CN2021142693W WO2022143815A1 WO 2022143815 A1 WO2022143815 A1 WO 2022143815A1 CN 2021142693 W CN2021142693 W CN 2021142693W WO 2022143815 A1 WO2022143815 A1 WO 2022143815A1
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antibody
seq
present
variable region
chain variable
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PCT/CN2021/142693
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French (fr)
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孙兵
伊春艳
孙晓玉
凌志洋
张亚光
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中国科学院分子细胞科学卓越创新中心
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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
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6839—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses
    • A61K47/6841—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting material from viruses the antibody targeting a RNA virus
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • 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 relates to the field of medicine, in particular to neutralizing antibodies against different epitopes in the receptor binding region of the novel coronavirus and applications thereof.
  • monoclonal antibodies have the potential to be used alone or as a supplement to vaccines.
  • monoclonal antibodies have potential utility in outbreak and post-exposure prophylaxis settings. Based on the broad potential use of monoclonal antibodies in prophylaxis, it is suitable for high-risk populations with known exposure or exposure to SARS-CoV-2 based on ongoing risk of occupational or housing status, and in patients with high-risk COVID-19 complications.
  • SARS-CoV-2 shares many similarities with the 2003 atypical pneumonia virus (SARS-CoV) in viral replication and host cell-infecting properties.
  • the surface spike (S) protein of the two viruses has a homology of up to 76%, and both bind to the angiotensin-converting enzyme 2 (ACE2) receptor, which is an important link for the virus to infect host cells.
  • the S protein of coronavirus consists of two functional domains, S1 and S2, which mediate the binding of the virus to the host cell ACE2 and the membrane fusion process, respectively.
  • the vast majority of neutralizing antibodies to coronaviruses target the S1 subunit or the receptor binding domain RBD on the S1 subunit.
  • the present invention provides a fully human monoclonal antibody capable of preventing and controlling novel coronavirus (SARS-CoV-2) infection.
  • a first aspect of the present invention provides a heavy chain variable region of an antibody, the heavy chain variable region comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the RBD binding affinity of SARS-CoV-2.
  • the heavy chain variable region further includes a human FR region or a murine FR region.
  • the heavy chain variable regions respectively have the amino acid sequences shown in SEQ ID NO.: 1-12.
  • the second aspect of the present invention provides a heavy chain of an antibody, wherein the heavy chain has the variable region of the heavy chain described in the first aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human origin, mouse origin or rabbit origin.
  • a third aspect of the present invention provides a light chain variable region of an antibody, the light chain variable region comprising the following three complementarity determining region CDRs:
  • amino acid sequence is CDR2' of EGT, AAS, DAS, EGS, EVS, or GAS, and
  • any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid derived sequence that retains the RBD binding affinity of SARS-CoV-2.
  • the light chain variable region further includes a human FR region or a murine FR region.
  • the light chain variable regions respectively have the amino acid sequences shown in SEQ ID NO.: 13-24.
  • the fourth aspect of the present invention provides a light chain of an antibody, the light chain has the light chain variable region according to the third aspect of the present invention.
  • the light chain of the antibody further includes a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • the fifth aspect of the present invention provides an antibody, the antibody has:
  • the antibody has: a heavy chain as described in the second aspect of the invention; and/or a light chain as described in the fourth aspect of the invention.
  • the antibody is a specific anti-SARS-CoV-2 antibody, preferably a specific anti-SARS-CoV-2 RBD protein antibody.
  • the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
  • the antibody is a diabody or a single-chain antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the antibody is in the form of a drug conjugate.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 13.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 2; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 14.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 3; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 15.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:4; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:16.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.:5; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.:17.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 6; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 18.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 7; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 19.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 8; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 20.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 9; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 21.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 10; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 22.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 11; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 23.
  • the heavy chain variable region sequence of the antibody is shown in SEQ ID NO.: 12; and the light chain variable region sequence of the antibody is shown in SEQ ID NO.: 24.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:25, CDR2 as shown in SEQ ID NO.:36 and as SEQ ID NO.:46; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:58, CDR2' as the amino acid sequence of EGT and CDR3' as shown in SEQ ID NO.:69.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:26, CDR2 as shown in SEQ ID NO.:37 and as SEQ ID NO.:47; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:59, CDR2' as amino acid sequence of AAS and CDR3' as shown in SEQ ID NO.:70.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:27, CDR2 as shown in SEQ ID NO.:38 and as SEQ ID NO.:48; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:60, CDR2' as the amino acid sequence of DAS and CDR3' as shown in SEQ ID NO.:71.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:28, CDR2 as shown in SEQ ID NO.:39 and as SEQ ID NO.:49; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:61, CDR2' as the amino acid sequence of EGS and CDR3' as shown in SEQ ID NO.:72.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:30, CDR2 as shown in SEQ ID NO.:41 and as SEQ ID NO.:51; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:63, CDR2' as amino acid sequence of AAS and CDR3' as shown in SEQ ID NO.:74.
  • the heavy chain variable region of the antibody comprises CDR1 as shown in SEQ ID NO.:31, CDR2 as shown in SEQ ID NO.:41 and as SEQ ID NO.:52; and
  • the light chain variable region comprises CDR1' as shown in SEQ ID NO.:64, CDR2' whose amino acid sequence is GAS and CDR3' as shown in SEQ ID NO.:75.
  • the sixth aspect of the present invention provides a recombinant protein, the recombinant protein has:
  • variable region of the heavy chain according to the first aspect of the present invention the heavy chain according to the second aspect of the present invention, the variable region of the light chain according to the third aspect of the present invention, the variable region of the light chain according to the fourth aspect of the present invention
  • the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.
  • the recombinant protein includes fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein specifically binds to SARS-CoV-2 RBD protein.
  • the seventh aspect of the present invention provides a CAR construct, the scFv segment of the antigen binding region of the CAR construct is specifically bound to the SARS-CoV-2 receptor binding region, and the scFv has the The heavy chain variable region according to the first aspect of the invention and the light chain variable region according to the third aspect of the invention.
  • the eighth aspect of the present invention provides a recombinant immune cell expressing the exogenous CAR construct according to the seventh aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (eg, mice).
  • the ninth aspect of the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising:
  • a conjugation moiety conjugated to the antibody moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pre- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or nanoparticles in any form, and the like.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the antibody moiety and the coupling moiety are coupled through chemical bonds or linkers.
  • the tenth aspect of the present invention provides the use of an active ingredient selected from the group consisting of the heavy chain variable region described in the first aspect of the present invention, the heavy chain variable region described in the second aspect of the present invention. chain, light chain variable region according to the third aspect of the invention, light chain according to the fourth aspect of the invention, or antibody according to the fifth aspect of the invention, recombinant according to the sixth aspect of the invention A protein, or a combination thereof, the active ingredient for use in the preparation of a medicament, reagent, assay panel or kit.
  • the reagent, detection plate or kit is used to detect novel coronavirus (SARS-CoV-2).
  • SARS-CoV-2 novel coronavirus
  • the medicament is used for the treatment or prevention of novel coronavirus (SARS-CoV-2) infection.
  • SARS-CoV-2 novel coronavirus
  • the reagent includes a chip and an antibody-coated immune particle.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region as described in the first aspect of the present invention, a heavy chain as described in the second aspect of the present invention, and a heavy chain as described in the third aspect of the present invention
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used for preventing and/or treating novel coronavirus (SARS-CoV-2) infection.
  • SARS-CoV-2 novel coronavirus
  • a twelfth aspect of the present invention provides a polynucleotide encoding a polypeptide selected from the group consisting of:
  • polynucleotides have the sequences shown in SEQ ID NO.: 81-92 and/or SEQ ID NO.: 93-104.
  • the thirteenth aspect of the present invention provides a vector, the vector containing the polynucleotide according to the twelfth aspect of the present invention.
  • the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector according to the thirteenth aspect of the present invention or integrates the vector according to the twelfth aspect of the present invention in the genome polynucleotides.
  • a fifteenth aspect of the present invention provides a method for detecting a novel coronavirus (SARS-CoV-2) in a sample, the method comprising the steps of:
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the present invention also provides a method for detecting SARS-CoV-2 RBD protein in a sample, the method comprising the steps of:
  • the detection is for non-therapeutic and non-diagnostic purposes.
