WO2021218947A1 - 一种抗新型冠状病毒的单克隆抗体及其应用 - Google Patents

一种抗新型冠状病毒的单克隆抗体及其应用 Download PDF

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WO2021218947A1
WO2021218947A1 PCT/CN2021/090146 CN2021090146W WO2021218947A1 WO 2021218947 A1 WO2021218947 A1 WO 2021218947A1 CN 2021090146 W CN2021090146 W CN 2021090146W WO 2021218947 A1 WO2021218947 A1 WO 2021218947A1
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seq
sequence
binding unit
abu
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French (fr)
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谢晓亮
曹云龙
孙文洁
张旭
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北京大学
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Priority to US17/921,965 priority Critical patent/US20230174628A1/en
Priority to CN202180031766.4A priority patent/CN115461364A/zh
Publication of WO2021218947A1 publication Critical patent/WO2021218947A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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

  • This article relates to the field of immunology and molecular virology, especially the diagnosis, prevention and treatment of the new coronavirus. Specifically, this article relates to antibodies against the novel coronavirus, and compositions containing the antibodies (for example, diagnostic agents and therapeutic agents). In addition, this article also relates to the use of the antibody.
  • the antibodies described herein can be used to diagnose, prevent, and/or treat a novel coronavirus infection and/or diseases caused by the infection (for example, novel coronavirus pneumonia).
  • coronavirus SARS-CoV-2 is the pathogen that causes the new type of coronavirus pneumonia (COVID-19). It is a single-stranded RNA virus. It is the same as the severe acute respiratory syndrome coronavirus (SARS-CoV ) And the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) that caused the outbreak in 2012 belong to the Coronavirus family (Coronaviridae). Coronavirus particles are round or elliptical, and also pleomorphic, with a diameter of 50-200nm, which is a relatively large virus. Coronavirus is an enveloped virus.
  • a lipid envelope is wrapped around the virus capsid, and a wide spike protein (Spike, S protein, SEQ ID No: 1460) is arranged on it, which is shaped like a sun halo.
  • Spike S protein, SEQ ID No: 1460
  • RBD receptor binding domain
  • ACE2 Enzyme 2
  • an antigen binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, the The light chain variable region includes VL CDR1, VL CDR2, and VL CDR3; wherein the VH CDR3 includes a sequence selected from SEQ ID NO: 1-360 and 2971-3005 or is similar to SEQ ID NO: 1-360 and 2971-3005 Compared with a sequence comprising one or more amino acid additions, deletions, or substitutions, and/or wherein the VL CDR3 comprises a sequence selected from SEQ ID NO: 361-720 and 3076-3110 or a sequence with SEQ ID NO: 361-720 and Compared with 3076-3110, a sequence containing one or more amino acid additions, deletions, or substitutions.
  • the antigen binding unit is less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM,
  • KD equilibrium dissociation constant
  • the equilibrium dissociation constant (KD) of less than 0.05 nM or less than 0.01 nM binds to the receptor binding domain (RBD) of the S protein of the new coronavirus (SARS-CoV-2).
  • the antigen binding unit is less than 20 ⁇ g/ml, less than 10 ⁇ g/ml, less than 9 ⁇ g/ml, less than 8 ⁇ g/ml, less than 7 ⁇ g/ml, less than 6 ⁇ g/ml, less than 5 ⁇ g/ml, less than 4 ⁇ g /ml, less than 3 ⁇ g/ml, less than 2 ⁇ g/ml, less than 1 ⁇ g/ml, less than 0.5 ⁇ g/ml, less than 0.25 ⁇ g/ml, less than 0.2 ⁇ g/ml, less than 0.1 ⁇ g/ml, less than 0.05 ⁇ g/ml, or less than The IC50 of 0.001 ⁇ g/ml neutralizes the new coronavirus (SARS-CoV-2).
  • SARS-CoV-2 new coronavirus
  • the VH CDR1 of the antigen-binding unit comprises a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935 or comprises one or more sequences compared with SEQ ID NO: 1461-1820 and 2901-2935 A sequence of multiple amino acid additions, deletions, or substitutions.
  • the VH CDR1 of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935.
  • the VH CDR1 of the antigen-binding unit comprises an amino acid that contains 5, 4, 3, 2 or 1 amino acid additions, deletions, or substitutions compared with SEQ ID NOs: 1461-1820 and 2901-2935 sequence.
  • the VH CDR1 of the antigen binding unit includes the same sequence as the CDR1 included in SEQ ID NOs: 721-1080 and 3111-3145.
  • the VH CDR2 of the antigen binding unit includes a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970 or includes one or more sequences compared with SEQ ID NO: 1821-2180 and 2936-2970. A sequence of multiple amino acid additions, deletions, or substitutions.
  • the VH CDR2 of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970.
  • the VH CDR2 of the antigen-binding unit includes an amino acid that contains 5, 4, 3, 2 or 1 amino acid additions, deletions, or substitutions compared with SEQ ID NOs: 1821-2180 and 2936-2970. sequence.
  • the VH CDR2 of the antigen binding unit comprises the same sequence as the CDR2 contained in SEQ ID NOs: 721-1080 and 3111-3145.
  • the VL CDR1 of the antigen binding unit comprises a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040 or comprises one or more sequences compared with SEQ ID NO: 2181-2540 and 3006-3040. A sequence of multiple amino acid additions, deletions, or substitutions.
  • the VL CDR1 of the antigen binding unit comprises a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040.
  • the VL CDR1 of the antigen-binding unit includes an amino acid that contains 5, 4, 3, 2 or 1 amino acid addition, deletion, or substitution compared with SEQ ID NO: 2181-2540 and 3006-3040. sequence.
  • the VL CDR1 of the antigen binding unit includes the same sequence as the CDR1 included in SEQ ID NOs: 1081-1440 and 3146-3180.
  • the VL CDR2 of the antigen binding unit includes a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075 or includes one or more sequences compared with SEQ ID NO: 2541-2900 and 3041-3075. A sequence of multiple amino acid additions, deletions, or substitutions.
  • the VL CDR2 of the antigen binding unit comprises a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075.
  • the VL CDR2 of the antigen-binding unit includes an amino acid addition, deletion, or substitution of 5, 4, 3, 2 or 1 compared with SEQ ID NO: 2541-2900 and 3041-3075. sequence.
  • the VL CDR2 of the antigen binding unit includes the same sequence as the CDR2 included in SEQ ID NOs: 1081-1440 and 3146-3180.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145 or contains one or more sequences compared with SEQ ID NO: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 compared with SEQ ID NOs: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145 that has at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180 or contains one or more sequences compared with SEQ ID NO: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 compared with SEQ ID NOs: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180 that has at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • an antigen binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, so
  • the light chain variable region includes VL CDR1, VL CDR2, and VL CDR3;
  • the VH CDR1 includes a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935, and SEQ ID NO: 1461-1820 and 2901-2935 Compared with a sequence containing one or more amino acid additions, deletions, or substitutions, or the same sequence as the CDR1 contained in SEQ ID NO: 721-1080 and 3111-3145
  • the VH CDR2 includes a sequence selected from SEQ ID NO :1821-2180 and 2936-2970 sequence, compared with SEQ ID NO: 1821-2180 and 2936-2970 containing one or more amino acid additions, deletions, or substitutions, or with SEQ ID NO: 721-10
  • an antigen binding unit comprising a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3, so
  • the light chain variable region includes VL CDR1, VL CDR2, and VL CDR3;
  • the VH CDR1 includes a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935 or a sequence that is identical to SEQ ID NO: 1461-1820 and 2901-2935 Compared with a sequence comprising one or more amino acid additions, deletions, or substitutions
  • the VH CDR2 comprises a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970 or a sequence with SEQ ID NO: 1821-2180 and 2936 Compared with 2970, a sequence comprising one or more amino acid additions, deletions, or substitutions
  • the VH CDR3 comprises a sequence selected from SEQ ID NO: 1-360 and 2971
  • the VL CDR2 contains a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075 or is compared with SEQ ID NO: 2541 Compared with 3041-3075, the VL CDR3 contains a sequence of one or more amino acid additions, deletions, or substitutions.
  • the VL CDR3 includes a sequence selected from SEQ ID NO: 361-720 and 3076-3110 or a sequence that is different from SEQ ID NO: 361. Compared with 3076-3110, -720 contains one or more amino acid additions, deletions, or substitutions.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145 or contains one or more sequences compared with SEQ ID NO: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 compared with SEQ ID NOs: 721-1080 and 3111-3145.
  • the VH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 721-1080 and 3111-3145 that has at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180 or contains one or more sequences compared with SEQ ID NO: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 compared with SEQ ID NOs: 1081-1440 and 3146-3180.
  • the VL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1081-1440 and 3146-3180 that has at least 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the antigen binding unit is less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM.
  • KD equilibrium dissociation constant
  • the equilibrium dissociation constant (KD) of less than 0.05nM, or less than 0.01nM binds to the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) S protein.
  • the antigen binding unit is less than 20 ⁇ g/ml, less than 10 ⁇ g/ml, less than 9 ⁇ g/ml, less than 8 ⁇ g/ml, less than 7 ⁇ g/ml, less than 6 ⁇ g/ml, less than 5 ⁇ g/ml, less than 4 ⁇ g /ml, less than 3 ⁇ g/ml, less than 2 ⁇ g/ml, less than 1 ⁇ g/ml, less than 0.5 ⁇ g/ml, less than 0.25 ⁇ g/ml, less than 0.2 ⁇ g/ml, less than 0.1 ⁇ g/ml, less than 0.05 ⁇ g/ml, or less than The IC50 of 0.001 ⁇ g/ml neutralizes the new coronavirus (SARS-CoV-2).
  • SARS-CoV-2 new coronavirus
  • an antigen binding unit which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a compound selected from SEQ ID NO: 721-1080 and 3111
  • the sequence of 3145 has a sequence of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity, and / Or wherein the VL contains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 99% identity sequence.
  • the antigen binding unit is less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM.
  • KD equilibrium dissociation constant
  • the equilibrium dissociation constant (KD) of less than 0.05nM, or less than 0.01nM binds to the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) S protein.
  • the antigen binding unit is less than 20 ⁇ g/ml, less than 10 ⁇ g/ml, less than 9 ⁇ g/ml, less than 8 ⁇ g/ml, less than 7 ⁇ g/ml, less than 6 ⁇ g/ml, less than 5 ⁇ g/ml, less than 4 ⁇ g /ml, less than 3 ⁇ g/ml, less than 2 ⁇ g/ml, less than 1 ⁇ g/ml, less than 0.5 ⁇ g/ml, less than 0.25 ⁇ g/ml, less than 0.2 ⁇ g/ml, less than 0.1 ⁇ g/ml, less than 0.05 ⁇ g/ml, or less than The IC50 of 0.001 ⁇ g/ml neutralizes the new coronavirus (SARS-CoV-2).
  • SARS-CoV-2 new coronavirus
  • the antigen binding unit further comprises a heavy chain constant region (CH).
  • the CH of the antigen binding unit includes the sequence of SEQ ID NO: 1457 or a sequence of one or more amino acid additions, deletions, or substitutions compared with SEQ ID NO: 1457.
  • the CH of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1457.
  • the CH of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid addition, deletion, Or substituted sequence.
  • the CH of the antigen binding unit includes a sequence selected from SEQ ID NO: 1457 that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the antigen binding unit further comprises a light chain constant region (CL).
  • the CL of the antigen binding unit includes the sequence of SEQ ID NO: 1458 or a sequence of one or more amino acid additions, deletions, or substitutions compared with SEQ ID NO: 1458.
  • the CL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1458.
  • the CL of the antigen binding unit includes 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid addition, deletion, Or substituted sequence.
  • the CL of the antigen binding unit comprises a sequence selected from SEQ ID NO: 1458 that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • nucleic acid molecule that encodes an antigen binding unit as described herein as defined above.
  • a vector comprising an isolated nucleic acid molecule as defined above.
  • the vector described herein can be a cloning vector or an expression vector.
  • the vectors described herein are, for example, plasmids, cosmids, or bacteriophages and the like.
  • a host cell comprising the isolated nucleic acid molecule or vector described herein.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the cells described herein can also be cell lines, such as HEK293 cells.
  • a method for preparing the antigen-binding unit described herein which comprises culturing the host cell described herein under suitable conditions, and recovering the antigen-binding unit described herein from the cell culture.
  • composition comprising the antigen binding unit, isolated nucleic acid molecule, vector or host cell as described above.
  • kits comprising the antigen binding unit described herein.
  • the antigen binding unit described herein further includes a detectable label.
  • the kit further includes a second antibody, which specifically recognizes the antigen binding unit described herein.
  • the second antibody further includes a detectable label.
  • detectable labels are well known to those skilled in the art, and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances, enzymes (such as horseradish peroxidase) and the like.
  • this document provides a method for detecting the presence or level of a novel coronavirus or its S protein or S protein RBD in a sample, which includes using the antigen binding unit described herein.
  • the antigen binding unit described herein further includes a detectable label.
  • the method further comprises using a second antibody carrying a detectable label to detect the antigen binding unit described herein.
  • the method can be used for diagnostic purposes (for example, the sample is a sample from a patient), or for non-diagnostic purposes (for example, the sample is a cell sample, not a sample from a patient).
  • this document provides a method for diagnosing whether a subject is infected with a novel coronavirus, which includes: using the antigen binding unit described herein to detect whether the novel coronavirus or its S protein or RBD of the S protein is derived from the subject. Existence in the subject’s sample.
  • the antigen binding unit described herein further includes a detectable label.
  • the method further comprises using a second antibody carrying a detectable label to detect the antigen binding unit described herein.
  • the antigen-binding unit described herein in the preparation of a kit for detecting the presence or level of a novel coronavirus or its S protein or RBD in a sample, Or used to diagnose whether the subject is infected with the new coronavirus.
  • composition comprising the antigen binding unit described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • this document provides a method for neutralizing the virulence of a novel coronavirus in a sample, which includes contacting a sample containing the novel coronavirus with the antigen binding unit described herein.
  • Such methods can be used for therapeutic purposes, or non-therapeutic purposes (for example, the sample is a cell sample, not a patient or a sample from a patient).
  • an antigen binding unit as described above which is used to neutralize the virulence of the novel coronavirus in a sample.
  • the use of the antigen binding unit described herein in the preparation of a pharmaceutical composition for the prevention or treatment of a novel coronavirus infection in a subject or a disease related to a novel coronavirus infection E.g. new coronavirus pneumonia.
  • the antigen binding unit as described above which is used to prevent or treat a novel coronavirus infection in a subject or a disease related to a novel coronavirus infection (for example, novel coronavirus pneumonia).
  • this article provides a method for preventing or treating a novel coronavirus infection or a novel coronavirus infection-related disease (such as novel coronavirus pneumonia) in a subject, which includes, to a subject in need A prophylactic or therapeutically effective amount of the antigen binding unit described herein or the pharmaceutical composition described herein is administered.
  • the subject is a mammal, such as a human.
  • the antigen binding unit described herein or the pharmaceutical composition described herein can be administered to a subject by any appropriate route of administration.
  • administration routes include, but are not limited to, oral, buccal, sublingual, topical, parenteral, rectal, intrathecal, or nasal routes.
  • the drugs and pharmaceutical compositions provided herein can be used alone or in combination, and can also be used in combination with other pharmaceutically active agents (for example, antiviral drugs, such as favipiravir, remdesivir, interferon, etc.).
  • the pharmaceutical composition further contains a pharmaceutically acceptable carrier and/or excipient.
  • a conjugate comprising an antigen binding unit as described above, wherein the antigen binding unit is conjugated to a chemically functional moiety.
  • the chemically functional moiety is selected from radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors, and inhibitors.
  • Figures 1A-1C exemplarily show the SDS-PAGE detection results of the antigen binding units ABU-174, ABU-175 and ABU190.
  • Figures 2A-2E exemplarily show the use of SPR technology to detect the affinity of antigen-binding units ABU-174 (A), ABU-175 (B), ABU190 (C), ABU297 (D), and ABU367 (E) with S protein The measurement results.
  • Figures 3A-3C exemplarily show the assay results of the neutralization inhibitory activity of the antigen binding units ABU-174 (A), ABU-175 (B), and ABU190 (C) against SARS-CoV-2 pseudovirus.
  • Figure 4 exemplarily shows the CPE assay result of the neutralization inhibitory activity of the ABU-175 antibody against SARS-CoV-2 true virus.
  • Figure 5 exemplarily shows the PRNT assay results of the neutralization inhibitory activity of the antigen binding units ABU-174, ABU-175, and ABU190 against SARS-CoV-2 true virus.
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear, cyclic or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • amino acid polymers that have been modified, such as by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic treatment, phosphorylation, iso Pennylation, racemization, selenization, transfer RNA-mediated addition of amino acids to proteins (such as arginination), ubiquitination, or any other manipulations, such as conjugation with labeled components.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polypeptide or amino acid sequence "derived" from a given protein refers to the origin of the polypeptide.
  • the polypeptide has an amino acid sequence substantially identical to the amino acid sequence of the polypeptide encoded in the sequence, or a part thereof, wherein the part consists of at least 10-20 amino acids or at least 20-30 amino acids or at least 30-50 amino acids, or It can be identified immunologically with the polypeptide encoded in the sequence.
  • the term also includes polypeptides expressed from a designated nucleic acid sequence.
  • domain refers to a part of a protein that is physically or functionally distinguished from other parts of the protein or peptide.
  • Physically defined domains include amino acid sequences that are extremely hydrophobic or hydrophilic, such as those membrane-bound or cytoplasmic-bound sequences.
  • the domain can also be defined by internal homology caused by gene duplication, for example.
  • Functionally defined domains have different biological functions.
  • the antigen-binding domain refers to the part of the antigen-binding unit or antibody that binds to the antigen.
  • the functionally defined domain does not need to be encoded by a continuous amino acid sequence, and the functionally defined domain may contain one or more physically defined domains.
  • amino acid refers to natural and/or unnatural or synthetic amino acids, including but not limited to D or L optical isomers, as well as amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to designate amino acids. In this context, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain).
  • Antibody light chains can be classified into kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the isotype of the antibody is defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region is composed of 3 domains (CH1, CH2, and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of a domain CL.
  • the constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (C1q) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions with hyperdenaturation (called complementarity determining regions (CDR)), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antibody binding site.
  • the allocation of amino acids to each region or domain follows Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917 ; Chothia et al.
  • the CDR amino acid residue numbers in VH are 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3); the CDR amino acid residue numbers in VL are 24-34 (CDR1) ), 50-56 (CDR2) and 89-97 (CDR3).
  • the CDR amino acid numbers in VH are 26-32 (CDR1), 52-56 (CDR2) and 95-102 (CDR3); the amino acid residue numbers in VL are 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3).
  • CDR1 the numbering of CDR amino acid residues in VH is approximately 26-33 (CDR1), 51-56 (CDR2) and 93-102 (CDR3); and the numbering of CDR amino acid residues in VL is approximately 27-32 (CDR1), 50-51 (CDR2) and 89-97 (CDR3) (as disclosed in https://www.novoprolabs.com/tools/cdr).
  • antibody is not limited by any specific method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody It is also called “antigen binding part” for specific binding to antigen. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Recombinant DNA technology can be used. Or by enzymatic or chemical cleavage of intact antibodies to produce antigen-binding fragments of antibodies.