  • the sixteenth aspect of the present invention provides a detection plate, the detection plate comprises: a substrate (support plate) and a test strip, the test strip contains the antibody according to the fifth aspect of the present invention or the The immunoconjugate according to the ninth aspect of the present invention.
  • a seventeenth aspect of the present invention provides a kit comprising:
  • the kit contains the detection plate according to the sixteenth aspect of the present invention.
  • An eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
  • a nineteenth aspect of the present invention provides a method for treating a novel coronavirus infection, the method comprising: administering the antibody or the antibody-drug conjugate of the antibody according to the fifth aspect of the present invention to a subject in need substance, or CAR-T cells expressing the antibody, or a combination thereof.
  • Figure 1 shows the binding activity of 12 antibodies against SARS-CoV-2 verified by ELISA.
  • Figure 2 shows the ability of ELISA to verify the ability of 12 antibodies to block RBD and ACE2.
  • Figure 3 shows the pseudovirus experiments to verify the neutralizing activity of the 12 antibodies against the SARS-CoV-2 pseudovirus.
  • Figure 4 shows the microneutralizing activity of RBD-specific antibodies against the SARS-CoV-2 euvirus.
  • Figure 5 shows the affinity of six antibodies for RBD detected by the OCTET RED 96 instrument.
  • Figure 6 shows the ELISA epitope competition method to verify the epitope recognition of 12 antibodies.
  • treating refers to the administration of an internal or external therapeutic agent, comprising the monoclonal antibodies against the coronavirus RBD protein of the present invention, and compositions thereof, to a patient who has one or more disease symptoms and is known to have The therapeutic agent has a therapeutic effect on these symptoms.
  • a patient is administered to a patient in an amount of the therapeutic agent effective to alleviate one or more symptoms of the disease (therapeutically effective amount).
  • the terms “optional” or “optionally” mean that the subsequently described event or circumstance can, but need not, occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region of a specific sequence may but not necessarily have, and may be 1, 2 or 3.
  • Sequence identity refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions or deletions.
  • sequence identity between the sequences described in the present invention and the sequences with which they are identical may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.
  • SARS-CoV-2 Novel Coronavirus
  • 2019 Novel Coronavirus on January 12, 2020, the World Health Organization officially named it 2019-nCoV.
  • antibody or "immunoglobulin” is a heterotetraglycan protein of about 150,000 Daltons having the same structural characteristics, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions.
  • VH variable region
  • Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • VL variable domain
  • Particular amino acid residues form the interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable domains of native heavy and light chains each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs that form linking loops, and in some cases form a partially folded structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • immunoglobulins can be grouped into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be classified into different classes according to the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those in the art.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific to a single antigenic site. Also, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants, each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the benefit of monoclonal antibodies is that they are synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is characteristically obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • the present invention also includes the monoclonal antibody having the corresponding amino acid sequence of the SARS-CoV-2 RBD protein monoclonal antibody, the monoclonal antibody having the variable region chain of the SARS-CoV-2 RBD protein monoclonal antibody, And other proteins or protein conjugates and fusion expression products with these chains.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having light and heavy chains containing hypervariable regions (complementarity determining regions, CDRs), as long as the The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules and the SARS-CoV-2 RBD protein Conjugates formed by the binding of anti-monoclonal antibodies or fragments thereof.
  • the present invention also includes cell surface markers or antigens that bind to the anti-SARS-CoV-2 RBD protein monoclonal antibody or fragments thereof.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment of an antibody” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising A bivalent fragment of two Fab fragments linked by a disulfide bridge on the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv consisting of the VH and VL domains of the one-armed antibody Fragment.
  • Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the present invention includes not only complete monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; and antibody light chains.
  • immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; and antibody light chains.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation.
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, the antibody binds with an affinity (KD) of about less than 10-7 M, eg, about less than 10-8 M, 10-9 M, or 10-10 M or less.
  • KD affinity
  • antigenic determinant refers to a discrete three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "murine antibody” in the present invention is a monoclonal antibody against the SARS-CoV-2 RBD protein prepared according to the knowledge and skills in the art.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody Antibody produced in framework sequences.
  • Humanized antibodies can overcome the heterologous reaction induced by chimeric antibodies due to the large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • antibodies may be monospecific, bispecific, trispecific, or more multispecific.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the heavy chain variable region of the antibody comprises three complementarity determining region CDRs as shown in Table A below:
  • SGFTVSSNY (SEQ ID NO.: 34) IIYSGGST (SEQ ID NO.: 44) ARDRGGYCDY (SEQ ID NO.: 55) 24-2K SGFTVSSNY (SEQ ID NO.: 34) VIYSGGS (SEQ ID NO.: 45) ARTFGELYIDY (SEQ ID NO.: 56) 26-45K SGLIVSSNY (SEQ ID NO.: 35) VIYSGGS (SEQ ID NO.: 45) ARDLGPYGMDV (SEQ ID NO.: 57).
  • amino acid sequences of the heavy chain variable regions are shown in SEQ ID NO.: 1-12, respectively, wherein the underlined sequences are the amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3.
  • the nucleic acid coding sequences of the heavy chain variable region are respectively as SEQ ID NO.: 81 (25-C9), 82 (25-G7), 83 (25-B5), 84 (28- 8L), 85(25-F8), 86(24-11K), 87(26-40K), 88(25-C4), 89(25-C5), 90(25-C8), 91(24-2K) ), 92 (26-45K).
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
  • VL light chain variable region
  • the light chain variable region of the antibody according to the present invention has the CDRs of the complementarity determining region selected from the following table B:
  • amino acid sequences of the light chain variable regions are respectively shown in SEQ ID NO.: 13-24, wherein the double underlines are the light chain variable region CDR1', CDR2', CDR3' in turn amino acid sequence.
  • the nucleic acid coding sequences of the light chain variable region are respectively as SEQ ID NO.: 93(25-C9), 94(25-G7), 95(25-B5), 96(28- 8L), 97(25-F8), 98(24-11K), 99(26-40K), 100(25-C4), 101(25-C5), 102(25-C8), 103(24-2K) ), 104 (26-45K).
  • the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
  • the function of the antibody of the present invention is determined by the specific gene sequences of the light chain and heavy chain variable region genes of the antibody, can specifically bind to the RBD protein of SARS-CoV-2, and can prevent SARS-CoV-2 from infecting susceptible cells .
  • this antibody variable region gene or complementarity determining region (CDR) gene different forms of genetically engineered antibodies can be engineered and produced in any expression system using prokaryotic and eukaryotic cells.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to antibodies that specifically bind to SARS-CoV-2 RBD protein, such as those with variable heavy chain region (amino acid sequences encoded by the nucleotide sequences shown in SEQ ID NO.: 81-92) and/or light chain variable regions (encoded by the nucleotide sequences shown in SEQ ID NO.: 93-104) amino acid sequence) proteins or polypeptides. They may or may not contain the starting methionine.
  • the antibody is a mouse or human-mouse chimeric monoclonal antibody against SARS-CoV-2 RBD protein, and its heavy chain constant region and/or light chain constant region can be humanized Heavy chain constant region or light chain constant region. More preferably, the humanized heavy chain constant region or light chain constant region is the heavy chain constant region or light chain constant region of human IgG1, IgG2 and the like.
  • variable regions which are separated into four framework regions (FRs), four
  • FRs framework regions
  • the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen.
  • the present invention includes those molecules having CDR-bearing monoclonal antibody light and heavy chain variable regions, as long as their CDRs are greater than 90% (preferably greater than 95%, optimally 98%) of the CDRs identified herein above) homology.
  • the present invention includes not only complete monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region with SARS-CoV-2 RBD protein binding activity.
  • the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
  • the present invention also includes fragments of the antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 60 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table C.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • the coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO.: 81-104 or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence encoding the same amino acid sequence as the polypeptide of the present invention, but differing from the coding region sequence shown in SEQ ID NO.: 81-104 .
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%. And, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO.: 1-12 and/or SEQ ID NO.: 13-24.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable products enzyme.
  • Therapeutic agents that can be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptides, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind the novel coronavirus RBD protein molecule, thus can be used to prolong the half-life of the drug, in addition, other therapeutic agents can be used at the same time.
  • the pharmaceutical composition of the present invention contains the above-mentioned monoclonal antibody (or its conjugate) of the present invention in a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 10 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 1 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • Antibodies of the invention can be used in detection applications, eg, in detection of samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsies known to those skilled in the art. Biopsy used in the present invention may thus include tissue samples prepared, for example, by endoscopic methods or needle or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or its fragment) of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the fully human monoclonal antibody of the present invention can specifically recognize and bind to the RBD protein of the coronavirus SARS-2-CoV, effectively block the binding of RBD and ACE2, and has a high neutralization effect on the coronavirus SARS-2-CoV. and activity.
  • the fully human monoclonal antibody of the present invention can recognize different epitopes of the coronavirus SARS-2-CoV RBD protein, and the combination of monoclonal antibodies targeting two different epitopes has a synergistic effect, and antibodies targeting different epitopes are used The combination can reduce the occurrence of drug resistance.
  • the different antibodies isolated by the present invention can form a cocktail therapy, enhance the antiviral effect and solve the drug resistance problem caused by the continuous mutation of the virus, and can also be used as a basic scientific research and clinical diagnostic tool.
  • the present invention is a fully human monoclonal antibody, does not contain mouse-derived parts, has lower immunogenicity and higher safety for humans, and can avoid human anti-mouse and other species-derived antibodies mediated immunity Rejection.
  • the following describes the process of preparing fully human monoclonal antibodies capable of neutralizing different epitopes of the coronavirus RBD protein and analyzing the characteristics of the antibodies.
  • Example 1 Obtaining antibody genes and antibody expression by single-cell RT-PCR method
  • PBMC peripheral blood mononuclear cells
  • Peripheral blood was drawn from healthy volunteers, using conventional Ficoll-Paque (manufactured by (CEDARLANE) density gradient centrifugation to obtain more than 10 7 peripheral blood mononuclear cells (PBMC).
  • CEDARLANE peripheral blood mononuclear cells
  • BD Horizon TM Fixable Viability Stain 780 was used to remove dead cells, and specific B cells were obtained by flow cytometry to 96-well RT-PCR plate , one cell per well, to obtain SARA-CoV-2 RBD protein-specific memory B cells.
  • RBD protein was expressed by mammalian cell CHO expression system; refer to Invitrogen ExpiCHO-S TM Expression System manual; RBD protein sequence refer to nCoV-SH01 (GenBank: MT121215.1), and the whole gene synthesis was carried out in Shanghai Jierui Company. The whole gene synthesis was carried out in Shanghai Jierui Company, and it was constructed on the expression vector of invitrogen pcDNA3.1;
  • Biotin labeling of RBD protein No-Weigh Sulfo-NHS-LC-Biotin (purchased from PIERCE, refer to EZ-Link Sulfo-NHS-LC-Biotin Biotin Labeling Protocol of PIERCE Company) 10mM reagent; Two markers, FITC-CD19 and APC-IgG, were purchased from BD Bioscience; SA Streptavidin PE and streptavidin BV 421 (purchased from BD) were used to detect biotin-labeled SARA-CoV-2 RBD protein;
  • PBMC cells are grouped into groups, experimental group + control group, markers are added according to the number of cells, stained in the dark, labeled, resuspended in PBS, and filtered through a 40 ⁇ m BD falcon filter;
  • Sorting of specific B cells use BD FACS Influx screening, screen lymphocytes from PBMC according to the forward angle and lateral angle, and then compensate by the adjustment of different control groups to obtain SARA-CoV-2 RBD protein specific memory B cells were sorted into 96-well plates for RT-PCR (reverse transcription PCR), one cell per well, and the plates were placed on dry ice.
  • RT-PCR reverse transcription PCR
  • the obtained single memory B cells were obtained by RT-PCR to obtain cDNA, and then the variable region of the antibody gene was obtained by nested-PCR, and the agarose nucleic acid gel was run to recover and sequence the gel blocks where the heavy and light chains could be paired.
  • Antibody gene sequences were retrieved through IgBLAST (https://www.ncbi.nlm.nih.gov/projects/igblast/).
  • the antibody genes were linked to corresponding IgH, Ig ⁇ and Ig ⁇ expression vectors through AgeI and SalI restriction sites, AgeI and BsiwI restriction sites, AgeI and XhoI restriction sites, respectively.
  • the fully human antibody expression vectors IgH, Ig ⁇ and Ig ⁇ (respectively express antibody heavy chain, kappa chain, lambda chain) were donated by Patrick Wilson laboratory.
  • CHO cells were transiently transfected for fully human antibody expression.
  • ExpiCHO-S TM cells were split to a final density of 3 x 106 - 4 x 106 viable cells/mL, and the cells were allowed to grow overnight.
  • viable cell density and percent viability were determined. The cell density should reach approximately 7 x 10 6 -10 x 10 6 viable cells/mL. Viability should be 95–99% before proceeding with transfection.
  • Cells were diluted to a final density of 6 x 106 viable cells/mL.
  • Expi Fectamine TM CHO/plasmid DNA complexes were prepared using pre-chilled reagents (4°C).
  • Antibodies were purified using Protein G Agarose 4FF media (purchased from GE). First, the collected CHO cell suspension was centrifuged at 4000rpm at 4°C for 30min, and the collected supernatant was filtered with a 0.45um filter for purification. Take a gravity-type spin column, add Protein G Agarose 4FF packing, stabilize the packing with 3 column volumes of 20% ethanol, then equilibrate the column with 5 column volumes of binding buffer, load the sample, and use 10 column volumes of binding The column was equilibrated with buffer, and finally the column was eluted with 3 column volumes of elution buffer, and neutralization buffer was added to the eluted antibody solution to make the pH of the eluted sample reach about 7.5. The antibody solution was dialyzed three times in 5L 1 ⁇ PBS, and the antibody was concentrated and stored at -80°C.
  • Single-cell RT-PCR and Nested-PCR methods were used to obtain matching antibody heavy and light chain variable region genes. Agarose gels were recovered and sequenced. Compare the sequencing results at http://www.ncbi.nlm.nih.gov/igblast and http://www.imgt.org/ to obtain antibody germline gene information and antibody heavy and light chain gene hypervariable regions information, and carry out expression vector construction and subsequent expression purification. Finally, through this technology, multiple strains of fully human monoclonal antibodies that neutralize the new coronavirus were successfully obtained.
  • amino acid sequence of the heavy chain variable region of the fully human antibody of the present invention is as follows, wherein the sequences of heavy chain amino acids CDR1, CDR2, and CDR3 are marked with underlining in order.
  • amino acid sequence of the light chain variable region of the fully human antibody of the present invention is as follows, wherein the sequences of light chain amino acid CDR1', CDR2' and CDR3' are marked with underlining in order.
  • ELISA was used to detect whether the expressed antibody recognized the RBD protein.
  • Coated RBD protein ELISA plate 0.5 ⁇ g/mL, 100 ⁇ L per well, overnight at 4°C. The next day, the plate was washed 3 times with PBST. Blocked with 2% BSA, 200 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST again.
  • RBD-specific antibodies were tested at 6 concentrations, 3-fold serial dilution, 2 duplicate wells, and the initial test concentration was 30 ⁇ g/ml. Loading, 100 ⁇ L per well, 37°C, 2h. Wash the plate 3 times with PBST.
  • Goat Anti-Human IgG (Fc specific)-Peroxidase antibody (sigma), diluted 1:5000, 100 ⁇ L per well, 37°C, 1h. Wash the plate 3 times with PBST. Add substrate TMB 100 ⁇ L/well to develop color, if the color is lighter, react at 37°C for 15min in the dark. Reactions were stopped by adding 2M H2SO4 , 50 ⁇ L per well. OD450 was determined and data processed.