  • antigen-binding fragments include Fab, Fab′, F(ab′) 2 , Fd, Fv, dAb and complementarity determining regions (CDR) Fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDR complementarity determining regions
  • the antigen binding of the antibody Fragments are single-chain antibodies (e.g., scFv) in which the VL and VH domains pair to form a monovalent molecule by allowing them to be produced as a linker of a single polypeptide chain (see, e.g., Bird et al., Science 242: 423 426 (1988) And Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879 5883 (1988)).
  • scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL- COOH.
  • a suitable prior art linker consists of a repetitive GGGGS amino acid sequence or a variant thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc . Natl. Acad. Sci. USA 90: 6444-6448).
  • Other linkers that can be used as described herein are described by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001) , Cancer Immunol. description.
  • the antigen-binding fragments of antibodies are diabodies, ie, diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow two structures in the same chain Pairing between the domains, thereby forcing the domain to pair with the complementary domain of the other chain and creating two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444 6448 ( 1993), and Poljak RJ et al., Structure 2: 1121 1123 (1994)).
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • the term “antigen-binding unit” includes the above-defined antibody and antigen-binding fragments thereof.
  • the term "monoclonal antibody” refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibodies, except for natural mutations that may occur spontaneously. Antibody molecule.
  • the monoclonal antibody has high specificity for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which usually include at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen.
  • Monoclonal antibodies can usually be obtained by hybridoma technology first reported by Kohler et al. (Nature, 256: 495, 1975), but can also be obtained by recombinant DNA technology (for example, see Journal of virological methods, 2009, 158(1-2): 171-179).
  • neutralizing antibody refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence (for example, the ability to infect cells) of the target virus.
  • sequence is the sequence of amino acids in the polypeptide in the direction from the amino terminal to the carboxy terminal, wherein the residues adjacent to each other in the sequence are in the primary structure of the polypeptide.
  • the middle is continuous.
  • sequence can also be a linear sequence of a part of a polypeptide known to contain additional residues in one or two directions.
  • identity refers to the difference between two or more polynucleotide sequences or between two or more polypeptide sequences Sequence similarity or interchangeability.
  • sequence identity refers to the difference between two or more polynucleotide sequences or between two or more polypeptide sequences Sequence similarity or interchangeability.
  • programs such as Emboss Needle or BestFit to determine the sequence identity, similarity or homology between two different amino acid sequences, you can use the default settings, or you can select an appropriate scoring matrix, such as blosum45 or blosum80, to optimize Identity, similarity, or homology score.
  • homologous polynucleotides are those that hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably compared to these sequences A polynucleotide with a sequence identity of 97%, more preferably 98%, and even more preferably 99%.
  • homologous polypeptides preferably have at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity, or at least 99% sequence identity.
  • percentage of sequence identity is defined as after aligning the sequences and introducing gaps if necessary to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions.
  • sequence identity the percentage of amino acid residues in the query sequence that are identical to the amino acid residues of the second, reference polypeptide sequence or part thereof.
  • the alignment aimed at determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. .
  • the percent identity can be measured over the length of the entire defined polypeptide sequence, or can be measured over a shorter length, for example, the length of a fragment taken from a larger, defined polypeptide sequence, such as Fragments of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues.
  • Fragments of at least 5, at least 10, at least 15, at least 20, at least 50, at least 100, or at least 200 consecutive residues.
  • the antigen binding unit described herein may have one or more modifications relative to the reference sequence.
  • the modification may be deletion, insertion or addition, or substitution or substitution of amino acid residues.
  • “Deletion” refers to a change in the amino acid sequence due to the lack of one or more amino acid residues.
  • “Insertion” or “addition” refers to an amino acid sequence change that results in the addition of one or more amino acid residues compared to a reference sequence.
  • substitution or “substitution” refers to the replacement of one or more amino acids with different amino acids.
  • the mutation of the antigen-binding unit relative to the reference sequence can be determined by comparing the antigen-binding unit with the reference sequence. The optimal alignment of the sequences for comparison can be performed according to any known method in the art.
  • antigen refers to a substance that is recognized and specifically bound by an antigen binding unit.
  • Antigens can include peptides, proteins, glycoproteins, polysaccharides and lipids; parts thereof, and combinations thereof.
  • Non-limiting exemplary antigens include proteins from coronaviruses such as SARS-CoV-2, and their other homologs.
  • isolated refers to separation from cellular and other components, wherein in nature, polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof are normally associated with them. Associated. Those skilled in the art know that non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof do not need to be “isolated” to distinguish them from their naturally occurring counterparts.
  • concentrate in addition, “concentrated”, “isolated” or “diluted” polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are distinguishable from their naturally occurring counterparts because of the concentration or number of molecules per unit volume Greater than (“concentrated”) or less than from its naturally occurring counterpart ("isolated”). Enrichment can be measured based on absolute amounts, such as the weight of solution per unit volume, or it can be measured relative to the second, potentially interfering species present in the source mixture.
  • polynucleotide refers to polymeric forms of nucleotides of any length (whether deoxyribonucleotides or ribonucleotides) or their analogs.
  • a polynucleotide can have any three-dimensional structure, and can perform any known or unknown function.
  • polynucleotides coding or non-coding regions of genes or gene fragments, loci determined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomes RNA, ribozyme, cDNA, recombinant polynucleotide, branched chain polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer, oligonucleotide or synthetic DNA .
  • Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polymer.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation with a labeling component.
  • polynucleotide When applied to a polynucleotide, "recombinant" means that the polynucleotide is a variety of other procedures for cloning, restriction digestion, and/or ligation steps, and other procedures that produce constructs different from those found in nature. The product of the combination.
  • gene or “gene fragment” are used interchangeably herein. They refer to polynucleotides containing at least one open reading frame that can encode a specific protein after transcription and translation.
  • the gene or gene fragment can be genomic, cDNA or synthetic, as long as the polynucleotide contains at least one open reading frame, which can cover the entire coding region or a segment thereof.
  • operably connected or “effectively connected” refer to juxtaposition, where the components so described are in a relationship that allows them to function in their intended manner. For example, if a promoter sequence promotes transcription of a coding sequence, the promoter sequence is operably linked to the coding sequence.
  • expression refers to the process by which polynucleotides are transcribed into mRNA, and/or the process by which transcribed mRNA (also referred to as “transcripts") is subsequently translated into peptides, polypeptides or proteins.
  • the transcript and the encoded polypeptide are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.
  • the term "vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda Bacteriophage or M13 phage and animal virus etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillary viruses.
  • Polyoma vacuole virus (such as SV40).
  • a vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, and fungal cells such as yeast cells or Aspergillus , Such as insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • biological sample includes a variety of sample types obtained from living organisms, and can be used in diagnostic or monitoring tests.
  • the term includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures, or cells derived therefrom and their progeny.
  • the term includes samples that have been processed in any way after they have been obtained, for example by treatment with reagents, solubilization, or enrichment for certain components.
  • the term includes clinical samples, and also includes cells in cell cultures, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
  • the terms "recipient”, “individual”, “subject”, “host” and “patient” are used interchangeably herein, and refer to a person who wishes to diagnose, treat, or treat Any mammalian subject, especially humans.
  • treatment As used herein, the terms “treatment”, “treatment”, etc. are used herein to generally refer to obtaining the desired pharmacological and/or physiological effects.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms, and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or adverse reactions attributed to the disease.
  • Treatment encompasses any treatment of a disease in a mammal, such as mice, rats, rabbits, pigs, primates, including humans and other apes, especially humans, and The term includes: (a) preventing a disease or symptom from occurring in subjects who may be susceptible to the disease or symptom but not yet diagnosed; (b) inhibiting the symptoms of the disease; (C) preventing the development of the disease; (d) alleviating the disease Symptoms; (e) cause the disease or symptoms to subside; or any combination thereof.
  • specific binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • an antibody that specifically binds to a certain antigen means that the antibody has a concentration of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, The affinity (KD) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds to the antigen.
  • KD refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • KD is defined as the ratio of two kinetic rate constants Ka/Kd, where "Ka” refers to the rate constant of antibody binding to antigen, and “Kd” refers to the rate constant of antibody dissociation from the antibody/antigen complex .
  • Ka refers to the rate constant of antibody binding to antigen
  • Kd refers to the rate constant of antibody dissociation from the antibody/antigen complex .
  • KD dissociation equilibrium constant
  • the specific binding properties between two molecules can be measured using methods known in the art, for example, using surface plasmon resonance (SPR) in a BIACORE instrument.
  • SPR surface plasmon resonance
  • neutralizing activity refers to the ability of an antibody or antibody fragment to bind to the antigen protein on the virus, thereby preventing the virus from infecting cells and/or the maturation of the virus progeny and/or the release of the virus progeny Antibodies or antibody fragments with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating the infection of the virus.
  • the neutralizing activity of the antibody or antibody fragment by viruses in IC 50 inhibition “Half maximal inhibitory concentration” (IC 50) is a measure of antibody drugs in inhibiting biological or biochemical function (e.g., viral potency) of. IC 50 is calculated herein on the virus (e.g.
  • the antigen binding unit described herein is particularly suitable for diagnosing, preventing and treating novel coronavirus infection or diseases related to novel coronavirus infection (such as novel coronavirus pneumonia).
  • the antigen binding unit described herein includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region includes VH CDR1, VH CDR2, and VH CDR3, and the light
  • the chain variable region includes VL CDR1, VL CDR2 and VL CDR3.
  • the VH of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 721-1080 and 3111-3145, which includes one or more sequences selected from SEQ ID NO: 721-1080 and 3111-3145 A sequence of amino acid addition, deletion, or substitution, or a sequence selected from SEQ ID NO: 721-1080 and 3111-3145 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VH of the antigen-binding unit described herein may have one, two, three, or three, compared with the reference polypeptide. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Add, delete or replace.
  • the VH of the antigen-binding unit described herein may have more than 1, 2, 3, and 3 compared with the reference polypeptide.
  • the VH of the antigen-binding unit described herein may have fewer than 2, 3, 4 compared to the reference polypeptide. 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 add , Delete or replace.
  • the VH CDR1 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, which includes one or A sequence of multiple amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VH CDR1 of the antigen-binding unit described herein may have 1, 2, 3, or 3 as compared with the reference polypeptide. One, four, or five additions, deletions or substitutions.
  • there are amino acid additions, deletions or substitutions in the VH CDR1 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be more than one or two VH CDR1s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • VH CDR1 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be less than 2, 3 VH CDR1s of the antigen-binding unit described herein compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VH CDR2 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, which includes one or more sequences compared with a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970.
  • the VH CDR2 of the antigen-binding unit described herein may have 1, 2, 3, and 3 compared with the reference polypeptide. One, four, or five additions, deletions or substitutions.
  • there are amino acid additions, deletions or substitutions in the VH CDR2 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be more than one or two VH CDR2s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • VH CDR2 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be fewer than 2, 3 VH CDR2s of the antigen-binding unit described herein compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VH CDR3 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1-360 and 2971-3005, which includes one or A sequence of multiple amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 1-360 and 2971-3005 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VH CDR3 of the antigen-binding unit described herein may have 1, 2, 3, and 3 compared with the reference polypeptide. One, four, or five additions, deletions or substitutions.
  • there are amino acid additions, deletions or substitutions in the VH CDR3 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be more than one or two VH CDR3s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • the VH CDR3 of the antigen-binding unit described herein may have less than 2, 3 compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VL of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1081-1440 and 3146-3180, which includes one or more sequences selected from SEQ ID NO: 1081-1440 and 3146-3180.
  • the VL of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be 1, 2, 3, 1, 2, 3 VLs in the VL of the antigen-binding unit described herein compared with the reference polypeptide. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 Add, delete or replace.
  • the VL of the antigen-binding unit described herein may have more than 1, 2, 3, and 3 compared with the reference polypeptide.
  • the VL of the antigen-binding unit described herein may have fewer than 2, 3, 4 compared to the reference polypeptide. 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 add , Delete or replace.
  • the VL CDR1 of the antigen binding unit described herein may comprise a sequence selected from SEQ ID NO.: 2181-2540 and 3006-3040, compared with a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040.
  • deletions or substitutions in the VL CDR1 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be 1, 2, 3, or 3 in the VL CDR1 of the antigen-binding unit described herein compared with the reference polypeptide.
  • One, four, or five additions, deletions or substitutions when there are amino acid additions, deletions or substitutions in the VL CDR1 of the antigen-binding unit described herein compared with the reference polypeptide sequence, there may be more than one or two VL CDR1s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • VL CDR1 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be fewer than 2, 3 VL CDR1s of the antigen-binding unit described herein compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VL CDR2 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 2541-2900 and 3041-3075, which includes one or more sequences compared to a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075 A sequence of multiple amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence.
  • the VL CDR2 of the antigen-binding unit described herein may have 1, 2, 3, and 3 compared with the reference polypeptide. One, four, or five additions, deletions or substitutions.
  • there are amino acid additions, deletions or substitutions in the VL CDR2 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be more than one or two VL CDR2s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • the VL CDR2 of the antigen-binding unit described herein may have fewer than 2, or 3 compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VL CDR3 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 361-720 and 3076-3110, which includes one or more sequences than the sequence selected from SEQ ID NO: 361-720 and 3076-3110.
  • deletions or substitutions in the VL CDR3 of the antigen-binding unit described herein compared with the reference polypeptide sequence there may be 1, 2, 3, or 3 in the VL CDR3 of the antigen-binding unit described herein compared with the reference polypeptide.
  • One, four, or five additions, deletions or substitutions when there are amino acid additions, deletions or substitutions in the VL CDR3 of the antigen-binding unit described herein compared with the reference polypeptide sequence, there may be more than one or two VL CDR3s of the antigen-binding unit described herein compared with the reference polypeptide. , 3, 4, or 5 additions, deletions or substitutions.
  • the VL CDR3 of the antigen-binding unit described herein may have fewer than 2, or 3 compared with the reference polypeptide. , 4, or 5 additions, deletions or substitutions.
  • the VH CDR1 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1461-1820 and 2901-2935, which includes one or A sequence of multiple amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 1461-1820 and 2901-2935 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence; and the VL CDR1 of the antigen binding unit described herein may comprise SEQ ID NO.: 2181-2540 and 3006
  • the sequence of -3040, compared with a sequence selected from SEQ ID NO: 2181-2540 and 3006-3040 contains one or more amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 2181-2540 and
  • the sequence of 3006-3040 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
  • the VH CDR2 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1821-2180 and 2936-2970, which includes one or more sequences compared with a sequence selected from SEQ ID NO: 1821-2180 and 2936-2970.
  • the sequence of 3041-3075 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence .
  • the VH CDR3 of the antigen-binding unit described herein may include a sequence selected from SEQ ID NO.: 1-360 and 2971-3005, which includes one or A sequence of multiple amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 1-360 and 2971-3005 with at least 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or 99% identity sequence; and the VL CDR3 of the antigen binding unit described herein may comprise SEQ ID NO.: 361-720 and 3076
  • the sequence of -3110, compared with the sequence selected from SEQ ID NO: 361-720 and 3076-3110, contains one or more amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO: 361-720 and
  • the sequence of 3076-3110 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • the VH of the antigen binding unit described herein may include VH CDR1, VH CDR2, and VH CDR3, wherein the VH CDR1 is selected from the sequence of SEQ ID NO.: 1461-1820 and 2901-2935, and is selected from the sequence of SEQ ID NO: 1461.
  • the sequence contains one or more amino acid additions, deletions, or substitutions, or has at least 80%, 85%, Sequences with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% identity; wherein the VH CDR2 is selected from SEQ ID NO.:
  • the sequence of 1821-2180 and 2936-2970 compared with the sequence selected from SEQ ID NO: 1821-2180 and 2936-2970, contains one or more amino acid addition, deletion, or substitution sequence, or the sequence selected from SEQ ID NO :
  • the sequence of 1821-2180 and 2936-2970 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99%
  • the VH CDR3 is selected from the sequence of SEQ ID NO.: 1-360 and 2971-3005, compared with the sequence selected from SEQ ID NO: 1-
  • the VL of the antigen binding unit described herein may include VL CDR1, VL CDR2, and VL CDR3, wherein the VL CDR1 is selected from the sequence of SEQ ID NO.: 2181-2540 and 3006-3040, and is selected from the sequence of SEQ ID NO: 2181 Compared with the sequence of -2540 and 3006-3040, the sequence contains one or more amino acid additions, deletions, or substitutions, or has at least 80%, 85%, and the sequence selected from SEQ ID NO: 2181-2540 and 3006-3040.
  • VL CDR2 is selected from SEQ ID NO.:
  • the sequence of 2541-2900 and 3041-3075 compared with a sequence selected from SEQ ID NO: 2541-2900 and 3041-3075, contains one or more amino acid additions, deletions, or substitutions, or a sequence selected from SEQ ID NO :
  • the sequence of 2541-2900 and 3041-3075 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99%
  • the VL CDR3 is selected from the sequence of SEQ ID NO.: 361-720 and 3076-3110, compared with the sequence selected from SEQ ID NO: 361-720 and 3076-3110, it contains one or A sequence of multiple amino acid additions, deletions, or substitutions, or at least 80%, 85%, 90%,
  • the VH of the antigen binding unit described herein may comprise a sequence selected from a combination of the following CDR1, CDR2, and CDR3:
  • the VH CDR1 of the antigen binding unit described herein may include the same sequence as the CDR1 contained in SEQ ID NOs: 721-1080 and 3111-3145; the VH CDR2 of the antigen binding unit described herein may It contains the same sequence as the CDR2 contained in SEQ ID NO: 721-1080 and 3111-3145; the VH CDR3 of the antigen binding unit described herein may contain the same sequence as that in SEQ ID NO: 721-1080 and 3111-3145
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the heavy chain variable region CDR1 amino acid sequence SEQ ID NO: 1634, SEQ ID NO: 1635, SEQ ID NO: 1650, SEQ ID NO: 1757, and SEQ ID NO: 2907;
  • Heavy chain variable region CDR2 amino acid sequence SEQ ID NO: 1994, SEQ ID NO: 1995, SEQ ID NO: 2010, SEQ ID NO: 2117, and SEQ ID NO: 2942;
  • the heavy chain variable region CDR3 amino acid sequence SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 190, SEQ ID NO: 297, and SEQ ID NO: 2977.
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:
  • CDR3 amino acid sequence of light chain variable region SEQ ID NO: 550;
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:
  • CDR1 amino acid sequence of light chain variable region SEQ ID NO: 2477;
  • the antibodies provided herein comprise one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following amino acid sequences:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antibodies provided herein comprise one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • the antigen binding unit described herein can bind to the new coronavirus (SARS-CoV-2) S protein.
  • the antigen binding unit described herein can be combined with the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) S protein.
  • RBD receptor binding domain
  • the binding ability of the antigen binding unit to the RBD can be characterized or represented by any method known in the art.
  • binding can be characterized by binding affinity, which can be the strength of the interaction between the antigen binding unit and the antigen.
  • the binding affinity can be determined by any method known in the art, such as an in vitro binding test.
  • the binding affinity of an antigen-binding unit herein can be expressed in terms of KD.
  • KD is defined as the ratio of two kinetic rate constants Ka/Kd, where “Ka” refers to the rate constant of antibody binding to antigen, and “Kd” refers to the ratio of antibody from antibody/Kd.
  • the antigen binding unit as disclosed herein specifically binds to the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) S protein with a KD in the range of about 10 ⁇ M to about 1 fM.