  • the 96-well plate was coated with 0.5 ⁇ g/ml recombinant SARS-CoV-2 RBD protein, refrigerated at 4°C overnight, 50 ⁇ g/ml blocking antibody was added to the microplate, incubated at 37°C for 2h, and then biotin-labeled detection antibody was added, 37 Incubate at °C for 2 h, and wash three times with PBST.
  • the secondary antibody was added with HRP-labeled SA (1:200), incubated at 37°C for 1 h, washed 3 times, TMB developed color, and reacted in the dark at room temperature for 2 min. After the reaction was terminated with 1 M HCl, the absorbance of the sample at A450 was determined by a microplate reader.
  • This experiment was performed using the OCTET RED 96 instrument at the Molecular Platform of the Institute of Biochemical Cells.
  • the full-length SARS-2-CoV S protein plasmid and pNL4-3 plasmid were co-transfected into HEK293T cells in a 10 cm dish, and the medium was changed for 6 hours. The supernatant was collected after 48 hours and diluted with complete medium.
  • the antibodies were serially diluted, mixed with an equal volume of virus, and incubated at 37°C for 1 h. The antibody and virus mixture was transferred into HEK293T cells stably expressing human ACE2. Incubate for 48 hours at 37°C. Remove the supernatant and add lysis buffer to the cells to fully lyse. Luciferase activity was assayed (Promega). Neutralization efficiency was calculated by comparing the luciferase value of the experimental group with the luciferase value of the virus-only control group. Calculated as follows:
  • IC50 values were calculated by Prism software.
  • Seeding cells Take Vero-E6 cells in logarithmic growth phase and seed them into a 96-well plate, 100 ⁇ l per well, 4 ⁇ 10 4 cells per well.
  • Neutralization experiment dilution of the test sample: as shown in Table 1, add 60 ⁇ l of 10 ⁇ g/ml pre-diluted sample in column 1 (rows B-G), and add 60 ⁇ l of virus diluent to the final antibody concentration of 5 ⁇ g/ml), and add the remaining wells in sequence 60 ⁇ l of 3-fold diluted sample.
  • the first row of cell control (Cell Control, CC) was added with 120 ⁇ l of serum-free medium
  • the eighth row of virus control (Virus Control, VC) was added with 60 ⁇ l of serum-free medium.
  • the virus stock titer is 2.5 ⁇ 10 5 TCID50/ml, take 200 ⁇ l of virus stock solution, add 25 ml of serum-free medium, mix well, and dilute the virus to 100 TCID50/50 ⁇ l.
  • Dropping virus vertically drop the virus (except cell control) into a 96-well plate, the sample volume is 60 ⁇ l/well, and the final virus-antibody mixture is 120 ⁇ l.
  • Neutralization Mix the added cell culture plate on a shaker, place it in a 37°C incubator, and neutralize for 1 hour. After neutralization, the supernatant of the culture plate inoculated with cells was aspirated, and then 100 ⁇ l/well of virus-serum mixture was added to it, and cultured in a 37°C CO 2 incubator for 1 hour for infection. After the virus infection was over, the supernatant from the culture plate was aspirated, and the plaque formation experimental samples were added to the maintenance medium containing 1% methylcellulose (DMEM medium containing 2% FBS), and placed in a 37°C CO2 incubator. Incubate for 72-96 hours.
  • DMEM medium containing 2% FBS maintenance medium containing 1% methylcellulose
  • RBD-specific antibodies can effectively block the binding of RBD to receptor ACE2
  • ACE2-FC recombinant protein was coated on 96 microwell plates, and different serial dilutions of antibodies were incubated with biotinylated RBD protein before adding onto pre-coated ACE2-FC microplates. Detection was performed with HRP-conjugated streptavidin. The results are shown in Figure 2. All 12 RBD-specific antibodies can effectively inhibit the binding of RBD to ACE2, and show a dose-dependent effect.
  • the 12 neutralizing antibodies were divided into two independent epitopes by ELISA epitope competition experiment. Among them, 26-40K, 24-11K, 25-C4, 25-C8, 26-45K, 24-2K, 25-G7, 28-8L and 25-B5 compete with each other, suggesting that the epitopes recognized by these antibodies are There is overlap or steric hindrance. In addition, there is no epitope competition between 25-C9 and the above antibodies, indicating that 25-C9 is far away from the first type of epitope and belongs to an independent epitope.
  • the inventors used single-cell RT-PCR technology to isolate multiple neutralizing antibodies from the peripheral blood PBMC of recovered volunteers infected with the new crown. Affinity, can effectively block the binding of RBD and ACE2.
  • the multiclonal antibodies can effectively inhibit the infection of host cells by SARS-CoV-2 pseudovirus and true virus, and the best antibody IC 50 was at the nanogram level.
  • the epitopes of these antibodies are diverse, and different antibodies can be combined to deal with the problem of drug resistance due to the rapid formation of SARS-CoV-2 mutation. 2 has potential application value.

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Abstract

一种抗新冠病毒受体结合区域(RBD)不同表位的中和抗体及其应用。具体地，涉及针对新冠病毒SARS-CoV-2的RBD蛋白的全人单克隆抗体、编码抗体和抗体片段的核酸序列及其制备方法。体外实验证实所述抗体能够识别SARS-CoV-2 RBD蛋白的不同表位，通过协同作用阻断SARS-CoV-2 RBD与其受体宿主受体血管紧张素转化酶2(ACE2)的相互作用，从而有效地预防及控制SARS-CoV-2感染，并减少耐药的发生。