  • the antigen binding unit may be less than about 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 50 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 1 pM, 0.1 pM, 10 fM. , 1fM, 0.1fM or less than 0.1fM KD specifically binds to the receptor binding domain (RBD) of the new coronavirus (SARS-CoV-2) S protein.
  • RBD receptor binding domain
  • SARS-CoV-2 new coronavirus
  • the antigen binding unit disclosed herein can be less than 100 nM, less than 50 nM, less than 20 nM, less than 15 nM, less than 10 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, less than 0.05 nM, Or the equilibrium dissociation constant (KD) of less than 0.01 nM binds to the receptor binding domain (RBD) of the S protein of the new coronavirus (SARS-CoV-2).
  • KD equilibrium dissociation constant
  • the antigen binding unit described herein has neutralizing activity against the new coronavirus (SARS-CoV-2).
  • the neutralizing activity of the antigen binding unit described herein against the novel coronavirus (SARS-CoV-2) can be analyzed by pseudovirus.
  • Pseudovirus has cell infection characteristics similar to herpes virus, can simulate the early process of true virus infection of cells, and can be detected and analyzed safely and quickly.
  • the neutralizing activity of the antigen-binding unit described herein against the novel coronavirus (SARS-CoV-2) can be detected by methods known in the art, such as a microporous cell neutralization test, refer to Temperton N J et al., Emerge Infect Dis, 2005, 11(3), 411-416.
  • the neutralizing activity of the antigen binding unit described herein against the novel coronavirus (SARS-CoV-2) can be tested using experimental cells such as Huh-7 cells and the pseudovirus SARS-CoV-2 virus.
  • the antigen binding unit described herein can be, for example, less than 100 ⁇ g/ml, less than 50 ⁇ g/ml, less than 20 ⁇ g/ml, less than 10 ⁇ g/ml, less than 9 ⁇ g/ml, less than 8 ⁇ g/ml, less than 7 ⁇ g/ml, less than 6 ⁇ g/ml, Less than 5 ⁇ g/ml, less than 4 ⁇ g/ml, less than 3 ⁇ g/ml, less than 2 ⁇ g/ml, less than 1 ⁇ g/ml, less than 0.5 ⁇ g/ml, less than 0.25 ⁇ g/ml, less than 0.2 ⁇ g/ml, less than 0.1 ⁇ g/ml, less than 0.05 ⁇ g/ml, less
  • the neutralizing activity of the antigen-binding unit described herein against the novel coronavirus (SARS-CoV-2) can be detected by the SARS-CoV-2 true virus through Plaque Reduction Neutralization Test (PRNT), and passed The reduction of plaque after incubation was calculated to calculate the IC50 of the antigen binding unit described herein and the SARS-CoV-2 true virus.
  • SARS-CoV-2 true virus through Plaque Reduction Neutralization Test (PRNT), and passed The reduction of plaque after incubation was calculated to calculate the IC50 of the antigen binding unit described herein and the SARS-CoV-2 true virus.
  • PRNT Plaque Reduction Neutralization Test
  • the antigen binding unit described herein can be, for example, less than 100 ⁇ g/ml, less than 50 ⁇ g/ml, less than 20 ⁇ g/ml, less than 10 ⁇ g/ml, less than 9 ⁇ g/ml, less than 8 ⁇ g/ml, less than 7 ⁇ g/ml, less than 6 ⁇ g/ml, Less than 5 ⁇ g/ml, less than 4 ⁇ g/ml, less than 3 ⁇ g/ml, less than 2 ⁇ g/ml, less than 1 ⁇ g/ml, less than 0.5 ⁇ g/ml, less than 0.25 ⁇ g/ml, less than 0.2 ⁇ g/ml, less than 0.1 ⁇ g/ml, less than 0.05 ⁇ g/ml, less than 1ng/ml, less than 0.5ng/ml, less than 0.25ng/ml, less than 0.2ng/ml, less than 0.1ng/ml, less than 50pg/ml, less than
  • the method comprises culturing a host cell expressing the antigen-binding unit under conditions suitable for expression of the antigen-binding unit, and isolating the antigen-binding unit expressed by the host cell.
  • the expressed antigen binding unit can be isolated using a variety of protein purification techniques known in the art. Generally, antigen binding units are isolated from the culture medium as secreted polypeptides, although they can also be recovered from host cell lysates or bacterial periplasm when they are directly produced without signal peptides. If the antigen-binding units are membrane-bound, they can be dissolved by a suitable detergent solution commonly used by those skilled in the art.
  • the recovered antigen-binding unit can be subjected to salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, running on a cation or anion exchange column at neutral pH and eluting with a step gradient of increasing ionic strength), gel filtration Chromatography (including gel filtration HPLC) and tag affinity column chromatography, or affinity resins such as protein A, protein G, hydroxyapatite and anti-immunoglobulin for further purification.
  • salt precipitation for example, with ammonium sulfate
  • ion exchange chromatography for example, running on a cation or anion exchange column at neutral pH and eluting with a step gradient of increasing ionic strength
  • gel filtration Chromatography including gel filtration HPLC
  • tag affinity column chromatography or affinity resins such as protein A, protein G, hydroxyapatite and anti-immunoglobulin for further purification.
  • derivatized immunoglobulins added with the following moieties can be used: chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, chemiluminescent moieties, specific binding moieties such as streptavidin Harmonin, avidin or biotin, or drug conjugate.
  • an antigen binding unit conjugated to a chemically functional moiety is also provided herein.
  • this part is a label capable of generating a detectable signal.
  • conjugated antigen binding units can be used in, for example, detection systems, such as the severity of viral infections, imaging of infected foci, and the like.
  • labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compound substrate cofactors, and inhibitors.
  • This part can be covalently linked to the antigen binding unit, recombinantly linked or conjugated to the antigen binding unit via a second reagent such as a second antibody, protein A or a biotin-avidin complex.
  • the signal peptide is a short amino acid sequence that directs the newly synthesized protein through the cell membrane (usually the endoplasmic reticulum in eukaryotic cells) and the inner or both inner and outer membranes of bacteria.
  • the signal peptide can be located in the N-terminal part of the polypeptide or the C-terminal part of the polypeptide, and the signal peptide can be enzymatically removed from the cell between the biosynthesis and secretion of the polypeptide.
  • Such peptides can be introduced into the antigen binding unit to allow the secretion of synthetic molecules.
  • Reagents that enhance immune reactivity include, but are not limited to, bacterial superantigens.
  • Reagents that facilitate coupling to the solid support include, but are not limited to, biotin or avidin.
  • the immunogenic carrier includes, but is not limited to, any physiologically acceptable buffer.
  • Biological response modifiers include cytokines, particularly tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor and gamma-interferon.
  • the chemically functional part can be prepared recombinantly, for example, by generating a fusion gene encoding the antigen binding unit and the functional part.
  • the antigen binding unit can be chemically bonded to the moiety by any of a variety of well-known chemical procedures.
  • the moiety is a protein
  • the connection can be through a heterobifunctional crosslinking agent, such as SPDP, carbodiimide glutaraldehyde, and the like.
  • This part can be covalently linked or conjugated via a second reagent, such as a second antibody, protein A or biotin-avidin complex.
  • Paramagnetic moieties and their conjugation to antibodies are well known in the art. See, for example, Miltenyi et al. (1990) Cytometry 11:231-238.
  • an isolated polynucleotide encoding the antigen binding unit described herein.
  • the nucleotide sequence corresponding to each region of the L or H chain of an existing antibody can be easily obtained and sequenced using conventional techniques (including but not limited to hybridization, PCR, and DNA sequencing).
  • Hybridoma cells that produce monoclonal antibodies are used as a preferred source of antibody nucleotide sequences.
  • Large numbers of hybridoma cells that produce a range of monoclonal antibodies can be obtained from public or private repositories. The largest storage institution is the American Type Culture Collection, which provides a variety of well-characterized hybridoma cell lines.
  • antibody nucleotides can be obtained from immunized or unimmunized rodents or humans, and from organs such as the spleen and peripheral blood lymphocytes. Specific techniques suitable for the extraction and synthesis of antibody nucleotides are described in Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86: 3833-3837; Larrick et al. 1989) biochem. Biophys. Res. Commun. 160: 1250-1255; Sastry et al. (1989) Proc. Natl. Acad. Sci., USA 86: 5728-5732; and U.S. Patent No. 5,969,108.
  • the antibody nucleotide sequence can also be modified, for example, by replacing the homologous non-human sequence by substituting the coding sequence for the human heavy and light chain constant regions. In this way, chimeric antibodies are prepared that retain the binding specificity of the original antibody.
  • the polynucleotides encoding the heavy chain and/or light chain of the antigen-binding unit can be codon-optimized to achieve optimized expression of the subject's antigen-binding unit in the desired host cell.
  • codon optimization method natural codons are replaced by the most common codons from a reference genome, where the codon translation rate of each amino acid is designed to be higher.
  • Additional exemplary methods for generating codon-optimized polynucleotides for expression of the desired protein are described in Kanaya et al., Gene, 238:143-155 (1999), Wang et al., Mol. Biol. Evol. , 18(5): 792-800 (2001), US Patent No. 5,795,737, US Publication No. 2008/0076161 and WO 2008/000632, the method can be applied to the heavy chain and/or light chain of the antigen binding unit.
  • polynucleotides herein include polynucleotides that encode functional equivalents of exemplary polypeptides and fragments thereof.
  • nucleotides of the L and H sequences and the heterodimerization sequences suitable for constructing the polynucleotides and vectors described herein can have considerable variation. These variations are included in the content of this article.
  • a method of using the antigen binding unit described herein to prevent or treat a novel coronavirus (SARS-CoV-2) infection in a subject comprising administering the antigen binding unit described herein to the subject.
  • SARS-CoV-2 novel coronavirus
  • telaprevir boceprevir
  • semiprevir sofosbuvir
  • daclastavir analpine Asunaprevir
  • lamivudine lamivudine
  • adefovir adefovir
  • entecavir tenofovir
  • telbivudine interferon alpha and pegylated interferon ⁇ .
  • the second agent may be selected from hydroxychloroquine, chloroquine, favilavir, Gimsilumab, AdCOVID (University of Alabama at Birmingham), AT-100 (Airway Therapeutics), TZLS-501 (Tiziana Life Science) , OYA1 (OyaGen), BPI-002 (BeyondSpring), INO-4800 (Inovio Pharmaceutical), NP-120 (ifenprodil), Redecive (GS-5734), Actemra (Roche), Galidevir (BCX4430) , SNG001 (Synairgen Research), or a combination thereof.
  • the second agent may be an agent for alleviating the symptoms of an inflammatory condition complicated by the subject.
  • the anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAID) and corticosteroids.
  • NSAID includes, but is not limited to, salicylate, such as acetylsalicylic acid; diflunisal, salicylic acid, and salicylate; propionic acid derivatives, such as ibuprofen; naproxen; Ketoprofen, flurbiprofen, oxaprazine, fenoprofen, loxoprofen, and ketoprofen; acetic acid derivatives, such as indomethacin, diclofenac, tolmetin, aceclofenac, sulphur Linic acid, nabumetone, etodolac and ketorolac; enolic acid derivatives such as piroxicam, lornoxicam, meloxicam, isoxicam, tenoxicam,
  • the second agent may be an immunosuppressive agent.
  • Immunosuppressive agents that can be used in combination with the antigen binding unit include, but are not limited to, hydroxychloroquine, sulfasalazine, leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, Oral gold compounds, injectable gold compounds (intramuscular injection), minocycline, gold sodium thiomalate, auranofin, D-penicillamine, clobenzari, bucillamine, and acotaril , Cyclophosphamide, azathioprine, methotrexate, mizoribine, cyclosporine and tacrolimus.
  • the specific dosage will vary according to the specific antigen-binding unit selected, the dosing schedule to be followed, whether to be administered in combination with other agents, the time of administration, the tissue to be administered, and the physical delivery system carrying the specific antigen-binding unit.
  • the specific antigen-binding unit selected, the dosing schedule to be followed, whether to be administered in combination with other agents, the time of administration, the tissue to be administered, and the physical delivery system carrying the specific antigen-binding unit.
  • An antigen binding unit in the range of 66, 67, 68, 69 or 70 mg.
  • Antigen binding unit in the range of 55mg.
  • antigen binding in the range of about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 mg is administered to the subject weekly unit.
  • the antigen binding unit is administered to the subject in an amount.
  • the antigen binding unit is administered to the subject in an average amount of about 6 to 10 mg, about 6.5 to 9.5 mg, about 6.5 to 8.5 mg, about 6.5 to 8 mg, or about 7 to 9 mg per day.
  • the dosage of the antigen binding unit can be about, at least about, or at most about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000mg or mg/kg, Or any range within which it can be derived.
  • the expected dose of mg/kg refers to the mg amount of antigen binding unit per kilogram of the subject's total body weight. It is expected that when multiple doses are given to a patient, the doses may vary in amount or they may be the same.
  • composition comprising a subject antibody or functional fragment thereof and a pharmaceutically acceptable carrier, excipient or stabilizer, including but not limited to inert solid diluents and fillers, diluents, sterile Aqueous solution and various organic solvents, penetration enhancers, solubilizers and adjuvants.
  • a pharmaceutically acceptable carrier including but not limited to inert solid diluents and fillers, diluents, sterile Aqueous solution and various organic solvents, penetration enhancers, solubilizers and adjuvants.
  • the pharmaceutical composition may be in unit dosage form suitable for single administration of precise dosages.
  • the pharmaceutical composition may further include an antigen binding unit as an active ingredient, and may include conventional pharmaceutical carriers or excipients. In addition, it may include other drugs or medicaments, carriers, adjuvants, and the like.
  • Exemplary parenteral administration forms include solutions or suspensions of active polypeptides and/or PEG-modified polypeptides in sterile aqueous solutions, such as aqueous propylene glycol solutions or dextrose solutions. If necessary, such dosage forms may be suitably buffered with salts such as histidine and/or phosphate.
  • the composition may further include one or more pharmaceutically acceptable additives and excipients.
  • additives and excipients include, but are not limited to, anti-sticking agents, defoamers, buffers, polymers, antioxidants, preservatives, chelating agents, viscosity modifiers (viscomodulator), tonicity modifiers, flavoring agents, coloring agents, Flavoring agents, sunscreens, suspending agents, binders, fillers, plasticizers, lubricants and mixtures thereof.
  • the kit described herein comprises the antigen binding unit described herein or a conjugate thereof as described herein. Also provided is the use of the antigen binding unit described herein in the preparation of a kit for detecting the presence or level of a novel coronavirus or its S protein or RBD in a sample, or for diagnosis Whether the subject is infected with the new coronavirus.
  • the sample includes, but is not limited to, excrement, oral or nasal secretions, alveolar lavage fluid, etc. from a subject (such as a mammal, preferably a human).
  • the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescence immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competition method, and similar detection methods.
  • ELISA enzyme-linked immunosorbent assay
  • enzyme immunoassay enzyme immunoassay
  • chemiluminescence immunoassay chemiluminescence immunoassay
  • radioimmunoassay radioimmunoassay
  • fluorescence immunoassay immunochromatography
  • competition method and similar detection methods.
  • the molecular biology experimental methods and immunoassays used in this article basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Compiled Molecular Biology Experiment Guide, 3rd Edition, John Wiley & Sons, Inc., 1995; the restriction enzymes are used in accordance with the conditions recommended by the product manufacturer. If no specific conditions are indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer.
  • the reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.
  • Chromium Single Cell V(D)J Reagent Kits purchased from 10X genomics, catalog number: 100006 were used to perform single-cell transcriptome VDJ sequencing on the above-mentioned enriched memory B cells.
  • the sequencing results were analyzed, and 360 strains of antigen binding units were obtained, which were named ABU 1-395.
  • the sequence information of the obtained antigen-binding unit is shown in Table 1 below.
  • nucleic acid molecules encoding the heavy and light chains of an antibody are synthesized in vitro, and then cloned into expression vectors, respectively, to obtain recombinant expression vectors encoding the heavy and light chains of the antibody, respectively.
  • the recombinant expression vectors encoding the heavy chain and light chain of the antibody obtained above were co-transfected into HEK293 cells. After 4-6 hours of transfection, the cell culture medium was replaced with a serum-free medium, and the culture was continued at 37°C for 6 days. After the culture is completed, the antibody protein expressed by the cells is purified from the culture through an affinity purification column. Subsequently, the purified target protein was detected by reducing and non-reducing SDS-PAGE.
  • the recombinantly expressed S protein RBD was used as the coating antigen, and horseradish peroxidase (HRP)-labeled Goat anti-human IgG Fc was used as the secondary antibody, and the antigen reaction of the purified antibody to be tested was detected by ELISA experiment sex.
  • HRP horseradish peroxidase
  • a 96-well plate is coated with recombinantly expressed S protein RBD (its amino acid sequence is shown in SEQ ID NO: 1459, and the concentration is 0.01 ⁇ g/ml or 1 ⁇ g/ml), and then the 96-well plate is processed with blocking solution Closed.
  • the monoclonal antibodies to be tested (control antibody, ABU-174, ABU-175 and ABU190; concentrations of 0.1 ⁇ g/ml respectively) were added and incubated. After washing with ELISA washing solution, horseradish peroxidase (HRP) labeled Goat anti-human IgG Fc was added as a secondary antibody (diluted at 1:500), and the incubation continued. Then, the ELISA plate was washed with PBST, and a color developer was added to develop the color. Then read the absorbance value of OD450nm on the microplate reader. The results are shown in Table 2. It can be seen from Table 2 that ABU-174, ABU-175 and ABU190 can specifically recognize and bind to S protein RBD.
  • HRP horseradish peroxidase
  • Table 2 The reactivity of ABU-174, ABU-175 and ABU190 antigen binding units with S protein RBD (OD450 reading) detected by ELISA
  • the surface plasmon resonance technology is used to detect the affinity between the antibody and the RBD region of the Spike protein.
  • SPR surface plasmon resonance technology
  • Biacore T200 for measurement, first couple the biotin-labeled SARS-COV-2RBD domain to the SA chip (GE Company), and the signal resonance unit RU value increased by 100 units.
  • the running buffer is PBS with pH 7.4 plus 0.005% P20 to ensure that the buffer in the analyte (such as antibody) is consistent with the running buffer. Dilute the purified antibody in a 3-fold gradient to make the concentration between 50-0.78125nM.
  • the measurement results are analyzed using Biacore Evaluation software.
  • Table 3 lists the binding affinity of exemplary antigen-binding units herein to the RBD region of Spike protein, wherein the KD value of each antigen-binding unit is less than 20 nM.
  • FIG. 2A-2E further exemplarily show the binding affinity of ABU-174, ABU-175, ABU190, ABU297, and ABU367 to the RBD region of Spike protein.
  • the KD value of ABU-174 is 0.29nM
  • the KD value of ABU-175 is 0.039nM
  • the KD value of ABU190 is 2.8nM
  • the KD value of ABU297 is 0.824nM
  • the KD value of ABU is 0.18. nM.
  • Figures 2A-2E show that ABU-174, ABU-175, ABU190, ABU297 and ABU367 all have good affinity with the new coronavirus S protein.
  • Example 5 Evaluation of the ability of the antigen binding unit herein to neutralize the SARS-CoV-2 pseudovirus
  • the microporous cell neutralization method was used to detect the effect of the antigen binding unit in this article on SARS- Neutralizing activity of CoV-2 pseudovirus.