Description

抗新冠病毒受体结合区域表位中和抗体及其应用 技术领域
本发明涉及医药领域，具体地涉及抗新冠病毒受体结合区域不同表位的中和抗体及其应用。
背景技术
于2019年底爆发的新型冠状病毒肺炎(COVID-19)疫情已成为全球性的公共卫生大事件，给全球的人们生命健康和经济造成了极大的冲击。针对这样一种具有较高的传染性和致病性的突发病毒，开发有效的防治手段刻不容缓。单克隆抗体临床干预在预防和治疗病毒感染方面具有较好的疗效，临床上已成功用于预防呼吸道合胞病毒的感染。
在预防措施方面，单克隆抗体有可能单独使用或作为疫苗的补充。半衰期延长的强效单抗可安全可靠地保护长达12个月，可作为疫苗替代品，用于由于免疫缺陷对疫苗响应差的个体。与疫苗不同的是，单克隆抗体在暴发和暴露后的预防环境中具有潜在的效用。基于单克隆抗体在预防方面的广泛潜在用途，适用于已知接触或基于职业或住房状况的持续风险而暴露于SARS-CoV-2的高危人群，以及高风险的COVID-19并发症患者。
在早期感染环境中，最有可能从单克隆抗体治疗中获益的人包括老年人、免疫功能低下者和其他最易发生COVID-19并发症的人。这种治疗的目的是减少住院、重症监护病房的入院和死亡，以及缩短疾病的持续时间。晚期疾病的大部分病理学可能是由于宿主的免疫反应加上潜在的共病条件的恶化需要抗病毒单抗和治疗免疫失调的药物组成的联合治疗。
SARS-CoV-2与2003年流行的非典型性肺炎病毒(SARS-CoV)在病毒复制和侵染宿主细胞特性有很多相似性。两种病毒表面刺突(Spike，S)蛋白同源性高达76％，均与血管紧张素转换酶2(ACE2)受体结合，是病毒感染宿主细胞的重要环节。冠状病毒的S蛋白由S1和S2两个功能域组成，分别介导病毒与宿主细胞ACE2的结合及膜融合过程。绝大部分冠状病毒的中和抗体都是靶向S1亚基或S1亚基上的受体结合域RBD。已有研究报道揭示与SARS相比，SARS-CoV-2 RBD某些关键的抗原表位已经发生改变，意味着基于SARS的开发的疫苗或者中和抗体极有可能失去了对新冠的保护活性。
因此，寻找靶向人类冠状病毒的有效中和抗体对预防和治疗SARS-CoV-2在内的冠状病毒意义重大。
发明内容
本发明提供了一种能够预防及控制新型冠状病毒(SARS-CoV-2)感染的全人单克隆抗体。
本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个 互补决定区CDR：
(i)分别如SEQ ID NO.:25-35所示的CDR1，
(ii)分别如SEQ ID NO.:36-45所示的CDR2，和
(iii)分别如SEQ ID NO.:46-57所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能够保留SARS-CoV-2的RBD结合亲和力的衍生序列。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区分别具有SEQ ID NO.:1-12所示的氨基酸序列。
本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
(i)分别如SEQ ID NO.:58-68所示的CDR1’，
(ii)氨基酸序列为EGT、AAS、DAS、EGS、EVS、或GAS的CDR2’，和
(iii)分别如SEQ ID NO.:69-80所示的CDR3’。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能够保留SARS-CoV-2的RBD结合亲和力的衍生序列。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区分别具有SEQ ID NO.:13-24所示的氨基酸序列。
本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第四方面所述的 轻链。
在另一优选例中，所述的抗体为特异性抗SARS-CoV-2的抗体，较佳地为特异性抗SARS-CoV-2 RBD蛋白的抗体。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体、或多克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:1所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:13所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:2所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:14所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:3所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:15所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:4所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:16所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:5所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:17所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:6所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:18所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:7所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:19所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:8所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:20所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:9所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:21所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:10所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:22所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:11所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:23所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:12所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:24所示。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:25所示的CDR1，如SEQ ID NO.:36所示的CDR2和如SEQ ID NO.:46；以及轻链可变区包含如SEQ ID NO.:58所示的 CDR1’，氨基酸序列为EGT的CDR2’和如SEQ ID NO.:69所示的CDR3’。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:26所示的CDR1，如SEQ ID NO.:37所示的CDR2和如SEQ ID NO.:47；以及轻链可变区包含如SEQ ID NO.:59所示的CDR1’，氨基酸序列为AAS的CDR2’和如SEQ ID NO.:70所示的CDR3’。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:27所示的CDR1，如SEQ ID NO.:38所示的CDR2和如SEQ ID NO.:48；以及轻链可变区包含如SEQ ID NO.:60所示的CDR1’，氨基酸序列为DAS的CDR2’和如SEQ ID NO.:71所示的CDR3’。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:28所示的CDR1，如SEQ ID NO.:39所示的CDR2和如SEQ ID NO.:49；以及轻链可变区包含如SEQ ID NO.:61所示的CDR1’，氨基酸序列为EGS的CDR2’和如SEQ ID NO.:72所示的CDR3’。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:30所示的CDR1，如SEQ ID NO.:41所示的CDR2和如SEQ ID NO.:51；以及轻链可变区包含如SEQ ID NO.:63所示的CDR1’，氨基酸序列为AAS的CDR2’和如SEQ ID NO.:74所示的CDR3’。
在另一优选例中，所述抗体的重链可变区包含如SEQ ID NO.:31所示的CDR1，如SEQ ID NO.:41所示的CDR2和如SEQ ID NO.:52；以及轻链可变区包含如SEQ ID NO.:64所示的CDR1’，氨基酸序列为GAS的CDR2’和如SEQ ID NO.:75所示的CDR3’。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签、GGGS序列、FLAG标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
在另一优选例中，所述的重组蛋白特异性结合SARS-CoV-2 RBD蛋白。
本发明的第七方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于SARS-CoV-2受体结合区域，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
本发明的第八方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第七方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第九方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或如本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或其组合，所述活性成分用于制备药剂、试剂、检测板或试剂盒。
在另一优选例中，所述试剂、检测板或试剂盒用于检测新型冠状病毒(SARS-CoV-2)。
在另一优选例中，所述药剂用于治疗或预防新型冠状病毒(SARS-CoV-2)感染。
在另一优选例中，所述的试剂包括芯片、包被抗体的免疫微粒。
本发明的第十一方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第八方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于预防和/或治疗新型冠状病毒(SARS-CoV-2)感染。
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第 三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；
(3)如本发明第七方面所述的CAR构建物。