  • the SARS-CoV-2 pseudovirus used in this example is provided by the China Institute for Food and Drug Control. It has cell infection characteristics similar to true viruses, can simulate the early process of true viruses infecting cells, and carries the reporter gene luciferase. Quick and easy detection and analysis. The operation of pseudovirus is highly safe, and the neutralization experiment can be completed in the P2 laboratory to detect the neutralization titer of the antibody.
  • the specific steps of the experimental method are as follows.
  • the wells of A2-H2 are set as cell control wells (CC), which contain only experimental cells; the wells of A3-H3 are set as Virus control wells (VV), which contain experimental cells and pseudoviruses; the wells of A4-A11, B4-B11, C4-C11, D4-D11, E4-E11, F4-F11, G4-G11, and H4-H11 are set as Experimental wells, which contain experimental cells, pseudoviruses, and different concentrations of antibodies to be tested; the remaining wells are set as blanks.
  • the experimental cells and pseudoviruses used in this example are Huh-7 cells and SARS-CoV-2 virus (both provided by the China Institute for Food and Drug Control).
  • Inhibition rate [1-(average value of luminous intensity of experimental hole-average value of luminous intensity of CC hole)/(average value of luminous intensity of VV hole-average value of luminous intensity of CC hole)] ⁇ 100%.
  • Table 5 lists the IC50 of the exemplary antigen-binding units herein and the SARS-CoV-2 pseudovirus, wherein the IC50 value of each antigen-binding unit is less than 1 ⁇ g/ml.
  • ABU-322 ⁇ 0.1 ABU-340 ⁇ 0.5 ABU-341 ⁇ 0.1 ABU-344 ⁇ 1 ABU-349 ⁇ 0.1 ABU-351 ⁇ 0.1 ABU-352 ⁇ 0.1 ABU-354 ⁇ 0.1 ABU-355 ⁇ 0.1 ABU-356 ⁇ 0.1 ABU-357 ⁇ 1 ABU-358 ⁇ 0.1 ABU-359 ⁇ 0.1 ABU-360 ⁇ 0.1 ABU-361 ⁇ 0.5 ABU-362 ⁇ 0.5 ABU-365 ⁇ 0.1 ABU-367 ⁇ 0.1 ABU-368 ⁇ 0.5 ABU-369 ⁇ 0.1 ABU-371 ⁇ 1 ABU-372 ⁇ 0.5 ABU-373 ⁇ 0.5 ABU-375 ⁇ 0.1 ABU-376 ⁇ 0.1 ABU-377 ⁇ 0.5 ABU-379 ⁇ 0.5 ABU-380 ⁇ 0.1 ABU-381 ⁇ 0.1 ABU-382 ⁇ 0.1 ABU-386 ⁇ 0.1 ABU-391 ⁇ 1 ABU-392 ⁇ 0.1 ABU-395 ⁇ 0.1
  • Figures 3A-3C further exemplarily show the neutralizing activity of ABU-174, ABU-175 and ABU190 against SARS-CoV-2 pseudovirus. It can be seen from Figure 3A-3C that ABU-174, ABU-175 and ABU190 all have good neutralizing activity, with IC50 of 0.026 ⁇ g/ml (ABU-174), 0.0086 ⁇ g/ml (ABU-175), 0.039 ⁇ g, respectively /ml(ABU190).
  • Example 6 Evaluation of the ability of the antigen binding unit herein to neutralize SARS-CoV-2 true virus
  • the neutralizing activity of the antibody to be tested was evaluated by the cytopathic (CPE) assay and the plaque reduction neutralization test (PRNT), respectively.
  • CPE cytopathic
  • PRNT plaque reduction neutralization test
  • SARS-CoV-2 virus provided by the Military Medical Academy
  • the titer the TCID50 of 10 5 / ml, and all experimental procedures were completed in BSL-3 laboratories.
  • CPE cytopathic
  • step (3) After the completion of the culture in step (1), discard the cell culture medium in the 96-well culture plate, and add the mixed solution (200 ⁇ l) containing the test antibody and the true virus prepared in step (2) as the experimental group. After incubating for 1 hour, aspirate the supernatant from the wells and add 200 ⁇ l DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) to each well.
  • DMEM medium containing 2% antibiotics and 16 ⁇ g/ml trypsin
  • a cell control group and a virus control group were set up in parallel.
  • the cell control group (4 multiple wells)
  • 200 ⁇ l DMEM medium containing 2% antibiotics and 16 ⁇ g/ml trypsin
  • the virus control group after discarding the cell culture medium in the wells, add 100 TCID50 true virus (100 ⁇ l) to each well, and incubate at 37°C for 1 hour; after the incubation, aspirate the supernatant from the wells , Add 200 ⁇ l DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) to each well.
  • CPE cytopathic
  • the test results of the antigen-binding unit ABU-174 are shown in Table 6 below. The results show that the antigen-binding unit ABU-174 has an inhibitory effect on the virus on cells, and the neutralizing antibody titer is 1.6 ng/ ⁇ l.
  • the detection results of the antigen-binding unit ABU-175 are shown in Table 7 and Figure 4 below. The results show that the antigen-binding unit ABU-175 has an inhibitory effect on the virus on cells, and the neutralizing antibody titer is 0.7 ng/ ⁇ l.
  • step (3) After the completion of the culture in step (1), discard the cell culture medium in the 96-well culture plate, and add the mixed solution (200 ⁇ l) containing the test antibody and the true virus prepared in step (2) as the experimental group. After incubating for 1 hour, aspirate the supernatant from the wells, and add 200 ⁇ l DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) to each well.
  • a cell control group and a virus control group were set up in parallel.
  • the cell control group after discarding the cell culture medium in the wells, add 200 ⁇ l DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) to each well.
  • the virus control group (4 multiple wells), after discarding the cell culture medium in the wells, add 100 TCID50 true virus (100 ⁇ l) to each well, and incubate at 37°C for 1 hour; after the incubation, aspirate the supernatant from the wells , Add 200 ⁇ l DMEM medium (containing 2% antibiotics and 16 ⁇ g/ml trypsin) to each well.
  • Figure 5 shows the dose-response curves of the exemplary antigen binding units ABU-174, ABU-175 and ABU190 herein. It can be seen from Figure 5 that the antigen binding units ABU-174, ABU-175 and ABU190 all have good neutralizing activity against SARS-CoV-2 true virus, and can effectively inhibit virus infection and cell invasion, with IC50 of 0.5 ⁇ g/ml ( ABU-174), 0.3 ⁇ g/ml (ABU-175) and 0.8 ⁇ g/ml (ABU-190).
  • SARS-CoV-2 infects cells through interaction with hACE2 receptor.
  • the neutralizing effect of the antigen-binding unit in the article on SARS-CoV-2 in vivo was evaluated in two different animal models.
  • hACE2 transgenic mice were used as animal models, and treatments were performed in two different modes: pre-exposure prevention or post-exposure prevention. Specifically, hACE2 transgenic mice were intranasally infected with SARS-CoV-2 viruses (2019-nCoV Beta CoV/Wuhan/AMMS01/2020) at a dose of 105 TCID50.
  • SARS-CoV-2 viruses 2019-nCoV Beta CoV/Wuhan/AMMS01/2020
  • the antigen-binding unit herein was injected intraperitoneally into hACE2 transgenic mice at a dose of 20 mg/kg, and the antigen-binding unit was tested as a pre-exposure preventive intervention The effect of.
  • the antigen binding unit at a dose of 20 mg/kg was injected into mice.
  • HG1K IgG1 antibody against H7N9 virus
  • the body weight that can reflect the health of the infected mice was recorded every day for 5 consecutive days.
  • hamster In the second model, hamster (Mesocricetus auratus) was used as an animal model, and treatment was performed in two different modes: pre-exposure prevention or post-exposure prevention. Specifically, similar to hACE2 transgenic mice, hamsters were infected intranasally with SARS-CoV-2 provirus (SARS-COV-2/WH-09/human/020/CHN) at a dose of 105 TCID50.
  • SARS-CoV-2 provirus SARS-CoV-2 provirus
  • the antigen binding unit herein at a dose of 20 mg/kg is injected into the hamster.
  • animals were injected with PBS 2 hours after infection.
  • the antigen-binding unit herein is injected intraperitoneally into the hamster at different doses (including 20, 10, 5, and 2 mg/kg) according to body weight.
  • hamsters injected with phosphate buffered saline (PBS) were used as controls.
  • the weight of the infected hamster was recorded every day for 7 consecutive days.
  • the hamsters were sacrificed 7 days after infection, and the lungs were collected for viral load analysis.

Abstract

本文涉及免疫学领域和分子病毒学领域，特别是新型冠状病毒的诊断、预防和治疗领域。具体而言，本文涉及抗新型冠状病毒的单克隆抗体，以及包含所述抗体的组合物(例如，诊断剂和治疗剂)。此外，本文还涉及所述抗体的用途。本文所述的抗体可用于诊断、预防和/或治疗新型冠状病毒的感染和/或由所述感染引起的疾病(例如，新型冠状病毒肺炎)。

Description

一种抗新型冠状病毒的单克隆抗体及其应用 技术领域
本文涉及免疫学领域和分子病毒学领域，特别是新型冠状病毒的诊断、预防和治疗领域。具体而言，本文涉及抗新型冠状病毒的抗体，以及包含所述抗体的组合物(例如，诊断剂和治疗剂)。此外，本文还涉及所述抗体的用途。本文所述的抗体可用于诊断、预防和/或治疗新型冠状病毒的感染和/或由所述感染引起的疾病(例如，新型冠状病毒肺炎)。
背景技术
新型冠状病毒SARS-CoV-2是导致新型冠状病毒肺炎(COVID-19)的病原体，是一种单链RNA病毒，它与2002-2003年引发疫情的重症急性呼吸综合征冠状病毒(SARS-CoV)以及2012年引发疫情的中东呼吸综合征冠状病毒(MERS-CoV)同属冠状病毒科(Coronaviridae)。冠状病毒颗粒呈圆形或椭圆形，亦为多形性，直径50-200nm，属于尺寸较大的病毒。冠状病毒是包膜病毒，病毒衣壳外面包裹着脂质包膜，其上排列较宽的刺突蛋白(Spike，S蛋白，SEQ ID No：1460)，形状如太阳光环。已有研究证实，新型冠状病毒SARS-CoV-2的病毒表面具有S蛋白，其能够在病毒感染宿主的过程中通过其包含的受体结合结构域(RBD)结合宿主细胞受体血管紧张素转换酶2(ACE2)分子，从而启动病毒膜与宿主细胞膜发生融合，导致宿主细胞感染病毒。
迄今为止，中和抗体已被证明是治疗病毒性疾病的有效方法。一般来说，患者体内的B淋巴细胞受到抗原的刺激后，会被活化，进而转变和分化成多种不同的细胞，并产生抗体。据现有的研究报道，新型冠状病毒肺炎康复者的外周血内抗新型冠状病毒的抗体，它们由经活化的B细胞产生和分泌。然而，康复者血浆内存在着多种B细胞，并且不同的B细 胞所产生的抗体的结合活性和中和效价也有所不同。到目前为止，尚无研究报道具有高结合活性和/或高中和活性的抗新型冠状病毒的抗体。
因此，需要开发能够抗新型冠状病毒SARS-CoV-2的具有高结合活性和/或高中和活性的抗体，以提供有效的诊断、预防和/或治疗新型冠状病毒感染的手段。
发明内容
本文所提供的下述技术方案满足了上述需求，并且提供了相关的优点。
一方面，本文提供一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列或者与SEQ ID NO：1-360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列，和/或其中所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列或者与SEQ ID NO：361-720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列。
在一些实施方案中，所述抗原结合单元以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
在一些实施方案中，所述的抗原结合单元以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC50中和新型冠状病毒(SARS-CoV-2)。
在一些实施方案中，所述抗原结合单元的所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列或者与SEQ ID NO：1461-1820 和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR1包含与SEQ ID NO：1461-1820和2901-2935相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR1包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR1相同的序列。
在一些实施方案中，所述抗原结合单元的所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列或者与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR2包含与SEQ ID NO：1821-2180和2936-2970相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH CDR2包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR2相同的序列。
在一些实施方案中，所述抗原结合单元的所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列或者与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR1包含与SEQ ID NO：2181-2540和3006-3040相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR1包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR1相同的序列。
在一些实施方案中，所述抗原结合单元的所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列或者与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR2包含选自SEQ ID  NO：2541-2900和3041-3075的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR2包含与SEQ ID NO：2541-2900和3041-3075相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL CDR2包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR2相同的序列。
在一些实施方案中，所述抗原结合单元的所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列或者与SEQ ID NO：721-1080和3111-3145相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列。在一些实施方案中，所述抗原结合单元的所述VH包含与SEQ ID NO：721-1080和3111-3145相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在一些实施方案中，所述抗原结合单元的所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列或者与SEQ ID NO：1081-1440和3146-3180相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列。在一些实施方案中，所述抗原结合单元的所述VL包含与SEQ ID NO：1081-1440和3146-3180相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
另一方面，本文提供一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列、与 SEQ ID NO：1461-1820和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR1相同的序列，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列、与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR2相同的序列，其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列、与SEQ ID NO：1-360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR3相同的序列；和/或其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列、与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR1相同的序列，所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列、与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR2相同的序列，所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列、与SEQ ID NO：361-720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR3相同的序列。
另一方面，本文提供一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列或者与SEQ ID NO：1461-1820和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列或者与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列，其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列或者与SEQ ID NO：1- 360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列；和/或其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列或者与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列，所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列或者与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列，所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列或者与SEQ ID NO：361-720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列。
在一些实施方案中，所述抗原结合单元的所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列或者与SEQ ID NO：721-1080和3111-3145相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列。在一些实施方案中，所述抗原结合单元的所述VH包含与SEQ ID NO：721-1080和3111-3145相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在一些实施方案中，所述抗原结合单元的所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列或者与SEQ ID NO：1081-1440和3146-3180相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列。在一些实施方案中，所述抗原结合单元的所述VL包含与SEQ ID NO：1081-1440和3146-3180相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在一些实施方案中，所述的抗原结合单元以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