在另一优选例中，所述的多核苷酸具有SEQ ID NO.:81-92和/或SEQ ID NO.:93-104所示的序列。
本发明的第十三方面，提供了一种载体，所述的载体含有如本发明第十二方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十三方面所述的载体或基因组中整合有如本发明第十二方面所述的多核苷酸。
本发明的第十五方面，提供了一种检测样品中新型冠状病毒(SARS-CoV-2)的方法，所述方法包括步骤：
(1)将样品与本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在SARS-CoV-2。
在另一优选例中，所述检测为非治疗非诊断目的。
本发明还提供了一种检测样品中SARS-CoV-2 RBD蛋白的方法，所述方法包括步骤：
(1)将样品与本发明的第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在SARS-CoV-2RBD蛋白。
在另一优选例中，所述检测为非治疗非诊断目的。
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第九方面所述的免疫偶联物。
本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十六方面所述的检测板。
本发明的第十八方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
本发明的第十九方面，提供了一种治疗新型冠状病毒感染的方法，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了ELISA验证12株抗体与SARS-CoV-2的结合活性。
图2显示了ELISA验证12株抗体阻断RBD与ACE2的能力。
图3显示了假病毒实验验证12株抗体对SARS-CoV-2假病毒的中和活性。
图4显示了RBD特异性抗体对SARS-CoV-2真病毒的微中和活性。
图5显示了OCTET RED 96仪器检测6个抗体对RBD的亲和力。
图6显示了ELISA表位竞争法验证12株抗体的表位识别情况。
具体实施方式
本发明人通过广泛而深入的研究，首次意外地研发了一种针对新型冠状病毒(SARS-CoV-2)的全人源单克隆抗体制备方法和多株编码抗体和抗体片段的核酸及氨基酸序列。体外假病毒和真病毒中和实验能均能证明这类抗体能有效地中和假病毒和真病毒感染的宿主细胞，而且是通过阻断SARS-CoV-2受体结合区域(RBD)与其受体宿主受体血管紧张素转化酶2(ACE2)的相互作用而发挥中和作用，并且本发明的多株抗体靶向RBD的不同区域，具有协同作用。在此基础上，完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普 通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的针对冠状病毒RBD蛋白的单克隆抗体及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、插入或缺失等突变的情况下最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
新型冠状病毒(SARS-CoV-2)
2019新型冠状病毒，2020年1月12日，世界卫生组织正式将其命名为2019-nCoV。
抗体
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA,IgD,IgE,IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1,IgG2,IgG3,IgG4,IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
如本文所用，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明还包括具有所述的SARS-CoV-2 RBD蛋白单克隆抗体的相应氨基酸序列的单克隆抗体、具有所述的SARS-CoV-2 RBD蛋白单克隆抗体可变区链的单克隆抗体，以及具有这些链的其他蛋白质或蛋白质偶联物及融合表达产物。具体地，本发明包括具有含超变区(互补决定区，CDR)的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该超变区与本发明的轻链和重链的超变区相同或至少90％同源性，较佳地至少95％同源性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与所述的SARS-CoV-2 RBD蛋白抗单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗SARS-CoV-2 RBD蛋白单克隆抗体或其片段结合的细胞表面标记物或抗原。
术语“抗体的抗原结合片段”(或简称“抗体片段”)是指抗体的保持特异性结合抗原的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗体的抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab’) 2片段，包含通过较链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体片段，如Fab或(Fab’) 2片段；抗体重链；抗体轻链。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3,4,5,6,7,8,9,10,11,12,13,14或15个连续或非连续的氨基酸。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先 确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或l0 -10M或更小的亲和力(KD)结合。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的针对SARS-CoV-2 RBD蛋白的单克隆抗体。术语“嵌合抗体(chimeric antibody)”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括如下表A所示的三个互补决定区CDR：
表A
VH CDR1 CDR2 CDR3
25-C9 GYSSTSYW(SEQ ID NO.:25) IDPSDSYT(SEQ ID NO.:36) ASSEVGYSSLREFDP(SEQ ID NO.:46)
25-G7 GGSISSSSYY(SEQ ID NO.:26) MFYSGST(SEQ ID NO.:37) ARMLGMIDV(SEQ ID NO.:47)
25-B5 GGSLSSGRYY(SEQ ID NO.:27) MYYSGS(SEQ ID NO.:38) AREPLVVPAALPPYGMDV(SEQ ID NO.:48)
28-8L GYTFSSFG(SEQ ID NO.:28) ISPYNGNT(SEQ ID NO.:39) ARDWELLGRFDY(SEQ ID NO.:49)
25-F8 GYTFTNYY(SEQ ID NO.:29) INPSGAS(SEQ ID NO.:40) ASWGGNIPLYYYYGMDV(SEQ ID NO.:50)
24-11K SGITVSSNY(SEQ ID NO.:30) VIYSGGST(SEQ ID NO.:41) ARDLSELGIDY(SEQ ID NO.:51)
26-40K SEFTVSRNY(SEQ ID NO.:31) VIYSGGST(SEQ ID NO.:41) ARDWGEYYFDY(SEQ ID NO.:52)
25-C4 SGLTVSSNY(SEQ ID NO.:32) VIYAGGST(SEQ ID NO.:42) ARESYGMDV(SEQ ID NO.:53)
25-C5 SGFIVSSNY(SEQ ID NO.:33) LIYSGGST(SEQ ID NO.:43) ARTYGDYYFDY(SEQ ID NO.:54)
25-C8 SGFTVSSNY(SEQ ID NO.:34) IIYSGGST(SEQ ID NO.:44) ARDRGGYCDY(SEQ ID NO.:55)
24-2K SGFTVSSNY(SEQ ID NO.:34) VIYSGGS(SEQ ID NO.:45) ARTFGELYIDY(SEQ ID NO.:56)
26-45K SGLIVSSNY(SEQ ID NO.:35) VIYSGGS(SEQ ID NO.:45) ARDLGPYGMDV(SEQ ID NO.:57)。
在另一优选例中，所述重链可变区的氨基酸序列分别如SEQ ID NO.:1-12所示，其中下划线标注的依次为重链可变区CDR1，CDR2，CDR3的氨基酸序列。
在另一优选例中，所述重链可变区的核酸编码序列分别如SEQ ID NO.:81(25-C9)、82(25-G7)、83(25-B5)、84(28-8L)、85(25-F8)、86(24-11K)、87(26-40K)、88(25-C4)、89(25-C5)、90(25-C8)、91(24-2K)、92(26-45K)所示。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区，所述重链恒定区可以为鼠源或人源。
如本文所用，术语“轻链可变区”与“VL”可互换使用。
在本发明的一个优选的实施方式中，根据本发明的抗体的轻链可变区，具有选自下表B的互补决定区CDR：
表B
VL CDR1’ CDR2’ CDR3’
25-C9 SSNIGSYNL(SEQ ID NO.:58) EGT CSYAGTSTFESYV(SEQ ID NO.:69)
25-G7 QGISNS(SEQ ID NO.:59) AAS QQYYSTPPLT(SEQ ID NO.:70)
25-B5 QSVSSY(SEQ ID NO.:60) DAS QQRSNWPPVT(SEQ ID NO.:71)
28-8L SSDVGNYNL(SEQ ID NO.:61) EGS CSYAGSSTWV(SEQ ID NO.:72)
25-F8 SSDVGGYNY(SEQ ID NO.:62) EVS TSYAGSNIWV(SEQ ID NO.:73)
24-11K SQGISSY(SEQ ID NO.:63) AAS QQLNSYLYT(SEQ ID NO.:74)
26-40K QTVSSSY(SEQ ID NO.:64) GAS QQYGSSPRT(SEQ ID NO.:75)
25-C4 QSISSSY(SEQ ID NO.:65) GAS QQYGSLYT(SEQ ID NO.:76)
25-C5 QGISNY(SEQ ID NO.:66) AAS QKDYSAPPGFT(SEQ ID NO.:77)
25-C8 QGFGSW(SEQ ID NO.:67) AAS QQTNSFPGYT(SEQ ID NO.:78)
24-2K QGISSY(SEQ ID NO.:68) AAS QHLRG(SEQ ID NO.:79)
26-45K QGISSY(SEQ ID NO.:68) AAS QQLNSYPT(SEQ ID NO.:80)。
在另一优选例中，所述轻链可变区的氨基酸序列分别如SEQ ID NO.:13-24所示，其中双下划线标注的依次为轻链可变区CDR1’，CDR2’，CDR3’的氨基酸序列。
在另一优选例中，所述轻链可变区的核酸编码序列分别如SEQ ID NO.:93(25-C9)、94(25-G7)、95(25-B5)、96(28-8L)、97(25-F8)、98(24-11K)、99(26-40K)、100(25-C4)、101(25-C5)、102(25-C8)、103(24-2K)、104(26-45K)所示。
在本发明的一个优选的实施方式中，所述抗体的轻链包括上述轻链可变区和轻链恒定区，所述轻链恒定区可以为鼠源或人源。