在一些实施方案中，所述的抗原结合单元以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC50中和新型冠状病毒(SARS-CoV-2)。
另一方面，本文提供一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，其中所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列，和/或其中所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在一些实施方案中，所述的抗原结合单元以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
在一些实施方案中，所述的抗原结合单元以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC50中和新型冠状病毒(SARS-CoV-2)。
在一些实施方案中，所述抗原结合单元进一步包含重链恒定区(CH)。在一些实施方案中，所述抗原结合单元的所述CH包含SEQ ID NO：1457 的序列或者与SEQ ID NO：1457相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述CH包含选自SEQ ID NO：1457的序列。在一些实施方案中，所述抗原结合单元的所述CH包含与SEQ ID NO：1457相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述CH包含与选自SEQ ID NO：1457的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在一些实施方案中，所述抗原结合单元进一步包含轻链恒定区(CL)。在一些实施方案中，所述抗原结合单元的所述CL包含SEQ ID NO：1458的序列或者与SEQ ID NO：1458相比包含一个或多个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述CL包含选自SEQ ID NO：1458的序列。在一些实施方案中，所述抗原结合单元的所述CL包含与SEQ ID NO：1458相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。在一些实施方案中，所述抗原结合单元的所述CL包含与选自SEQ ID NO：1458的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
在另一个方面，本文提供了分离的核酸分子，其编码如上文所定义的本文所述的抗原结合单元。
在另一个方面，本文提供了一种载体，其包含如上文所定义的分离的核酸分子。本文所述的载体可以是克隆载体，也可以是表达载体。在一些实施方案中，本文所述的载体是例如质粒、粘粒、或噬菌体等等。
在另一个方面，还提供了包含本文所述的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞、昆虫细胞、植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本文所述的细胞还可以是细胞系，例如HEK293细胞。
在另一个方面，还提供了制备本文所述的抗原结合单元的方法，其包括，在合适的条件下培养本文所述的宿主细胞，和从细胞培养物中回收本文所述的抗原结合单元。
在另一个方面，本文提供了一种组合物，其包含如上文所描述的抗原结合单元、分离的核酸分子、载体或宿主细胞。
在另一个方面，本文提供了一种试剂盒，其包括本文所述的抗原结合单元。在一些实施方案中，本文所述的抗原结合单元还包括可检测的标记。在一些实施方案中，所述试剂盒还包括第二抗体，其特异性识别本文所述的抗原结合单元。优选地，所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。
在另一个方面，本文提供了检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平的方法，其包括，使用本文所述的抗原结合单元。在一些实施方案中，本文所述的抗原结合单元还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本文所述的抗原结合单元。所述方法可以用于诊断目的(例如，所述样品是来自患者的样品)，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，本文提供了诊断受试者是否感染了新型冠状病毒的方法，其包括：使用本文所述的抗原结合单元检测新型冠状病毒或其S蛋白或S蛋白的RBD在来自所述受试者的样品中的存在。在一些实施方案中，本文所述的抗原结合单元还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本文所述的抗原结合单元。
在另一个方面，提供了本文所述的抗原结合单元在制备试剂盒中的用途，所述试剂盒用于检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平，或用于诊断受试者是否感染了新型冠状病毒。
在另一个方面，本文提供了一种药物组合物，其包含本文所述的抗原结合单元，以及药学上可接受的载体和/或赋形剂。
在另一个方面，本文提供了用于中和样品中新型冠状病毒的毒力的方法，其包括，将包含新型冠状病毒的样品与本文所述的抗原结合单元接触。此类方法可以用于治疗目的，或非治疗目的(例如所述样品是细胞样品，而不是患者或来自患者的样品)。
在另一个方面，提供了本文所述的抗原结合单元用于制备药物的用途，所述药物用于中和样品中新型冠状病毒的毒力。在另一个方面，本文提供了如上文所描述的抗原结合单元，其用于中和样品中新型冠状病毒的毒力。
在另一个方面，提供了本文所述的抗原结合单元在制备药物组合物中的用途，所述药物组合物用于预防或治疗受试者的新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。在另一个方面，本文提供了如上文所描述的抗原结合单元，其用于预防或治疗受试者的新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。
在另一个方面，本文提供了用于预防或治疗受试者的新型冠状病毒感染或新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)的方法，其包括，给有此需要的受试者施用预防或治疗有效量的本文所述的抗原结合单元，或者本文所述的药物组合物。
在一些实施方案中，所述受试者是哺乳动物，例如人。
可通过任何适当的施用途径来将本文所述的抗原结合单元或者本文所述的药物组合物施用给受试者。此类施用途径包括但不限于，口服、口腔、舌下、局部、肠胃外、直肠、叶鞘内、或鼻腔途径。
本文所提供的药物和药物组合物可以单独使用或联合使用，也可以与其他药学活性剂(例如抗病毒药物，如法匹拉韦、瑞德西韦和干扰素等)联合使用。在一些实施方案中，所述药物组合物还含药学上可接受的载体和/或赋形剂。
在另一个方面，本文提供了缀合物，其其包含如上文所描述的抗原结合单元，其中所述抗原结合单元与化学功能部分缀合。在一些实施方案中， 所述化学功能部分选自射性同位素、酶、荧光化合物、化学发光化合物、生物发光化合物底物辅因子和抑制剂。
附图说明
图1A-1C示例性地显示了抗原结合单元ABU-174、ABU-175和ABU190的SDS-PAGE检测结果。
图2A-2E示例性地显示了使用SPR技术检测抗原结合单元ABU-174(A)、ABU-175(B)、ABU190(C)、ABU297(D)和ABU367(E)与S蛋白的亲和力的测定结果。
图3A-3C示例性地显示了抗原结合单元ABU-174(A)、ABU-175(B)、ABU190(C)对SARS-CoV-2假病毒的中和抑制活性的测定结果。
图4示例性地显示了ABU-175抗体对SARS-CoV-2真病毒的中和抑制活性的CPE测定结果。
图5示例性地显示了抗原结合单元ABU-174、ABU-175、和ABU190对SARS-CoV-2真病毒的中和抑制活性的PRNT测定结果。
具体实施方案
虽然本文已经示出并描述了本文所述的优选实施方案，但对于本领域技术人员显而易见的是，这样的实施方案只是通过示例的方式提供的。本领域技术人员在不脱离本文所述的情况下现将想到许多变化、改变和替代。应当理解，在实施本文所述的过程中可以采用本文所述的本文实施方案的各种替代方案。旨在用以下权利要求限定本文所述的范围，因此涵盖这些权利要求范围内的方法和结构及其等同物。
当提供数值范围时，应当理解，在此范围的上限与下限之间的每个中间值(精确到下限单位的十分之一，除非上下文另有明确规定)以及在所述范围内的其它任何所指出的或中间的值都包含在本发明内。这些较小范围的上限和下限可独立地包括在较小范围中，而且也包含在本发明内，除了所述范围内任何具体排除的限值。当所述范围包含一个或两个限值时，除去任一或两个包含在内的限值的范围也包含在本发明中。
如本文中所使用的，术语“多肽”、“肽”和“蛋白质”在本文中可互换使用，以指任何长度的氨基酸聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，并且可以被非氨基酸中断。该术语还包括已经被修饰的氨基酸聚合物，该修饰例如是通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解处理、磷酸化、异戊烯化、外消旋化、硒化、转移RNA介导的氨基酸向蛋白质的添加(如精氨酸化)、遍在蛋白化，或其它任何操作，如与标记组分的缀合。如本文所用的，术语“氨基酸”是指天然和/或非天然或合成的氨基酸，包括甘氨酸和D或L光学异构体，以及氨基酸类似物和拟肽。由指定蛋白质“衍生”的多肽或氨基酸序列是指多肽的起源。优选地，多肽具有与序列中编码的多肽的氨基酸序列基本相同的氨基酸序列，或其部分，其中该部分由至少10-20个氨基酸或至少20-30氨基酸或至少30-50个氨基酸组成，或者其可用序列中编码的多肽免疫学地标识。该术语还包括从指定核酸序列表达的多肽。如本文中所使用的，术语“结构域”是指蛋白质的一部分，它在物理上或功能上与该蛋白质或肽的其它部分区分开。物理上定义的结构域包括极具疏水性或亲水性的氨基酸序列，如那些膜结合的或细胞质结合的序列。结构域还可以通过例如由基因复制引起的内部同源性来定义。功能上定义的结构域具有不同的生物学功能。例如，抗原结合域是指抗原结合单元或抗体中与抗原结合的部分。功能上定义的结构域不需要由连续的氨基酸序列编码，且功能上定义的结构域可以含有一个或多个物理上定义的结构域。
如本文中所使用的，术语“氨基酸”是指天然的和/或非天然的或合成的氨基酸，包括但不限于D或L旋光异构体，以及氨基酸类似物和拟肽。标准的单字母或三字母代码用来指称氨基酸。在本文中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体 的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987 and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883，或IMGT(ImMunoGenTics)(Lefranc，M.-P.，The Immunologist，7，132-136(1999)；Lefranc，M.-P.et al.，Dev.Comp.Immunol.，27，55-77(2003))的定义。除非另有指出，本申请中抗体的VH和VL中的CDR均基于IMGT编码系统的定义。在Kabat编号系统下，VH中的CDR氨基酸残基编号为31-35(CDR1)，50-65(CDR2)和95-102(CDR3)；VL中的CDR氨基酸残基编号为24-34(CDR1)，50-56(CDR2)和89-97(CDR3)。在Chothia下，VH中的CDR氨基酸编号为26-32(CDR1)，52-56(CDR2)和95-102(CDR3)；VL中的氨基酸残基编号为24-34(CDR1)，50-56(CDR2)和89-97(CDR3)。在IMGT编号系统下，VH中CDR的氨基酸残基编号大约26-33(CDR1)，51-56(CDR2)和93-102(CDR3)；并且VL中的CDR氨基酸残基编号大约为27-32(CDR1)，50-51(CDR2)和89-97(CDR3)(如https：//www.novoprolabs.com/tools/cdr所公开)。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例 如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423 426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879 5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本文所述的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444 6448(1993)，和Poljak R.J.等人，Structure 2：1121 1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)可从给定的抗体(例如本文提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
在本文中，除非上下文明确指出，术语“抗原结合单元”包括上述定义的抗体及其抗原结合片段。
如本文中所使用的，术语“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见Journal of virological methods，2009，158(1-2)：171-179)。
如本文中所使用的，“中和抗体”是指，能清除或显著降低目标病毒的毒力(例如，感染细胞的能力)的抗体或抗体片段。
如本文中所使用的，在多肽的情况下，“序列”是多肽中的氨基酸在从氨基末端到羧基末端方向上的顺序，其中该序列中彼此相邻的残基在该多肽的一级结构中是连续的。序列也可以是已知在一个或两个方向上包含额外残基的多肽的一部分的线性序列。
如本文中所使用的，“同一性”、“同源性”或“序列同一性”是指在两个或更多个多核苷酸序列之间或在两个或更多个多肽序列之间的序列相似性或可互换性。当使用诸如Emboss Needle或BestFit等程序确定两个不同氨基酸序列之间的序列同一性、相似性或同源性时，可以使用默认设置，或者可以选择适当的打分矩阵，例如blosum45或blosum80，来优化同一性、相似性或同源性得分。优选地，同源的多核苷酸是那些在如本文所定义的严格条件下杂交并且与这些序列相比具有至少70％、优选至少80％、 更优选至少90％、更优选95％、更优选97％、更优选98％并且甚至更优选99％的序列同一性的多核苷酸。当对可比长度的序列进行最佳比对时，同源的多肽优选具有至少80％，或至少90％，或至少95％，或至少97％，或至少98％的序列同一性，或具有至少99％的序列同一性。
就本文所确定的抗原结合单元而言，“序列同一性百分比(％)”被定义为在比对序列并在必要情况下引入缺口以获得最大序列同一性百分比后，并且不把任何保守置换视为序列同一性的一部分，查询序列中与第二、参考多肽序列或其部分的氨基酸残基相同的氨基酸残基的百分比。可以以本领域技术内的各种方式，例如使用可公开获得的计算机软件，如BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件，来实现旨在确定氨基酸序列同一性百分比的比对。本领域技术人员可以确定用于测量比对的合适的参数，包括在所比较的序列的全长上获得最大比对所需的任何算法。同一性百分比可以在整个定义的多肽序列的长度上进行测量，或者可以在较短的长度上，例如，在从较大的、定义的多肽序列取得的片段的长度上进行测量，该片段例如是至少5、至少10、至少15、至少20、至少50、至少100或至少200个连续残基的片段。这些长度只是示例性的，并且应该理解，由本文的表格、附图或序列表中所示的序列支持的任何片段长度可以用来描述在其上可以测量同一性百分比的长度。
本文所述的抗原结合单元可具有相对于参考序列的一个或多个修饰。所述修饰可以是缺失、插入或添加，或氨基酸残基的取代或置换。“缺失”是指由于缺少一个或多个氨基酸残基而导致的氨基酸序列变化。“插入”或“添加”是指导致与参考序列相比添加一个或多个氨基酸残基的氨基酸序列变化。“取代”或“置换”是指一个或多个氨基酸被不同的氨基酸所取代。在本文中，可以通过将抗原结合单元与参考序列进行比较来确定抗原结合单元相对于参考序列的突变。用于比较的序列的最佳比对可以根据本领域的任何已知方法进行。
如本文中所使用的，术语“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质；其部分， 及其组合。非限制性的示例性抗原包括来自冠状病毒如SARS-CoV-2的蛋白，以及它们的其它同源物。
如本文中所使用的，术语“分离的”是指与细胞的和其它方面的成分分离的，其中在自然界中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在正常情况下与之相关联。本领域技术人员知晓，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”来与其天然存在的对应物区分开来。另外，“浓缩的”、“分离的”或“稀释的”多核苷酸、肽、多肽、蛋白质、抗体或其片段与其天然存在的对应物是可区分的，因为每单位体积的分子浓度或数目大于(“浓缩的”)或小于从其天然存在的对应物(“分离的”)。富集可基于绝对量来测量，例如每单位体积的溶液的重量，或者其可相对于来源混合物中存在的第二、可能干扰性的物质来测量。
术语“多核苷酸”、“核酸”、“核苷酸”和“寡核苷酸”可互换使用。它们是指任何长度的核苷酸(无论是脱氧核糖核苷酸还是核糖核苷酸)或其类似物的聚合形式。多核苷酸可具有任何三维结构，并且可以执行任何已知或未知的功能。以下是多核苷酸的非限制性实例：基因或基因片段的编码区或非编码区、从连锁分析确定的基因座、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、支链多核苷酸、质粒、载体、任意序列的分离的DNA、任意序列的分离的RNA、核酸探针、引物、寡核苷酸或合成的DNA。多核苷酸可以包含修饰的核苷酸，如甲基化核苷酸和核苷酸类似物。如果存在的话，对核苷酸结构的修饰可以在聚合物装配之前或之后赋予。核苷酸的序列可以被非核苷酸组分中断。多核苷酸可以在聚合后进一步修饰，例如通过与标记组分缀合。
当应用于多核苷酸时，“重组的”意味着该多核苷酸是克隆、限制酶切消化和/或连接步骤以及产生与自然界中发现的多核苷酸不同的构建体的其它程序的各种组合的产物。
术语“基因”或“基因片段”在本文中可互换使用。它们是指含有至少一个开放阅读框的多核苷酸，该开放阅读框能够在转录和翻译后编码特定蛋白质。基因或基因片段可以是基因组的、cDNA或合成的，只要该多核 苷酸含有至少一个开放阅读框，该开放阅读框可以覆盖整个编码区或其区段。
术语“可操作地连接”或“有效连接”是指并置，其中这样描述的组件所处的关系允许它们以其预期方式发挥作用。例如，如果启动子序列促进编码序列的转录，则该启动子序列与该编码序列可操作地连接。
如本文所用的，“表达”是指多核苷酸被转录为mRNA的过程，和/或转录的mRNA(也称为“转录物”)随后被翻译成肽、多肽或蛋白质的过程。转录物和被编码的多肽统称为基因产物。如果多核苷酸衍生自基因组DNA，则表达可以包括真核细胞中mRNA的剪接。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草芽孢杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“生物样品”包括多种从生物体获得的样品类型，并且可以在诊断或监测试验中使用。该术语包括血液和生物起源的其它液体样品，固体组织样品，如活检标本或组织培养物，或由其衍生的 细胞及其后代。该术语包括在获得后已经以任何方式进行处理的样品，例如通过用试剂处理、溶解或针对某些组分进行富集。该术语包括临床样品，并且还包括在细胞培养物、细胞上清液、细胞裂解物、血清、血浆、生物流体和组织样品中的细胞。
如本文中所使用的，术语“接受者”、“个体”、“受试者”、“宿主”和“患者”在本文中可互换使用，并且是指希望对其进行诊断、处理或治疗的任何哺乳动物受试者，特别是人类。
如本文中所使用的，术语“治疗”、“处理”等在本文中用来泛指获得所需的药理学和/或生理学效果。该效果就完全或部分防止疾病或其症状而言可以是预防性的，和/或就部分或完全稳定或治愈疾病和/或归因于该疾病的不良反应而言可以是治疗性的。如本文所用的“治疗”涵盖在哺乳动物中对疾病的任何治疗，该哺乳动物例如是小鼠、大鼠、兔、猪、灵长类动物，包括人类和其它猿类，特别是人，并且该术语包括：(a)防止疾病或症状在可能易患该疾病或症状但尚未发生诊断的受试者中发生；(b)抑制疾病症状；(C)阻止疾病的发展；(d)缓解疾病症状；(e)引起疾病或症状消退；或其任意组合。如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。在本文中，KD被定义为两个动力学速率常数Ka/Kd的比率，其中“Ka”指抗体与抗原结合的速率常数，“Kd”指抗体从抗体/抗原复合物中解离的速率常数。平衡解离常数KD越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M的解离平衡常数(KD)结合抗原。两分子间的特异性结合性质可使用本领域公知的方法进行测定，例如使用表面等离子体共振术(SPR)在BIACORE仪中测定。
如本文中所使用的，术语“中和活性”是指抗体或抗体片段具有与病毒上的抗原蛋白相结合，从而阻止病毒感染细胞和/或病毒子代的成熟和/或病毒子代的释放的功能活性，具有中和活性的抗体或抗体片段可以阻止病毒的扩增，从而抑制或消除病毒的感染。在一些实施方案中，所述中和活性通过抗体或抗体片段对病毒抑制的IC 50表示。“半数最大抑制浓度”(IC 50)是药物如抗体在抑制生物或生化功能等方面(如病毒效力)的量度。在本文中的IC 50根据抗原结合片段对病毒(例如假病毒或真病毒)感染细胞的中和抑制率，并利用Reed-Muench法计算。本文提供一种抗原结合单元，其能够特异性识别和靶向新型冠状病毒的S蛋白，特别是S蛋白的受体结合结构域(RBD)，并且显示出了高效的中和病毒的能力。因此，本文所述的抗原结合单元特别适合用于诊断、预防和治疗新型冠状病毒感染或与新型冠状病毒感染相关的疾病(例如新型冠状病毒肺炎)。
抗原结合单元
一方面，本文所述的抗原结合单元包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，且所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3。
本文所述的抗原结合单元的VH可包含选自SEQ ID NO.：721-1080和3111-3145的序列，与选自SEQ ID NO：721-1080和3111-3145的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VH与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH与参考多肽相比可存在1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个或20个添加、删除或取代。当本文所述的抗原结合单元的VH与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH与参考多肽相比可存在多于1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个 或20个添加、删除或取代。当本文所述的抗原结合单元的VH与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH与参考多肽相比可存在少于2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个或20个添加、删除或取代。
本文所述的抗原结合单元的VH CDR1可包含选自SEQ ID NO.：1461-1820和2901-2935的序列，与选自SEQ ID NO：1461-1820和2901-2935的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1461-1820和2901-2935的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VH CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR1与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR1与参考多肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR1与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VH CDR2可包含选自SEQ ID NO.：1821-2180和2936-2970的序列，与选自SEQ ID NO：1821-2180和2936-2970的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1821-2180和2936-2970的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VH CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR2与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR2与参考多 肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR2与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VH CDR3可包含选自SEQ ID NO.：1-360和2971-3005的序列，与选自SEQ ID NO：1-360和2971-3005的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1-360和2971-3005的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VH CDR3与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR3与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR3与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR3与参考多肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VH CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VH CDR3与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VL可包含选自SEQ ID NO.：1081-1440和3146-3180的序列，与选自SEQ ID NO：1081-1440和3146-3180的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VL与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL与参考多肽相比可存在1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个或20个添加、删除或取代。当本文所述的抗原结合单元的VL与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL与参 考多肽相比可存在多于1个、2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个或20个添加、删除或取代。当本文所述的抗原结合单元的VL与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL与参考多肽相比可存在少于2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个或20个添加、删除或取代。