本发明抗体的功能是由此抗体轻链和重链可变区基因特异性基因序列决定，可以特异性的结合SARS-CoV-2的RBD蛋白，能够阻止SARS-CoV-2侵染易感细胞。利用此抗体可变区基因或互补决定区(CDR)基因，可在利用原核和真核细胞的任何表达系统中改造和生产不同形式的基因工程抗体。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合SARS-CoV-2 RBD蛋白的抗体，例如具有重链可变区(如SEQ ID NO.: 81-92所示的核苷酸序列编码的氨基酸序列)和/或轻链可变区(如SEQ ID NO.:93-104所示的核苷酸序列编码的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
在另一优选例中，所述的抗体为抗SARS-CoV-2 RBD蛋白的鼠或人鼠嵌合单克隆抗体，它的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地，所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或轻链恒定区。
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链和/或轻链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的单克隆抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有SARS-CoV-2 RBD蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱 导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约60个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表C进行氨基酸替换而产生。
表C
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:81-104所示的编码区序列相同或者是简并的变异体。如本文所用，“简并的变异体”在本发明中是指编码具有与本发明的多肽相同的氨基酸序列，但与SEQ ID NO.:81-104所示的编码区序列有差别的核酸序列。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与SEQ ID NO.:1-12和/或SEQ ID NO.:13-24所示的成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如 果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可偶联的治疗剂包括但不限于：胰岛素、IL-2、干扰素、降钙素、GHRH肽、肠肽类似物、白蛋白、抗体片段、细胞因子、和激素。
此外还可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：口服、呼吸道、瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合新型冠状病毒RBD蛋白分子，因而可用于延长药物的半衰期，此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约10毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明的主要优点
(1)本发明的全人单克隆抗体能够特异性识别并结合冠状病毒SARS-2-CoV RBD蛋白，有效地阻断RBD与ACE2的结合，对冠状病毒SARS-2-CoV具有很高的中和活性。
(2)本发明的全人单克隆抗体能够识别冠状病毒SARS-2-CoV RBD蛋白的不同表位，靶向两个不同表位的单抗组合具有协同效应，使用靶向不同表位的抗体组合可以减少耐药的发生。
(3)本发明分离到的不同的抗体可以组成鸡尾酒疗法，增强抗病毒效果并能解决因为病毒不断突变而形成的耐药问题，同时也可以作为基础科研和临床上的诊断工具。
(4)本发明是全人单克隆抗体，不含鼠源部分，对人体来说具有更低的免疫原性和更高的安全性，可以避免人抗鼠等其它物种来源的抗体介导免疫排斥反应。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明实施例或测试例中未注明具体条件的实验，通常按常规条件进行，或按照原料/商品制造商建议的条件；未注明具体来源的试剂，为市场购买的常规试剂。
以下说明本发明能够中和冠状病毒RBD蛋白不同表位的全人单克隆抗体制备以及抗体特性分析过程。
实施例1单细胞RT-PCR法获得抗体基因以及抗体表达
1.1外周血单核细胞(PBMC)的获得
从健康的志愿者体内抽取外周血，采用常规Ficoll-Paque(厂家为
Figure PCTCN2021142693-appb-000001
(CEDARLANE)公司)密度梯度离心，得到10 7以上个外周血单核细胞(PBMC)。
Ficoll分离方法：
(1)收集血液，于50ml离心管(预含4％柠檬酸钠1ml)，收集全血20ml，颠倒混匀8-10次。(即使柠檬酸钠终浓度为0.4％)；
(2)加等体积RPMI1640(含柠檬酸钠)，混匀；
(3)用15ml透明离心管，铺3ml淋巴细胞分离液，在其上小心加6ml血样。形成分离界面(或4ml分离液加8ml血样)；
(4)室温离心800g，20min(2000rpm，20min)；
(5)小心吸取界面层细胞，转移至新管；
(6)加RPMI1640(含柠檬酸钠)，稀释减小液体密度。离心，800g/2000rpm，10min。去上清；
(7)RPMI1640洗细胞2-3次，备用
1.2新冠病毒特异性记忆B细胞的获得
使用FITC-CD19/APC-IgG/BV421和SARA-CoV-2 RBD蛋白为标志物，BD Horizon TM Fixable Viability Stain 780去掉死细胞，经流式细胞仪获得特异性B细胞至96孔RT-PCR板，每孔一个细胞，获得SARA-CoV-2 RBD蛋白特异性记忆B细胞。
(1)RBD蛋白由哺乳动物细胞CHO表达系统表达；参考Invitrogen ExpiCHO-S TM Expression System手册；RBD蛋白序列参考nCoV-SH01(GenBank:MT121215.1)，在上海捷瑞公司进行全基因合成。在上海捷瑞公司进行全基因合成，构建到invitrogen pcDNA3.1的表达载体上；
(2)RBD蛋白进行生物素(Biotin)标记：No-Weigh Sulfo-NHS-LC-Biotin(购自PIERCE，参考PIERCE公司EZ-Link Sulfo-NHS-LC-Biotin生物素标记Protocol)10mM试剂；另两个标志物FITC-CD19和APC-IgG均购自BD Bioscience公司；SA Streptavidin PE和streptavidin BV 421(购自BD公司)用来检测生物素标记的SARA-CoV-2 RBD蛋白；
(3)分选细胞的标记：PBMC细胞分组，实验组+对照组，按细胞数加入标志物，避光染色，进行标记，用PBS重悬后，使用40μm BD falcon滤膜过滤；
(4)特异性B细胞的分选：使用BD FACS Influx筛选，根据前向角和侧向角从PBMC中筛选到淋巴细胞，然后通过不同对照组的调节补偿，获得SARA-CoV-2 RBD蛋白的特异性记忆B细胞，将其分选到96孔板中进行RT-PCR(反转录PCR)，每孔一个细胞，板置于干冰上。
1.3抗体基因获得及载体构建
将获得的单个记忆B细胞通过RT-PCR获得cDNA,然后通过nested-PCR获得抗体基因可变区，跑琼脂糖核酸凝胶，将重轻链可以配对的胶块回收并测序。通过IgBLAST网(https://www.ncbi.nlm.nih.gov/projects/igblast/)检索获得抗体基因序列。接下来通过AgeI及SalI酶切位点，AgeI及BsiwI酶切位点，AgeI及XhoI酶切位点将抗体基因分别连接到相应的IgH，Igκ和Igλ表达载体上。全人抗体表达载体IgH，Igκ和Igλ(分别表 达抗体heavy链、kappa链、lambda链)由Patrick Wilson实验室馈赠，载体序列见NCBI GenBank:FJ475055、FJ475056和FJ517647。
1.4抗体表达和纯化
瞬时转染CHO细胞，进行全人抗体表达。转染前一天(第-1天)，分种ExpiCHO-S TM细胞，最终密度为3×10 6–4×10 6个活细胞/mL，使细胞过夜生长。次日(第0天)，测定活细胞密度和存活率百分比。细胞密度应达到约7×10 6–10×10 6个活细胞/mL。存活率应为95–99％，方可继续转染。将细胞稀释至最终密度为6×10 6个活细胞/mL。使用预冷的试剂(4℃)配制Expi Fectamine TM CHO/质粒DNA复合物。室温孵育Expi Fectamine TM CHO/质粒DNA复合物1–5分钟，然后将溶液慢慢转移至CHO细胞培养瓶中，在添加过程中轻轻晃动培养瓶。将细胞置于轨道摇床上(37℃培养箱含8％CO 2的湿化空气条件下)培养。培养7-11天，待细胞死亡一半时即可收上清，开始纯化抗体。
使用Protein G Agarose 4FF填料(购自GE)纯化抗体。首先将收集的CHO细胞悬液4000rpm,4℃离心30min，将收集的上清再用0.45um filter过滤，待纯化。取重力型离心柱，加入Protein G Agarose 4FF填料，使用3倍柱体积的20％乙醇稳定填料，之后用5倍柱体积的结合缓冲液平衡柱子，然后上样品，再用10倍柱体积的结合缓冲液平衡柱子，最后用3倍柱体积的洗脱缓冲液洗脱柱子，向洗脱下来的抗体溶液中加入中和buffer使洗脱下来的样品pH达7.5左右。将抗体溶液在5L 1×PBS中透析3次，即可将抗体浓缩保存至-80℃。
实验结果：
单细胞RT-PCR和Nested-PCR法获得匹配抗体重轻链可变区基因。琼脂糖割胶回收并进行测序。将测序结果在http://www.ncbi.nlm.nih.gov/igblast及http://www.imgt.org/进行比对，获得抗体胚系基因信息和抗体重轻链基因高可变区信息，并进行表达载体构建及后续表达纯化。最后，通过该技术，成功获得多株中和新冠病毒病毒的全人单克隆抗体。
本发明的全人抗体重链可变区氨基酸序列如下，其中使用下划线标注的依次为重链氨基酸CDR1，CDR2，CDR3序列。
25-C9重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000002
25-G7重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000003
25-B5重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000004
28-8L重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000005
25-F8重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000006
24-11K重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000007
26-40K重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000008
25-C4重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000009
25-C5重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000010
25-C8重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000011
24-2K重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000012
Figure PCTCN2021142693-appb-000013
26-45K重链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000014
本发明的全人抗体轻链可变区氨基酸序列如下，其中使用下划线标注的依次为轻链氨基酸CDR1’，CDR2’和CDR3’序列。
25-C9轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000015
25-G7轻链可变区氨基酸序列:
Figure PCTCN2021142693-appb-000016
25-B5轻链可变区氨基酸序列:
Figure PCTCN2021142693-appb-000017
28-8L轻链可变区氨基酸序列:
Figure PCTCN2021142693-appb-000018
25-F8轻链可变区氨基酸序列:
Figure PCTCN2021142693-appb-000019
24-11K轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000020
26-40K轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000021
25-C4轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000022
Figure PCTCN2021142693-appb-000023
25-C5轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000024
25-C8轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000025
24-2K轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000026
26-45K轻链可变区氨基酸序列：
Figure PCTCN2021142693-appb-000027
实施例2抗体特性分析
实验方法：
(1)ELISA检测抗体结合抗原的活性
使用ELISA检测表达的抗体是否识别RBD蛋白。包被RBD蛋白ELISA板，0.5μg/mL，每孔100μL，4℃过夜。次日PBST洗板3次。2％BSA封闭,每孔200μL，37℃,2h。再次PBST洗板3次。RBD特异性抗体测试6个浓度，3倍梯度稀释，2复孔，起始测试浓度为30μg/ml。上样，每孔100μL，37℃，2h。PBST洗板3次。Goat Anti-Human IgG(Fc specific)-Peroxidase antibody(sigma)，1:5000稀释，每孔100μL，37℃，1h。PBST洗板3次。加底物TMB 100μL/孔显色，若颜色较浅可37℃避光反应15min。加入2M H 2SO 4终止反应，每孔50μL。测定OD 450，并进行数据处理。
(2)受体阻断实验
包被5ug/ml的hACE2蛋白过夜；PBST洗板3次，2％BSA buffer封闭2h；不同梯度稀释的抗体与150ng/ml的生物素化的SARS-CoV-2 RBD蛋白孵育2h，不加抗体作为对照；加入预包被hACE2的微孔板中，孵育2h,PBST洗版三次，SA-HRP 40稀释孵育30min；TMB底物显色，检测OD450读值。阳性对照组是不加block抗体，但是加第二个检测抗体。
(3)ELISA表位竞争实验
96孔板包被0.5μg/ml重组SARS-CoV-2 RBD蛋白，4℃冰箱过夜，50μg/ml的 阻断抗体加入微孔板中，37℃孵育2h，随后加入biotin标记的检测抗体，37℃孵育2h，PBST洗涤3次。二抗加入HRP标记的SA(1:200)，37℃孵育1h，洗涤3次，TMB显色，室温避光反应2min，1M HCl终止反应后，酶标仪测定样品在A450的吸光值。
(4)BLI亲和力检测
本实验在生化细胞研究所的分子平台使用OCTET RED 96仪器完成。首先将AHC sensor放到有PBS的96孔黑板中浸泡至少10min，加样不同RBD抗体，浓度为20ug/ml；抗原设置浓度梯度，2倍梯度稀释，200nM-100nM-50nM-25nM-12.5nM-6.25nM；将抗体抗原及再生buffer及PBS buffer加到另一块黑板中，置于仪器上；设定程序，开始运行，程序为：baseline 120s→loading Ab→300s-baseline→240s-association→900s-disassociation→900s-regenaration→5s-baseline→5s-regenaration→5s-baseline→5s-regenaration→5s-baseline→5s；软件分析数据，计算抗原抗体亲和力KD值。
(5)假病毒中和实验
全长SARS-2-CoV S蛋白质粒和pNL4-3的质粒共转染HEK293T细胞于10cm培养皿中，6小时换液，48小时后收集上清液，使用完全培养基稀释。将抗体梯度稀释，并与等体积的病毒混合，37℃下培养1h。将抗体和病毒混合物转移到稳定表达的人ACE2的HEK293T细胞中。37℃下培养48小时。去掉上清，向细胞中加入裂解缓冲液，充分裂解。检测荧光素酶活性(Promega)。通过比较实验组的荧光素酶值与仅病毒对照组的荧光素酶值来计算中和效率。计算公式如下：
抑制活性百分比(％)＝(病毒对照平均值-测试孔读值)/(病毒对照平均值-细胞对照平均值)×100；
IC 50值通过Prism软件计算。
(6)真病毒微中和实验
接种细胞：取处于对数生长期的Vero-E6细胞，接种到96孔板中，每孔100μl，每孔细胞4×10 4个。
中和实验：供试品稀释：如表1所示，第1列(B-G行)加入10μg/ml预稀释样品60μl，加入病毒稀释液60μl后抗体终浓度为5μg/ml)，其余孔加入依次3倍稀释的样品60μl。第1行为细胞对照(Cell Control,CC)加入无血清培养基120μl，第8行为病毒对照(Virus Control,VC)加入无血清培养基60μl。
表1. 96孔板加样方法
Figure PCTCN2021142693-appb-000028
病毒稀释：病毒储存液滴度为2.5×10 5TCID50/ml，取病毒储存液200μl，加入25ml无血清培养基，充分混匀，将病毒稀释至100TCID50/50μl。
滴加病毒：垂直悬滴病毒(细胞对照除外)至96孔板内，加样体积60μl/孔，最终病毒-抗体混合液120μl。
中和：将加好样的细胞培养板在震荡器上混匀，置于37℃培养箱，中和1小时。中和结束后，吸去接种有细胞的培养板上清液，随后将病毒-血清混合液吸取100μl/孔加入其中，放入37℃CO 2培养箱中培养1小时，进行感染。病毒感染结束后，吸去培养板上清液，空斑形成实验样品加入含有1％的甲基纤维素的维持培养基(DMEM培养基含有2％FBS)，放入37℃CO 2培养箱中培72-96小时。用倒置显微镜于4天时观察并记录结果，弃去上清，空斑形成实验样品经甲醛固定后，结晶紫染色空斑计数分析。对于测试100&抑制率，用倒置显微镜于2天时观察CPE并记录结果。
实验结果：
2.1 ELISA验证分离抗体对新冠病毒RBD的结合活性
为了研究RBD特异性的抗体是否能结合SARS-2-CoV S蛋白的RBD区域，用ELISA的方法验证重组表达抗体对SARS-CoV-2蛋白RBD的结合活性。结果如图1所示,有12株抗体对SARS-CoV-2 RBD蛋白有较高的结合活性。
2.2 RBD特异性的抗体有效阻断RBD与受体ACE2的结合
为了进一步确认RBD特异性的抗体是否有效阻断RBD与受体ACE2的结合，在96微孔板上包被ACE2-FC重组蛋白，将不同梯度稀释的抗体与生物素化的RBD蛋白孵育后加入到预包被的ACE2-FC的微孔板上。用HRP偶联的链霉亲和素进行检测。结果如图2所示，12株RBD特异性的抗体均能有效地抑制RBD与ACE2的结合，并呈现剂量依赖效应。
2.3假病毒实验确认RBD特异性抗体中和活性
为了进一步确认12株RBD特异性抗体的功能，包装了SARS-CoV-2的假病毒，在稳转人ACE2的293T细胞上进行SARS-CoV-2假病毒中和实验，实验表明，12株抗体 以不同能力中和假病毒，半数有效抑制浓度IC 50在1-50ng/ml之间(图3)。
2.4真病毒实验验证RBD特异性抗体的中和活性
为了进一步确认25-C9，26-40K等抗体对SARS-CoV-2真病毒中和活性，在BSL-3实验室进行SARS-CoV-2真病毒中和活性检测。结果表明，检测的6株抗体可以有效抑制SARS-CoV-2真病毒，IC 50在26-150ng/ml之间(图4)。
2.5 OCTET RED 96仪器检测RBD特异性抗体对RBD蛋白的亲和力
为了研究25-C9，24-11K等抗体对RBD的亲和力，使用OCTET RED 96仪器检测6个抗体对RBD的亲和力。结果显示6株抗体对SARS-CoV-2 RBD呈现较高的亲和力，在10 -8-10 -10M之间(图5)。
2.6表位竞争实验
通过ELISA表位竞争实验，将12株中和抗体分成两个独立的表位。其中26-40K，24-11K，25-C4，25-C8，26-45K，24-2K，25-G7，28-8L及25-B5相互之间存在竞争，提示这些抗体识别的表位有重叠或者存在空间上的位阻，此外，25-C9与以上的抗体不存在表位竞争，说明25-C9与第一类表位空间距离较远，属于独立的表位。25-F8抗体与26-40K，24-11K，25-C4，25-C8，26-45K，24-2K，28-8L之间不存在表位竞争关系，但是与25-C9，25-B5，25-G7存在竞争关系，提示25-F8与这三株抗体存在部分表位重叠(如图6所示)。使用靶向不同表位的抗体组合可以减少耐药的发生。同时，靶向两个不同表位的单抗组合的另一优势是它们往往有协同效应。本发明分离到的不同的抗体可以组成鸡尾酒疗法，增强抗病毒效果并能解决因为病毒不断突变而形成的耐药问题。
发明人利用单细胞RT-PCR技术从新冠感染的康复志愿者的外周血PBMC中分离到多株中和抗体，这些抗体经过了体细胞高频突变的过程，对于新冠的RBD蛋白具有较高的亲和力，均能有效地阻断RBD与ACE2的结合。体外实验证实验证实多株抗体均能有效的抑制SARS-CoV-2假病毒和真病毒感染宿主细胞，表现最佳的抗体IC 50在纳克级别。此外。这些抗体的表位具有多样性，不同抗体可以组合，应对因为SARS-CoV-2突变较快形成的耐药问题，本发明的靶向新冠的全人中和抗体对预防和治疗SARS-CoV-2具有潜在的应用价值。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Figure PCTCN2021142693-appb-000029
Figure PCTCN2021142693-appb-000030

Claims (3)

  1. 要求4所述的轻链、或如权利要求5所述的抗体、如权利要求9所述的重组蛋白、或其组合，其特征在于，所述活性成分用于制备药剂、试剂、检测板或试剂盒。
  2. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求9所述的重组蛋白、如权利要求11所述的免疫细胞、如权利要求12所述的抗体药物偶联物、或其组合；以及
    (ii)药学上可接受的载体。
  3. 一种治疗新型冠状病毒感染的方法，所述方法包括：给需要的对象施用如权利要求5所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
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