本文所述的抗原结合单元的VL CDR1可包含选自选自SEQ ID NO.：2181-2540和3006-3040的序列，与选自SEQ ID NO：2181-2540和3006-3040的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2181-2540和3006-3040的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VL CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR1与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VL CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR1与参考多肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VL CDR1与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR1与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VL CDR2可包含选自SEQ ID NO.：2541-2900和3041-3075的序列，与选自SEQ ID NO：2541-2900和3041-3075的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2541-2900和3041-3075的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VL CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR2与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。 当本文所述的抗原结合单元的VL CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR2与参考多肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VL CDR2与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR2与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VL CDR3可包含选自SEQ ID NO.：361-720和3076-3110的序列，与选自SEQ ID NO：361-720和3076-3110的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：361-720和3076-3110的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。当本文所述的抗原结合单元的VL CDR3与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR3与参考多肽相比可存在1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VL CDR3与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR3与参考多肽相比可存在多于1个、2个、3个、4个、或5个添加、删除或取代。当本文所述的抗原结合单元的VL CDR3与参考多肽序列相比存在氨基酸添加、删除或取代时，本文所述的抗原结合单元的VL CDR3与参考多肽相比可存在少于2个、3个、4个、或5个添加、删除或取代。
本文所述的抗原结合单元的VH CDR1可包含选自SEQ ID NO.：1461-1820和2901-2935的序列，与选自SEQ ID NO：1461-1820和2901-2935的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1461-1820和2901-2935的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；并且本文所述的抗原结合单元的VL CDR1可包含选自SEQ ID NO.：2181-2540和3006-3040的序列，与选自SEQ ID NO：2181-2540和3006-3040的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2181-2540和3006-3040的序列具有至少80％、 85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
本文所述的抗原结合单元的VH CDR2可包含选自SEQ ID NO.：1821-2180和2936-2970的序列，与选自SEQ ID NO：1821-2180和2936-2970的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1821-2180和2936-2970的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；并且本文所述的抗原结合单元的VL CDR2可包含选自SEQ ID NO.：2541-2900和3041-3075的序列，与选自SEQ ID NO：2541-2900和3041-3075的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2541-2900和3041-3075的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
本文所述的抗原结合单元的VH CDR3可包含选自SEQ ID NO.：1-360和2971-3005的序列，与选自SEQ ID NO：1-360和2971-3005的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1-360和2971-3005的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；并且本文所述的抗原结合单元的VL CDR3可包含选自SEQ ID NO.：361-720和3076-3110的序列，与选自SEQ ID NO：361-720和3076-3110的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：361-720和3076-3110的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
本文所述的抗原结合单元的VH可包含VH CDR1、VH CDR2和VH CDR3，其中所述VH CDR1选自SEQ ID NO.：1461-1820和2901-2935的序列，与选自SEQ ID NO：1461-1820和2901-2935的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1461-1820和2901-2935的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；其中所述VH  CDR2选自SEQ ID NO.：1821-2180和2936-2970的序列，与选自SEQ ID NO：1821-2180和2936-2970的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1821-2180和2936-2970的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；且其中所述VH CDR3选自SEQ ID NO.：1-360和2971-3005的序列，与选自SEQ ID NO：1-360和2971-3005的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：1-360和2971-3005的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
本文所述的抗原结合单元的VL可包含VL CDR1、VL CDR2和VL CDR3，其中所述VL CDR1选自SEQ ID NO.：2181-2540和3006-3040的序列，与选自SEQ ID NO：2181-2540和3006-3040的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2181-2540和3006-3040的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；其中所述VL CDR2选自SEQ ID NO.：2541-2900和3041-3075的序列，与选自SEQ ID NO：2541-2900和3041-3075的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：2541-2900和3041-3075的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列；且其中所述VL CDR3选自SEQ ID NO.：361-720和3076-3110的序列，与选自SEQ ID NO：361-720和3076-3110的序列相比包含一个或多个氨基酸添加、删除、或取代的序列，或者与选自SEQ ID NO：361-720和3076-3110的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
本文所述的抗原结合单元的VH可包含选自以下CDR1、CDR2、和CDR3的组合的序列：
Figure PCTCN2021090146-appb-000001
Figure PCTCN2021090146-appb-000002
Figure PCTCN2021090146-appb-000003
Figure PCTCN2021090146-appb-000004
Figure PCTCN2021090146-appb-000005
Figure PCTCN2021090146-appb-000006
Figure PCTCN2021090146-appb-000007
Figure PCTCN2021090146-appb-000008
Figure PCTCN2021090146-appb-000009
Figure PCTCN2021090146-appb-000010
Figure PCTCN2021090146-appb-000011
Figure PCTCN2021090146-appb-000012
Figure PCTCN2021090146-appb-000013
Figure PCTCN2021090146-appb-000014
Figure PCTCN2021090146-appb-000015
Figure PCTCN2021090146-appb-000016
Figure PCTCN2021090146-appb-000017
Figure PCTCN2021090146-appb-000018
Figure PCTCN2021090146-appb-000019
Figure PCTCN2021090146-appb-000020
Figure PCTCN2021090146-appb-000021
Figure PCTCN2021090146-appb-000022
Figure PCTCN2021090146-appb-000023
Figure PCTCN2021090146-appb-000024
Figure PCTCN2021090146-appb-000025
Figure PCTCN2021090146-appb-000026
Figure PCTCN2021090146-appb-000027
Figure PCTCN2021090146-appb-000028
Figure PCTCN2021090146-appb-000029
Figure PCTCN2021090146-appb-000030
本文所述的抗原结合单元的所述的VH CDR1可包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR1相同的序列；本文所述的抗原结合单元的所述的VH CDR2可包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR2相同的序列；本文所述的抗原结合单元的所述的VH CDR3可包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR3相同的序列；所述抗原结合单元的所述VL CDR1可包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR1相同的序列；所述抗原结合单元的所述VL CDR2可包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR2相同的序列；和/或所述抗原结合单元的所述VL CDR3可包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR3相同的序列。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：2354、SEQ ID NO：2355、SEQ ID NO：2370、SEQ ID NO：2477、及SEQ ID NO：3012；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：2714、SEQ ID NO：2715、SEQ ID NO：2730、SEQ ID NO：2837、及SEQ ID NO：3047；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：534、SEQ ID NO：535、SEQ ID NO：550、SEQ ID NO：657、及SEQ ID NO：3082；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：1634、SEQ ID NO：1635、SEQ ID NO：1650、SEQ ID NO：1757、及SEQ ID NO：2907；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：1994、SEQ ID NO：1995、SEQ ID NO：2010、SEQ ID NO：2117、及SEQ ID NO：2942；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：174、SEQ ID NO：175、SEQ ID NO：190、SEQ ID NO：297、及SEQ ID NO：2977。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：2354；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：2714；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：534；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：1634；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：1994；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：174。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：2355；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：2715；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：535；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：1635；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：1995；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：175。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：2370；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：2730；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：550；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：1650；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：2010；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：190。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：2477；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：2837；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：657；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：1757；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：2117；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：297。
在一个实施方式中，本文提供的抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区CDR1氨基酸序列：SEQ ID NO：3012；
b.轻链可变区CDR2氨基酸序列：SEQ ID NO：3047；
c.轻链可变区CDR3氨基酸序列：SEQ ID NO：3082；
d.重链可变区CDR1氨基酸序列：SEQ ID NO：2907；
e.重链可变区CDR2氨基酸序列：SEQ ID NO：2942；及
f.重链可变区CDR3氨基酸序列：SEQ ID NO：2977。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：1377、及SEQ ID NO：3152；及
b.重链可变区氨基酸序列：SEQ ID NO：1017、及SEQ ID NO：3117。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：1254；及
b.重链可变区氨基酸序列：SEQ ID NO：894。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：1255；及
b.重链可变区氨基酸序列：SEQ ID NO：895。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：1270；及
b.重链可变区氨基酸序列：SEQ ID NO：910。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：1377；及
b.重链可变区氨基酸序列：SEQ ID NO：1017。
在一个实施方式中，本文提供的抗体包含一个、或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变区氨基酸序列：SEQ ID NO：3152；及
b.重链可变区氨基酸序列：SEQ ID NO：3117。
本文所述的抗原结合单元可与新型冠状病毒(SARS-CoV-2)S蛋白结合。本文所述的抗原结合单元可与新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。抗原结合单元与RBD的结合可的结合可通过本领域已知的任何方法来表征或表示。例如，结合可以用结合亲和力来表征，结合亲和力可以是抗原结合单元与抗原之间的相互作用的强度。结合亲和力可通过本领域中已知的任何方法，如体外结合试验来确定。本文抗原结合单元的结合亲和力可按照KD来表示，KD被定义为两个动力学速率常数Ka/Kd的比率，其中“Ka”指抗体与抗原结合的速率常数，“Kd”指抗体从抗体/抗原复合物中解离的速率常数。如本文公开的抗原结合单元以约10μM至约1fM范围内的KD特异性结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。例如，抗原结合单元可以以小于约10μM、1μM、0.1μM、50nM、20nM、15nM、10nM、5nM、4nM、3nM、2nM、1nM、0.5nM、0.1nM、50pM、10pM、1pM、0.1pM、10fM、1fM、0.1fM或小于0.1fM的KD特异性结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。本文公开的抗原结合单元可以以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
本文所述的抗原结合单元对新型冠状病毒(SARS-CoV-2)具有中和活性。本文所述的抗原结合单元对新型冠状病毒(SARS-CoV-2)的中和活性可通过假病毒进行分析。假病毒具有与疹病毒相似的细胞感染特点，能够模拟真病毒感染细胞的早期过程，并且可以安全、快速地进行检测分析。本文所述的抗原结合单元对新型冠状病毒(SARS-CoV-2)的中和活性可通过本领域已知的方法检测，如采用微孔细胞中和试验，参照Temperton N J等人，Emerg Infect Dis，2005，11(3)，411-416的描述进行。
本文所述的抗原结合单元对新型冠状病毒(SARS-CoV-2)的中和活性可采用实验细胞如Huh-7细胞以及假病毒SARS-CoV-2病毒检测。本文所述的抗原结合单元可以以例如小于100μg/ml、小于50μg/ml、小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、小于1ng/ml、小于0.5ng/ml、小于0.25ng/ml、小于0.2ng/ml、小于0.1ng/ml、小于50pg/ml、小于25pg/ml、小于20pg/ml、小于10pg/ml、小于5pg/ml、小于2.5pg/ml、小于2pg/ml、或小于1pg/ml的IC50中和新型冠状病毒(SARS-CoV-2)假病毒。
本文所述的抗原结合单元对新型冠状病毒(SARS-CoV-2)的中和活性可采用SARS-CoV-2真病毒通过空斑减少中和试验(Plaque Reduction Neutralization Test，PRNT)进行检测，通过孵育后空斑(plaque)的减少计算本文所述中的抗原结合单元中和SARS-CoV-2真病毒的IC50。本文所述的抗原结合单元可以以例如小于100μg/ml、小于50μg/ml、小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、小于1ng/ml、小于0.5ng/ml、小于0.25ng/ml、小于0.2ng/ml、小于0.1ng/ml、小于50pg/ml、小于25pg/ml、小于20pg/ml、小于10pg/ml、小于5pg/ml、小于2.5pg/ml、小于2pg/ml、或小于1pg/ml的IC50中和新型冠状病毒(SARS-CoV-2)真病毒。
抗原结合单元的制备
本文提供了产生任何本文公开的抗原结合单元的方法，其中该方法包括在适于表达抗原结合单元的条件下培养表达抗原结合单元的宿主细胞，以及分离由宿主细胞表达的抗原结合单元。
所表达的抗原结合单元可以使用本领域已知的多种蛋白质纯化技术分离。通常，抗原结合单元作为分泌的多肽从培养基中分离，尽管它们也可在无信号肽的情况下直接产生时从宿主细胞裂解物或细菌周质中回收。如果抗原结合单元是膜结合的，它们可以通过本领域技术人员常用的适当去污剂溶液溶解。回收的抗原结合单元可以通过盐沉淀(例如，用硫酸铵)、离子交换色谱法(例如在中性pH下在阳离子或阴离子交换柱上运行并用离子强度增加的步骤梯度洗脱)、凝胶过滤色谱法(包括凝胶过滤HPLC)以及标签亲和柱色谱法，或亲和树脂如蛋白A、蛋白G、羟基磷灰石和抗免疫球蛋白进一步纯化。
在本文所述的方法和组合物中可以使用添加了以下部分的衍生免疫球蛋白：化学连接体，可检测部分如荧光染料，酶，底物，化学发光部分，特异性结合部分如链霉亲和素、亲和素或生物素，或药物缀合物。
本文还提供与化学功能部分缀合的抗原结合单元。通常，该部分是能够产生可检测信号的标记。这些缀合的抗原结合单元可用于例如检测系统，如病毒感染严重程度、感染灶的成像等。此类标记是本领域已知的，并且包括但不限于放射性同位素、酶、荧光化合物、化学发光化合物、生物发光化合物底物辅因子和抑制剂。教导使用此类标签的专利的实例参见美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；和4,366,241。该部分可以与抗原结合单元共价连接、重组连接或通过第二试剂如第二抗体、蛋白A或生物素-亲和素复合物与抗原结合单元缀合。
其它功能部分包括信号肽、增强免疫反应性的试剂、促进与固体支持物偶联的试剂、疫苗载体、生物反应调节剂、顺磁标记和药物。 信号肽是短氨基酸序列，其引导新合成的蛋白质通过细胞膜(通常是真核细胞中的内质网)以及细菌的内膜或内膜和外膜二者。信号肽可以位于多肽的N-末端部分或多肽的C-末端部分，并且信号肽可以在多肽的生物合成与分泌之间从细胞酶促除去。这类肽可以被引入抗原结合单元中，以允许合成分子的分泌。
增强免疫反应性的试剂包括但不限于细菌超抗原。促进与固体支持物偶联的试剂包括但不限于生物素或亲和素。免疫原载体包括但不限于任何生理学上可接受的缓冲液。生物反应调节剂包括细胞因子，特别是肿瘤坏死因子(TNF)、白介素-2、白介素-4、粒细胞巨噬细胞集落刺激因子和γ-干扰素。
化学功能部分可以重组制备，例如通过产生编码抗原结合单元和功能部分的融合基因。或者，抗原结合单元可以通过各种熟知的化学程序中的任何一种化学键合到该部分。例如，当该部分是蛋白质时，连接可以通过异双官能交联剂，例如，SPDP、碳二亚胺戊二醛等。该部分可以通过第二试剂，如第二抗体、蛋白A或生物素-亲和素复合物共价连接或缀合。顺磁部分及其与抗体的缀合在本领域中是公知的。参见，例如，Miltenyi等人(1990)Cytometry 11：231-238。
核酸
在一个方面，本文提供了编码本文所述的抗原结合单元的分离的多核苷酸。对应于现有抗体的L或H链的各个区域的核苷酸序列可以使用常规技术(包括但不限于杂交、PCR和DNA测序)容易地获得并进行测序。产生单克隆抗体的杂交瘤细胞用作抗体核苷酸序列的优选来源。可以从公共或私人储存库获得产生一系列单克隆抗体的大量杂交瘤细胞。最大的储藏机构是美国典型培养物保藏中心(American Type Culture Collection)，它提供了多种不同的充分表征的杂交瘤细胞系。或者，抗体核苷酸可以从免疫的或未免疫的啮齿动物或人，以及从诸如脾和外周血淋巴细胞的器官获得。适用于提取和合成抗体核苷酸的具体技术描述于Orlandi等人(1989)Proc.Natl.Acad.Sci.U.S.A 86： 3833-3837；Larrick等人1989)biochem.Biophys.Res.Commun.160：1250-1255；Sastry等人(1989)Proc.Natl.Acad.Sci.，U.S.A.86：5728-5732；以及美国专利号5,969,108。
还可以修饰抗体核苷酸序列，例如，通过用编码序列取代人重链和轻链恒定区从而代替同源非人序列。以这种方式，制备嵌合抗体，其保留原始抗体的结合特异性。
另外，可以对编码抗原结合单元的重链和/或轻链的多核苷酸进行密码子优化，以实现受试者抗原结合单元在所需宿主细胞中的优化表达。例如，在一种密码子优化方法中，天然密码子被来自参考基因组的最常见密码子所取代，其中每种氨基酸的密码子翻译速率被设计得较高。用于生成用于表达所需蛋白质的密码子优化的多核苷酸的另外的示例性方法描述于Kanaya等人，Gene，238：143-155(1999)，Wang等人，Mol.Biol.Evol.，18(5)：792-800(2001)，美国专利号5,795,737，美国公开号2008/0076161和WO 2008/000632中，所述方法可应用于抗原结合单元的重链和/或轻链。
本文内容的多核苷酸包括编码示例性多肽的功能等同物及其片段的多核苷酸。
由于遗传密码的简并性，L和H序列的核苷酸以及适于构建本文所述的多核苷酸和载体的异二聚化序列可以有相当大的变异。这些变异包含在本文内容中。
治疗方法
本文提供了使用本文所述的抗原结合单元来预防或治疗受试者的新型冠状病毒(SARS-CoV-2)感染的方法，包括向所述受试者给予本文所述的抗原结合单元。
本文提供了使用本文所述的抗原结合单元与第二药剂联合治疗哺乳动物的疾病、病况或病症的方法。第二药剂可以与抗体一起、在抗体之前或之后施用。所述第二药剂可以是抗病毒剂。抗病毒剂包括但不限于特拉匹韦(telaprevir)、波普瑞韦(boceprevir)、西美瑞韦 (semiprevir)、索菲布韦(sofosbuvir)、达拉他韦(daclastavir)、阿那匹韦(asunaprevir)、拉米夫定(lamivudine)、阿德福韦(adefovir)、恩替卡韦(entecavir)、替诺福韦(tenofovir)、替比夫定(telbivudine)、干扰素α和PEG化干扰素α。所述第二药剂可以选自羟氯喹、氯喹、法维拉韦、金西单抗(Gimsilumab)、AdCOVID(University of Alabama at Birmingham)、AT-100(Airway Therapeutics)、TZLS-501(Tiziana Life Sciences)、OYA1(OyaGen)、BPI-002(BeyondSpring)、INO-4800(Inovio Pharmaceutical)、NP-120(ifenprodil)、瑞德西韦(GS-5734)、Actemra(Roche)、加利地韦(BCX4430)、SNG001(Synairgen Research)、或其组合。
所述第二药剂可以是用于缓解受试者并发的炎性病况的症状的药剂。所述抗炎剂包括非甾体抗炎药(NSAID)和皮质类固醇。NSAID包括但不限于水杨酸盐，如乙酰水杨酸；二氟尼柳、水杨酸和双水杨酯；丙酸衍生物，如布洛芬；萘普生；右布洛芬、右酮洛芬、氟比洛芬、奥沙普嗪、非诺洛芬、洛索洛芬和酮洛芬；乙酸衍生物，如吲哚美辛、双氯芬酸、托美汀、醋氯芬酸、舒林酸、萘丁美酮、依托度酸和酮咯酸；烯醇酸衍生物，如吡罗昔康、氯诺昔康、美洛昔康、伊索昔康、替诺昔康、苯基丁氮酮和屈恶昔康；邻氨基苯甲酸衍生物，如甲芬那酸、氟芬那酸、甲氯芬那酸和托芬那酸；选择性COX-2抑制剂，如塞来昔布、罗美昔布、罗非昔布、艾托考昔、伐地考昔、非罗考昔和帕瑞考昔；磺胺尼西林，如尼美舒利；和其他非甾体抗炎药如氯尼辛和利考非隆。皮质类固醇包括但不限于可的松、地塞米松、氢化可的松、甲泼尼龙、泼尼松和泼尼松龙。
所述第二药剂可以是免疫抑制剂。可与抗原结合单元组合使用的免疫抑制剂包括但不限于羟氯喹、柳氮磺胺吡啶、来氟米特、依那西普、英夫利昔单抗、阿达木单抗、D-青霉胺、口服金化合物、可注射金化合物(肌内注射)、米诺环素、硫代苹果酸金钠、金诺芬、D-青霉胺、氯苯扎利、布西拉明、阿克他利、环磷酰胺、硫唑嘌呤、甲氨蝶呤、咪唑立宾、环孢菌素和他克莫司。
具体剂量将根据所选择的特定抗原结合单元、要遵循的给药方案、是否与其他药剂组合施用、施用时间、施用的组织以及携带该特定抗原结合单元的物理递送系统而变化。在一些实施方案中，在治疗周期的过程中，平均每周向受试者施用约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69或70mg范围内的抗原结合单元。例如，每周向受试者施用约35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54或55mg范围内的抗原结合单元。在一些实施方案中，每周向受试者施用约40、41、42、43、44、45、46、47、48、49、50、51、52、53、54或55mg范围内的抗原结合单元。
在治疗周期的过程中，可以平均每天以大于1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5或10mg的量向受试者施用抗原结合单元。例如，在治疗周期的过程中，平均每天以约6至10mg、约6.5至9.5mg、约6.5至8.5mg、约6.5至8mg或约7至9mg的量向受试者施用抗原结合单元。
抗原结合单元的剂量可以是约、至少约或至多约0.1、0.5、1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、125、150、175、200、225、250、275、300、325、350、375、400、425、450、475、500、525、550、575、600、625、650、675、700、725、750、775、800、825、850、875、900、925、950、975、1000mg或mg/kg，或者其中可衍生的任何范围。预期mg/kg的剂量是指每千克受试者总体重的抗原结合单元mg量。预期当给予患者多剂量时，剂量可以在量上变化或者它们可以是相同的。
药物组合物
本文提供了药物组合物，其包含受试者抗体或其功能片段和药学上可接受的载体、赋形剂或稳定剂，其包括但不限于惰性固体稀释剂和填充剂、稀释剂、无菌水溶液和各种有机溶剂、渗透促进剂、增溶剂和佐剂。(Remington′s Pharmaceutical Sciences第16版，Osol，A.编著(1980))。
药物组合物可以是适合于单次施用精确剂量的单位剂型。药物组合物可进一步包含抗原结合单元作为活性成分，并且可包括常规的药物载体或赋形剂。此外，它可包括其他药物或药剂、载体、佐剂等。示例性的肠胃外施用形式包括活性多肽和/或PEG修饰的多肽在无菌水溶液中的溶液或悬浮液，例如丙二醇水溶液或右旋糖溶液。如果需要，此类剂型可以适当地用盐如组氨酸和/或磷酸盐缓冲。
所述组合物可进一步包括一种或多种药学上可接受的添加剂和赋形剂。这些添加剂和赋形剂包括但不限于防粘剂、消泡剂、缓冲剂、聚合物、抗氧化剂、防腐剂、螯合剂、粘度调节剂(viscomodulator)、张力调节剂、调味剂、着色剂、增味剂、遮光剂、悬浮剂、粘合剂、填充剂、增塑剂、润滑剂及其混合物。
试剂盒
本文所述的试剂盒包含本文所述的抗原结合单元或如本文所述的其缀合物。还提供了本文所述的抗原结合单元在制备试剂盒中的用途，所述试剂盒用于检测新型冠状病毒或其S蛋白或S蛋白的RBD在样品中的存在或其水平，或用于诊断受试者是否感染了新型冠状病毒。
在一些实施方案中，所述样品包括但不限于来自受试者(例如哺乳动物，优选人)的排泄物、口腔或鼻腔分泌物、肺泡灌洗液等。
使用抗体或其抗原结合片段来检测目标病毒或抗原(例如，新型冠状病毒或其S蛋白或S蛋白的RBD)在样品中的存在或其水平的一般方法是本领域技术人员所熟知的。在一些实施方案中，所述检测方法可以使用酶联免疫吸附(ELISA)、酶免疫检测、化学发光免疫检测、放射免疫检测、荧光免疫检测、免疫色谱法、竞争法及类似检测方法。
实施例
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本文中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：记忆B细胞的分离
采集感染SARS-CoV-2病毒且痊愈出院的人员的血液(由北京佑安医院提供)，并在P2+生物安全实验室中，利用STEMCELL SepMate TM-15(Stemcell Technologies，产品目录号：86415)进行PBMCs的提取。随后，根据制造商的说明书，使用STEMCELL EasySep Human Memory B Cell Isolation Kit(Stemcell Technologies，产品目录号：17864)对提取的PBMCs进行记忆B细胞的富集。
实施例2：抗原结合单元序列的获得与鉴定
根据制造商的说明书，使用Chromium Single Cell V(D)J Reagent Kits(购自10X genomics，产品目录号：100006)对上述富集后的记忆B细胞进行单细胞转录组的VDJ测序。对测序结果进行分析，获得360株抗原结合单元，分别命名为ABU 1-395。其中所获得的抗原结合单元的序列信息如下表1所示。
表1本文中获得的示例性抗原结合单元
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-1 721 1081
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-2 722 1082
ABU-3 723 1083
ABU-4 724 1084
ABU-5 725 1085
ABU-6 726 1086
ABU-7 727 1087
ABU-8 728 1088
ABU-9 729 1089
ABU-10 730 1090
ABU-11 731 1091
ABU-12 732 1092
ABU-13 733 1093
ABU-14 734 1094
ABU-15 735 1095
ABU-16 736 1096
ABU-17 737 1097
ABU-18 738 1098
ABU-19 739 1099
ABU-20 740 1100
ABU-21 741 1101
ABU-22 742 1102
ABU-23 743 1103
ABU-24 744 1104
ABU-25 745 1105
ABU-26 746 1106
ABU-27 747 1107
ABU-28 748 1108
ABU-29 749 1109
ABU-30 750 1110
ABU-31 751 1111
ABU-32 752 1112
ABU-33 753 1113
ABU-34 754 1114
ABU-35 755 1115
ABU-36 756 1116
ABU-37 757 1117
ABU-38 758 1118
ABU-39 759 1119
ABU-40 760 1120
ABU-41 761 1121
ABU-42 762 1122
ABU-43 763 1123
ABU-44 764 1124
ABU-45 765 1125
ABU-46 766 1126
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-47 767 1127
ABU-48 768 1128
ABU-49 769 1129
ABU-50 770 1130
ABU-51 771 1131
ABU-52 772 1132
ABU-53 773 1133
ABU-54 774 1134
ABU-55 775 1135
ABU-56 776 1136
ABU-57 777 1137
ABU-58 778 1138
ABU-59 779 1139
ABU-60 780 1140
ABU-61 781 1141
ABU-62 782 1142
ABU-63 783 1143
ABU-64 784 1144
ABU-65 785 1145
ABU-66 786 1146
ABU-67 787 1147
ABU-68 788 1148
ABU-69 789 1149
ABU-70 790 1150
ABU-71 791 1151
ABU-72 792 1152
ABU-73 793 1153
ABU-74 794 1154
ABU-75 795 1155
ABU-76 796 1156
ABU-77 797 1157
ABU-78 798 1158
ABU-79 799 1159
ABU-80 800 1160
ABU-81 801 1161
ABU-82 802 1162
ABU-83 803 1163
ABU-84 804 1164
ABU-85 805 1165
ABU-86 806 1166
ABU-87 807 1167
ABU-88 808 1168
ABU-89 809 1169
ABU-90 810 1170
ABU-91 811 1171
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-92 812 1172
ABU-93 813 1173
ABU-94 814 1174
ABU-95 815 1175
ABU-96 816 1176
ABU-97 817 1177
ABU-98 818 1178
ABU-99 819 1179
ABU-100 820 1180
ABU-101 821 1181
ABU-102 822 1182
ABU-103 823 1183
ABU-104 824 1184
ABU-105 825 1185
ABU-106 826 1186
ABU-107 827 1187
ABU-108 828 1188
ABU-109 829 1189
ABU-110 830 1190
ABU-111 831 1191
ABU-112 832 1192
ABU-113 833 1193
ABU-114 834 1194
ABU-115 835 1195
ABU-116 836 1196
ABU-117 837 1197
ABU-118 838 1198
ABU-119 839 1199
ABU-120 840 1200
ABU-121 841 1201
ABU-122 842 1202
ABU-123 843 1203
ABU-124 844 1204
ABU-125 845 1205
ABU-126 846 1206
ABU-127 847 1207
ABU-128 848 1208
ABU-129 849 1209
ABU-130 850 1210
ABU-131 851 1211
ABU-132 852 1212
ABU-133 853 1213
ABU-134 854 1214
ABU-135 855 1215
ABU-136 856 1216
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-137 857 1217
ABU-138 858 1218
ABU-139 859 1219
ABU-140 860 1220
ABU-141 861 1221
ABU-142 862 1222
ABU-143 863 1223
ABU-144 864 1224
ABU-145 865 1225
ABU-146 866 1226
ABU-147 867 1227
ABU-148 868 1228
ABU-149 869 1229
ABU-150 870 1230
ABU-151 871 1231
ABU-152 872 1232
ABU-153 873 1233
ABU-154 874 1234
ABU-155 875 1235
ABU-156 876 1236
ABU-157 877 1237
ABU-158 878 1238
ABU-159 879 1239
ABU-160 880 1240
ABU-161 881 1241
ABU-162 882 1242
ABU-163 883 1243
ABU-164 884 1244
ABU-165 885 1245
ABU-166 886 1246
ABU-167 887 1247
ABU-168 888 1248
ABU-169 889 1249
ABU-170 890 1250
ABU-171 891 1251
ABU-172 892 1252
ABU-173 893 1253
ABU-174 894 1254
ABU-175 895 1255
ABU-176 896 1256
ABU-177 897 1257
ABU-178 898 1258
ABU-179 899 1259
ABU-180 900 1260
ABU-181 901 1261
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-182 902 1262
ABU-183 903 1263
ABU-184 904 1264
ABU-185 905 1265
ABU-186 906 1266
ABU-187 907 1267
ABU-188 908 1268
ABU-189 909 1269
ABU-190 910 1270
ABU-191 911 1271
ABU-192 912 1272
ABU-193 913 1273
ABU-194 914 1274
ABU-195 915 1275
ABU-196 916 1276
ABU-197 917 1277
ABU-198 918 1278
ABU-199 919 1279
ABU-200 920 1280
ABU-201 921 1281
ABU-202 922 1282
ABU-203 923 1283
ABU-204 924 1284
ABU-205 925 1285
ABU-206 926 1286
ABU-207 927 1287
ABU-208 928 1288
ABU-209 929 1289
ABU-210 930 1290
ABU-211 931 1291
ABU-212 932 1292
ABU-213 933 1293
ABU-214 934 1294
ABU-215 935 1295
ABU-216 936 1296
ABU-217 937 1297
ABU-218 938 1298
ABU-219 939 1299
ABU-220 940 1300
ABU-221 941 1301
ABU-222 942 1302
ABU-223 943 1303
ABU-224 944 1304
ABU-225 945 1305
ABU-226 946 1306
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-227 947 1307
ABU-228 948 1308
ABU-229 949 1309
ABU-230 950 1310
ABU-231 951 1311
ABU-232 952 1312
ABU-233 953 1313
ABU-234 954 1314
ABU-235 955 1315
ABU-236 956 1316
ABU-237 957 1317
ABU-238 958 1318
ABU-239 959 1319
ABU-240 960 1320
ABU-241 961 1321
ABU-242 962 1322
ABU-243 963 1323
ABU-244 964 1324
ABU-245 965 1325
ABU-246 966 1326
ABU-247 967 1327
ABU-248 968 1328
ABU-249 969 1329
ABU-250 970 1330
ABU-251 971 1331
ABU-252 972 1332
ABU-253 973 1333
ABU-254 974 1334
ABU-255 975 1335
ABU-256 976 1336
ABU-257 977 1337
ABU-258 978 1338
ABU-259 979 1339
ABU-260 980 1340
ABU-261 981 1341
ABU-262 982 1342
ABU-263 983 1343
ABU-264 984 1344
ABU-265 985 1345
ABU-266 986 1346
ABU-267 987 1347
ABU-268 988 1348
ABU-269 989 1349
ABU-270 990 1350
ABU-271 991 1351
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-272 992 1352
ABU-273 993 1353
ABU-274 994 1354
ABU-275 995 1355
ABU-276 996 1356
ABU-277 997 1357
ABU-278 998 1358
ABU-279 999 1359
ABU-280 1000 1360
ABU-281 1001 1361
ABU-282 1002 1362
ABU-283 1003 1363
ABU-284 1004 1364
ABU-285 1005 1365
ABU-286 1006 1366
ABU-287 1007 1367
ABU-288 1008 1368
ABU-289 1009 1369
ABU-290 1010 1370
ABU-291 1011 1371
ABU-292 1012 1372
ABU-293 1013 1373
ABU-294 1014 1374
ABU-295 1015 1375
ABU-296 1016 1376
ABU-297 1017 1377
ABU-298 1018 1378
ABU-299 1019 1379
ABU-300 1020 1380
ABU-301 1021 1381
ABU-302 1022 1382
ABU-303 1023 1383
ABU-304 1024 1384
ABU-305 1025 1385
ABU-306 1026 1386
ABU-307 1027 1387
ABU-308 1028 1388
ABU-309 1029 1389
ABU-310 1030 1390
ABU-311 1031 1391
ABU-312 1032 1392
ABU-313 1033 1393
ABU-314 1034 1394
ABU-315 1035 1395
ABU-316 1036 1396
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-317 1037 1397
ABU-318 1038 1398
ABU-319 1039 1399
ABU-320 1040 1400
ABU-321 1041 1401
ABU-322 1042 1402
ABU-323 1043 1403
ABU-324 1044 1404
ABU-325 1045 1405
ABU-326 1046 1406
ABU-327 1047 1407
ABU-328 1048 1408
ABU-329 1049 1409
ABU-330 1050 1410
ABU-331 1051 1411
ABU-332 1052 1412
ABU-333 1053 1413
ABU-334 1054 1414
ABU-335 1055 1415
ABU-336 1056 1416
ABU-337 1057 1417
ABU-338 1058 1418
ABU-339 1059 1419
ABU-340 1060 1420
ABU-341 1061 1421
ABU-342 1062 1422
ABU-343 1063 1423
ABU-344 1064 1424
ABU-345 1065 1425
ABU-346 1066 1426
ABU-347 1067 1427
ABU-348 1068 1428
ABU-349 1069 1429
ABU-350 1070 1430
ABU-351 1071 1431
ABU-352 1072 1432
ABU-353 1073 1433
ABU-354 1074 1434
ABU-355 1075 1435
ABU-356 1076 1436
ABU-357 1077 1437
ABU-358 1078 1438
ABU-359 1079 1439
ABU-360 1080 1440
ABU-361 3111 3146
ABU No. VH SEQ ID No. VL SEQ ID NO.
ABU-362 3112 3147
ABU-363 3113 3148
ABU-364 3114 3149
ABU-365 3115 3150
ABU-366 3116 3151
ABU-367 3117 3152
ABU-368 3118 3153
ABU-369 3119 3154
ABU-370 3120 3155
ABU-371 3121 3156
ABU-372 3122 3157
ABU-373 3123 3158
ABU-374 3124 3159
ABU-375 3125 3160
ABU-376 3126 3161
ABU-377 3127 3162
ABU-378 3128 3163
ABU-379 3129 3164
ABU-380 3130 3165
ABU-381 3131 3166
ABU-382 3132 3167
ABU-383 3133 3168
ABU-384 3134 3169
ABU-385 3135 3170
ABU-386 3136 3171
ABU-387 3137 3172
ABU-388 3138 3173
ABU-389 3139 3174
ABU-390 3140 3175
ABU-391 3141 3176
ABU-392 3142 3177
ABU-393 3143 3178
ABU-394 3144 3179
ABU-395 3145 3180
实施例3：本文中抗原结合单元的制备和纯化
根据实施例2中获得的抗原结合单元的序列信息，委托北京义翘神州有限公司表达和纯化所获得的抗原结合单元，并检测了它们的抗原反应性。
简言之，在体外合成编码抗体重链和轻链的核酸分子，然后分别克隆至表达载体中，从而得到分别编码抗体重链和轻链的重组表达载体。将上 述得到的分别编码抗体重链和轻链的重组表达载体共转染HEK293细胞。转染4-6小时后，将细胞培养液更换成无血清的培养基，并且继续在37℃下培养6天。培养结束后，通过亲和纯化柱从培养物中纯化细胞所表达的抗体蛋白。随后，通过还原性和非还原性SDS-PAGE检测所纯化的目的蛋白。其中，以ABU-174、ABU-175和ABU190为例，其制备后的电泳结果分别如图1A-1C所示。结果显示，经纯化的ABU-174、ABU-175和ABU190的纯度分别为95.9％、96.4％、98.2％。
随后，使用重组表达的S蛋白RBD作为包被抗原，使用辣根过氧化物酶(HRP)标记的Goat anti-human IgG Fc作为二抗，通过ELISA实验，检测经纯化的待测抗体的抗原反应性。简言之，用重组表达的S蛋白RBD(其氨基酸序列如SEQ ID NO：1459所示，浓度为0.01μg/ml或1μg/ml)包被96孔板，随后用封闭液对96孔板进行封闭。然后，分别加入待测单抗(对照抗体、ABU-174、ABU-175和ABU190；浓度分别为0.1μg/ml)，并孵育。用ELISA洗涤液进行洗涤后，加入辣根过氧化物酶(HRP)标记的Goat anti-human IgG Fc作为二抗(以1：500稀释)，并继续孵育。然后，用PBST洗涤酶标板，并加入显色剂显色。随后在酶标仪上读取OD450nm的吸收值。结果如表2所示。由表2可知，ABU-174、ABU-175和ABU190均能够特异性识别并结合S蛋白RBD。
表2：通过ELISA检测的ABU-174、ABU-175和ABU190抗原结合单元与S蛋白RBD的反应性(OD450读数)
Figure PCTCN2021090146-appb-000031
实施例4：本文中的抗原结合单元与S蛋白的结合能力的评估
本实施例采用表面等离子体共振技术(SPR)检测抗体与Spike蛋白RBD区域的亲和力。使用Biacore T200进行测量，先将生物素标记的SARS-COV-2RBD结构域偶联到SA芯片(GE公司)上，信号共振单位RU值上升100个单位。运行缓冲液是PH 7.4的PBS加上0.005％P20，确保分析物(如抗体)中的缓冲液与运行缓冲液一致。3倍梯度稀释纯化好的抗体，使其浓度在50-0.78125nM间。测量结果使用Biacore Evaluation软件进行分析，1∶1模型拟合所有曲线，获得抗体与抗原结合的速率常数Ka以及抗体从抗体/抗原复合物中解离的速率常数Kd，并计算解离平衡常数KD，其中KD＝Kd/Ka，结果如下表3所示。
表3列举了本文中示例性抗原结合单元与Spike蛋白RBD区域的结合亲和力，其中各抗原结合单元的KD值均小于20nM。
表3.本文示例性抗原结合单元与Spike蛋白RBD区域的结合亲和力的KD值
Figure PCTCN2021090146-appb-000032
Figure PCTCN2021090146-appb-000033
Figure PCTCN2021090146-appb-000034
图2A-2E进一步示例性地显示了ABU-174、ABU-175、ABU190、ABU297和ABU367与Spike蛋白RBD区域的结合亲和力。由图2A-2E可见，ABU-174的KD值为0.29nM，ABU-175的KD值为0.039nM，ABU190的KD值为2.8nM，ABU297的KD值为0.824nM，以及ABU的KD值为0.18nM。图2A-2E表明，ABU-174、ABU-175、ABU190、ABU297和ABU367与新型冠状病毒S蛋白均具有良好的亲和力。
实施例5：本文抗原结合单元中和SARS-CoV-2假病毒的能力的评估
在本实施例中，参照Temperton N J等人，Emerg Infect Dis，2005，11(3)，411-416的描述，利用微孔细胞中和实验法，检测了本文中的抗原结合单元对SARS-CoV-2假病毒的中和活性。本实施例所应用的SARS-CoV-2假病毒由中国食品药品检定研究院提供，其具有与真病毒相似的细胞感染特点，能够模拟真病毒感染细胞的早期过程，并且携带报告基因luciferase，可以快速方便地进行检测分析。操作假病毒的安全性高，在P2级实验室内就可完成中和实验，检测抗体的中和活性(Neutralization titer)。实验方法的具体步骤如下。
1.平衡试剂
将保存于2-8℃的试剂(0.25％胰酶-EDTA，DMEM完全培养基)取出，室温平衡30分钟以上。
2.试验操作
(1)取96孔板，按照表4所示，设置样品的排布方式；其中，A2-H2的孔设置为细胞对照孔(CC)，其仅含有实验细胞；A3-H3的孔设置为病毒对照孔(VV)，其含有实验细胞和假病毒；A4-A11、B4-B11、C4-C11、D4-D11、E4-E11、F4-F11、G4-G11、H4-H11的孔设置为实验孔，其含有实验细胞、假病毒以及不同浓度的待测抗体；其余的孔设置为空白。本实施例中所使用的实验细胞和假病毒分别为Huh-7细胞和SARS-CoV-2病毒(均由中国食品药品检定研究院提供)。
表4.96孔板中样品的排布方式
1 2 3 4 5-10 11 12
A - CC VV 稀释度1 稀释度1 稀释度1 -
B - CC VV 稀释度2 稀释度2 稀释度2 -
C - CC VV 稀释度3 稀释度3 稀释度3 -
D - CC VV 稀释度4 稀释度4 稀释度4 -
E - CC VV 稀释度5 稀释度5 稀释度5 -
F - CC VV 稀释度6 稀释度6 稀释度6 -
G - CC VV 稀释度7 稀释度7 稀释度7 -
H - CC VV 稀释度8 稀释度8 稀释度8 -
(2)在细胞对照孔中加入100μl/孔的DMEM完全培养基(含有1％的抗生素，25mM HEPES，10％FBS)；在病毒对照孔中加入100μl/孔的DMEM完全培养基；并且，在实验孔中加入50μl/孔的指定浓度的、稀释于DMEM完全培养基中的待测抗体。表4中所使用的稀释度1-8的抗体浓度分别为1/30μg/μl，1/90μg/μl，1/270μg/μl，1/810μg/μl，1/2430μg/μl，1/7290μg/μl，1/21870μg/μl，1/65610μg/μl。
(3)用DMEM完全培养基将SARS-CoV-2假病毒稀释至约1.3×10 4/ml(TCID50)；然后向病毒对照孔和实验孔中添加50μl/孔的SARS-CoV-2假 病毒。
(4)将96孔板置于细胞培养箱中(37℃，5％CO 2)孵育1小时。
(5)用DMEM完全培养基将预先培养好的Huh-7细胞稀释至2×10 5个/ml。在前一步骤的孵育结束后，向细胞对照孔、病毒对照孔和实验孔中添加100μl/孔的细胞。
(6)将96孔板置于细胞培养箱中(37℃，5％CO 2)培养20-28小时。
(7)从细胞培养箱中取出96孔板，从每个孔中吸弃150μl上清，然后加入100μl荧光素酶检测试剂，室温避光反应2min。
(8)反应结束后，用移液器将各个孔中的液体反复吹吸6～8次，使细胞充分裂解。然后，从每孔中吸出150μl液体，转移至对应的96孔化学发光检测板中，用化学发光检测仪(Perkinelmer EnSight多功能酶标仪)读取发光值。
(9)计算中和抑制率：
抑制率＝[1-(实验孔的发光强度均值-CC孔的发光强度均值)/(VV孔的发光强度均值-CC孔的发光强度均值)]×100％。
(10)根据中和抑制率的结果，利用Reed-Muench法计算待测抗体的IC50。
表5列举了本文中示例性抗原结合单元中和SARS-CoV-2假病毒的IC50，其中各抗原结合单元的IC50值均小于1μg/ml。
表5.本文示例性抗原结合单元中和SARS-CoV-2假病毒的IC50
ABU No. IC50(μg/ml)
ABU-174 ＜0.1
ABU-175 ＜0.1
ABU-190 ＜0.1
ABU-207 ＜0.5
ABU-208 ＜0.5
ABU-257 ＜0.5
ABU-290 ＜0.1
ABU-291 ＜0.5
ABU-296 ＜0.1
ABU-297 ＜0.1
ABU-308 ＜0.5
ABU-322 ＜0.1
ABU-340 ＜0.5
ABU-341 ＜0.1
ABU-344 ＜1
ABU-349 ＜0.1
ABU-351 ＜0.1
ABU-352 ＜0.1
ABU-354 ＜0.1
ABU-355 ＜0.1
ABU-356 ＜0.1
ABU-357 ＜1
ABU-358 ＜0.1
ABU-359 ＜0.1
ABU-360 ＜0.1
ABU-361 ＜0.5
ABU-362 ＜0.5
ABU-365 ＜0.1
ABU-367 ＜0.1
ABU-368 ＜0.5
ABU-369 ＜0.1
ABU-371 ＜1
ABU-372 ＜0.5
ABU-373 ＜0.5
ABU-375 ＜0.1
ABU-376 ＜0.1
ABU-377 ＜0.5
ABU-379 ＜0.5
ABU-380 ＜0.1
ABU-381 ＜0.1
ABU-382 ＜0.1
ABU-386 ＜0.1
ABU-391 ＜1
ABU-392 ＜0.1
ABU-395 ＜0.1
图3A-3C进一步示例性地显示了ABU-174、ABU-175和ABU190对SARS-CoV-2假病毒的中和活性。由图3A-3C可见，ABU-174、ABU-175和ABU190均具有良好的中和活性，其IC50分别为0.026μg/ml(ABU-174)、0.0086μg/ml(ABU-175)、0.039μg/ml(ABU190)。
实施例6：本文中的抗原结合单元中和SARS-CoV-2真病毒的能力的评估
在本实施例中，分别通过细胞病变(CPE)测定和空斑减少中和试验(PRNT)来评估待测抗体的中和活性。所使用的SARS-CoV-2病毒由军事医学研究院提供，其滴度(TCID50)为10 5/ml，并且，所有实验操作均在BSL-3实验室内完成。
6.1细胞病变(CPE)测定
(1)以5×10 4/ml的浓度，向96孔培养板的每孔中加入100μl Vero E6细胞，并在37℃，5％CO 2的条件下培养24小时。
(2)将待测抗体稀释成10个浓度：为1/10μg/μl，1/30μg/μl，1/90μg/μl，1/270μg/μl，1/810μg/μl，1/2430μg/μl，1/7290μg/μl，1/21870μg/μl，1/65610μg/μl，1/196830μg/μl。取100μl指定浓度的待测抗体，加入等体积的SARS-CoV-2真病毒(100TCID50)，并在37℃，5％CO 2的条件下孵育1h。
(3)在步骤(1)的培养结束后，弃去96孔培养板中的细胞培养液，加入步骤(2)制备的含有待测抗体和真病毒的混合液(200μl)，作为实验组。孵育1h后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
在实验过程中，平行设置细胞对照组和病毒对照组。在细胞对照组(4个复孔)中，弃去孔中的细胞培养液后，每孔添加200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。在病毒对照组(3个复孔)中，弃去孔中的细胞培养液后，每孔添加100TCID50的真病毒(100μl)，并在37℃孵育1h；孵育结束后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
(4)在37℃，5％CO 2的条件下培养细胞4-5天。
(5)在光学显微镜下观察细胞病变(CPE)，并根据细胞病变情况，评估不同浓度的单抗对CPE的抑制活性。
抗原结合单元ABU-174的检测结果如下表6所示，结果表明抗原结合单元ABU-174在细胞上对病毒有抑制效果，中和抗体滴度为1.6ng/μl。
表6.抗原结合单元ABU-174对SARS-CoV-2的中和活性效果
Figure PCTCN2021090146-appb-000035
“+”为细胞有CPE变化，“-”为细胞无CPE变化或正常细胞形态
抗原结合单元ABU-175的检测结果如下表7及图4所示，结果表明抗原结合单元ABU-175在细胞上对病毒有抑制效果，中和抗体滴度为0.7ng/μl。
表7.抗原结合单元ABU-175对SARS-CoV-2的中和活性效果
Figure PCTCN2021090146-appb-000036
“+”为细胞有CPE变化，“-”为细胞无CPE变化或正常细胞形态
6.2空斑减少中和试验(PRNT)：
(1)以5×10 4/ml的浓度，向96孔培养板的每孔中加入100μl Vero E6细胞，并在37℃，5％CO 2的条件下培养24小时。
(2)将待测抗体稀释成5个浓度：为50μg/ml，10μg/ml，2μg/ml，0.4μg/ml，0.08μg/ml。
(3)在步骤(1)的培养结束后，弃去96孔培养板中的细胞培养液，加入步骤(2)制备的含有待测抗体和真病毒的混合液(200μl)，作为实验组。孵育1h后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
在实验过程中，平行设置细胞对照组和病毒对照组。在细胞对照组中，弃去孔中的细胞培养液后，每孔添加200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。在病毒对照组(4个复孔)中，弃去孔中的细胞培养液后，每孔添加100TCID50的真病毒(100μl)，并在37℃孵育1h；孵育结束后，从孔中吸出上清，每孔加入200μl DMEM培养基(含有2％抗生素和16μg/ml胰蛋白酶)。
(4)在37℃，5％CO 2的条件下培养细胞4天。
(5)细胞甲醛固定后，利用兔抗SARS-COV血清(sino生物)和过氧化物酶标记的山羊抗兔IgG(Dako)标记。利用TMB(True Blue，KPL)显色后观测空斑，计算抑制率并绘制剂量-反应曲线。
图5显示了本文示例性的抗原结合单元ABU-174、ABU-175和ABU190的剂量-反应曲线。由图5可见，抗原结合单元ABU-174、ABU-175和ABU190对SARS-CoV-2真病毒均具有良好的中和活性，能够有效抑制病毒感染和侵入细胞，IC50分别为0.5μg/ml(ABU-174)，0.3μg/ml(ABU-175)和0.8μg/ml(ABU-190)。
实施例7.本文中的抗原结合单元的体内效力
SARS-CoV-2通过与hACE2受体的相互作用感染细胞。在两种不同的动物模型中评估了文中的抗原结合单元在体内对SARS-CoV-2的中和效力。
7.1抗原结合单元在hACE2转基因小鼠体内的效力
在第一个模型中，使用了hACE2转基因小鼠作为动物模型，并以暴露前预防或暴露后预防2种不同模式进行了治疗。具体而言，将hACE2 转基因小鼠经鼻内感染SARS-CoV-2种病毒(2019-nCoV Beta CoV/Wuhan/AMMS01/2020)，剂量为105 TCID50。
在暴露前预防治疗模式中，在病毒感染前24小时，将20mg/kg剂量的本文中的抗原结合单元腹膜内注射到hACE2转基因小鼠中，并检测了所述抗原结合单元作为暴露前预防干预的功效。
在暴露后预防治疗模式中，病毒感染后2小时，将20mg/kg剂量的抗原结合单元注射至小鼠中。使用HG1K(抗H7N9病毒的IgG1抗体)作为阴性对照，在病毒感染后2小时以20mg/kg注射。连续5天每天记录能够反映受感染小鼠健康状况的体重。
7.2抗原结合单元在仓鼠体内的效力
在第二个模型中，使用仓鼠(Mesocricetus auratus)作为动物模型，并以暴露前预防或暴露后预防2种不同模式进行了治疗。具体而言，类似于hACE2转基因小鼠，在仓鼠鼻内感染SARS-CoV-2原病毒(SARS-COV-2/WH-09/human/020/CHN)，剂量为105 TCID50。
在仓鼠暴露前预防治疗模式中，在病毒感染前1天，将20mg/kg剂量的本文中的抗原结合单元注射到仓鼠中。对照组中，感染后2小时向动物注射PBS。
在仓鼠暴露后预防治疗模式中，感染后2小时，将本文中的抗原结合单元根据体重以不同剂量(包括20、10、5和2mg/kg)腹膜内注射到仓鼠中。同时将注射了磷酸盐缓冲液(PBS)的仓鼠作为对照。连续7天每天记录受感染仓鼠的体重。感染后7天将仓鼠处死，收集肺用于病毒载量分析。
序列信息
本文所涉及的部分序列的信息如下表8所示。
表8.序列表
Figure PCTCN2021090146-appb-000037
Figure PCTCN2021090146-appb-000038
Figure PCTCN2021090146-appb-000039
Figure PCTCN2021090146-appb-000040
Figure PCTCN2021090146-appb-000041
Figure PCTCN2021090146-appb-000042
Figure PCTCN2021090146-appb-000043
Figure PCTCN2021090146-appb-000044
Figure PCTCN2021090146-appb-000045
Figure PCTCN2021090146-appb-000046
Figure PCTCN2021090146-appb-000047
Figure PCTCN2021090146-appb-000048
Figure PCTCN2021090146-appb-000049
Figure PCTCN2021090146-appb-000050
Figure PCTCN2021090146-appb-000051
Figure PCTCN2021090146-appb-000052
Figure PCTCN2021090146-appb-000053
Figure PCTCN2021090146-appb-000054
Figure PCTCN2021090146-appb-000055
Figure PCTCN2021090146-appb-000056
Figure PCTCN2021090146-appb-000057
Figure PCTCN2021090146-appb-000058
Figure PCTCN2021090146-appb-000059
Figure PCTCN2021090146-appb-000060
Figure PCTCN2021090146-appb-000061
Figure PCTCN2021090146-appb-000062
Figure PCTCN2021090146-appb-000063
Figure PCTCN2021090146-appb-000064
Figure PCTCN2021090146-appb-000065
Figure PCTCN2021090146-appb-000066
Figure PCTCN2021090146-appb-000067
Figure PCTCN2021090146-appb-000068
Figure PCTCN2021090146-appb-000069
Figure PCTCN2021090146-appb-000070
Figure PCTCN2021090146-appb-000071
Figure PCTCN2021090146-appb-000072
Figure PCTCN2021090146-appb-000073
Figure PCTCN2021090146-appb-000074
Figure PCTCN2021090146-appb-000075
Figure PCTCN2021090146-appb-000076
Figure PCTCN2021090146-appb-000077
Figure PCTCN2021090146-appb-000078
Figure PCTCN2021090146-appb-000079
Figure PCTCN2021090146-appb-000080
Figure PCTCN2021090146-appb-000081
Figure PCTCN2021090146-appb-000082
Figure PCTCN2021090146-appb-000083
Figure PCTCN2021090146-appb-000084
Figure PCTCN2021090146-appb-000085
Figure PCTCN2021090146-appb-000086
Figure PCTCN2021090146-appb-000087
Figure PCTCN2021090146-appb-000088
Figure PCTCN2021090146-appb-000089
Figure PCTCN2021090146-appb-000090
Figure PCTCN2021090146-appb-000091
Figure PCTCN2021090146-appb-000092
Figure PCTCN2021090146-appb-000093
Figure PCTCN2021090146-appb-000094
Figure PCTCN2021090146-appb-000095
Figure PCTCN2021090146-appb-000096
Figure PCTCN2021090146-appb-000097
Figure PCTCN2021090146-appb-000098
Figure PCTCN2021090146-appb-000099
Figure PCTCN2021090146-appb-000100
Figure PCTCN2021090146-appb-000101
Figure PCTCN2021090146-appb-000102
Figure PCTCN2021090146-appb-000103
Figure PCTCN2021090146-appb-000104
Figure PCTCN2021090146-appb-000105
Figure PCTCN2021090146-appb-000106
Figure PCTCN2021090146-appb-000107
Figure PCTCN2021090146-appb-000108
Figure PCTCN2021090146-appb-000109
Figure PCTCN2021090146-appb-000110
Figure PCTCN2021090146-appb-000111
Figure PCTCN2021090146-appb-000112
Figure PCTCN2021090146-appb-000113
尽管本文所述的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本文所述的保护范围之内。本文所述的全部分为由所附权利要求及其任何等同物给出。

Claims (52)

  1. 一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列或者与SEQ ID NO：1-360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列，和/或其中所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列或者与SEQ ID NO：361-720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列。
  2. 如前述权利要求之一所述的抗原结合单元，其以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
  3. 如前述权利要求之一所述的抗原结合单元，其以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC 50中和新型冠状病毒(SARS-CoV-2)。
  4. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列或者与SEQ ID NO：1461-1820和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列。
  5. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列。
  6. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR1包含与SEQ ID NO：1461-1820和2901-2935相比包含5、4、3、2或1个 氨基酸添加、删除、或取代的序列。
  7. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR1包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR1相同的序列。
  8. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列或者与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列。
  9. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列。
  10. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR2包含与SEQ ID NO：1821-2180和2936-2970相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  11. 如前述权利要求之一所述的抗原结合单元，其中所述VH CDR2包含与SEQ ID NO：721-1080和3111-3145中所包含的CDR2相同的序列。
  12. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列或者与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列。
  13. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列。
  14. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR1包含与SEQ ID NO：2181-2540和3006-3040相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  15. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR1包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR1相同的序列。
  16. 如前述权利要求之一所述的抗原结合单元，其中所述VL  CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列或者与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列。
  17. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列。
  18. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR2包含与SEQ ID NO：2541-2900和3041-3075相比包含5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  19. 如前述权利要求之一所述的抗原结合单元，其中所述VL CDR2包含与SEQ ID NO：1081-1440和3146-3180中所包含的CDR2相同的序列。
  20. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列或者与SEQ ID NO：721-1080和3111-3145相比包含一个或多个氨基酸添加、删除、或取代的序列。
  21. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列。
  22. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含与SEQ ID NO：721-1080和3111-3145相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  23. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
  24. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列或者与SEQ ID NO：1081-1440和3146-3180相比包含一个或多个氨基酸添加、删除、或取代的序列。
  25. 如前述权利要求之一所述的抗原结合单元，其中所述VL包 含选自SEQ ID NO：1081-1440和3146-3180的序列。
  26. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含与SEQ ID NO：1081-1440和3146-3180相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  27. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
  28. 一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列、与SEQ ID NO：1461-1820和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR1相同的序列，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列、与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR2相同的序列，其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列、与SEQ ID NO：1-360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：721-1080和3111-3145中所包含的CDR3相同的序列；和/或其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列、与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR1相同的序列，所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列、与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR2相同的序列，所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列、与SEQ ID NO：361- 720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列、或者与SEQ ID NO：1081-1440和3146-3180中所包含的CDR3相同的序列。
  29. 一种抗原结合单元，其包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区包含VH CDR1、VH CDR2和VH CDR3，所述轻链可变区包含VL CDR1、VL CDR2和VL CDR3；其中所述VH CDR1包含选自SEQ ID NO：1461-1820和2901-2935的序列或者与SEQ ID NO：1461-1820和2901-2935相比包含一个或多个氨基酸添加、删除、或取代的序列，其中所述VH CDR2包含选自SEQ ID NO：1821-2180和2936-2970的序列或者与SEQ ID NO：1821-2180和2936-2970相比包含一个或多个氨基酸添加、删除、或取代的序列，其中所述VH CDR3包含选自SEQ ID NO：1-360和2971-3005的序列或者与SEQ ID NO：1-360和2971-3005相比包含一个或多个氨基酸添加、删除、或取代的序列；和/或其中所述VL CDR1包含选自SEQ ID NO：2181-2540和3006-3040的序列或者与SEQ ID NO：2181-2540和3006-3040相比包含一个或多个氨基酸添加、删除、或取代的序列，所述VL CDR2包含选自SEQ ID NO：2541-2900和3041-3075的序列或者与SEQ ID NO：2541-2900和3041-3075相比包含一个或多个氨基酸添加、删除、或取代的序列，所述VL CDR3包含选自SEQ ID NO：361-720和3076-3110的序列或者与SEQ ID NO：361-720和3076-3110相比包含一个或多个氨基酸添加、删除、或取代的序列。
  30. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列或者与SEQ ID NO：721-1080和3111-3145相比包含一个或多个氨基酸添加、删除、或取代的序列。
  31. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含选自SEQ ID NO：721-1080和3111-3145的序列。
  32. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含与SEQ ID NO：721-1080和3111-3145相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  33. 如前述权利要求之一所述的抗原结合单元，其中所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
  34. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列或者与SEQ ID NO：1081-1440和3146-3180相比包含一个或多个氨基酸添加、删除、或取代的序列。
  35. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含选自SEQ ID NO：1081-1440和3146-3180的序列。
  36. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含与SEQ ID NO：1081-1440和3146-3180相比包含10、9、8、7、6、5、4、3、2或1个氨基酸添加、删除、或取代的序列。
  37. 如前述权利要求之一所述的抗原结合单元，其中所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
  38. 如前述权利要求之一所述的抗原结合单元，其以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
  39. 如前述权利要求之一所述的抗原结合单元，其以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC 50中和新型冠状病毒(SARS-CoV-2)。
  40. 一种抗原结合单元，其包含重链可变区(VH)和轻链可变区 (VL)，其中所述VH包含与选自SEQ ID NO：721-1080和3111-3145的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列，和/或其中所述VL包含与选自SEQ ID NO：1081-1440和3146-3180的序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99％的同一性的序列。
  41. 如前述权利要求之一所述的抗原结合单元，其以小于100nM、小于50nM、小于20nM、小于15nM、小于10nM、小于5nM、小于4nM、小于3nM、小于2nM、小于1nM、小于0.5nM、小于0.1 nM、小于0.05nM、或小于0.01nM的平衡解离常数(KD)结合新型冠状病毒(SARS-CoV-2)S蛋白的受体结合结构域(RBD)。
  42. 如前述权利要求之一所述的抗原结合单元，其以小于20μg/ml、小于10μg/ml、小于9μg/ml、小于8μg/ml、小于7μg/ml、小于6μg/ml、小于5μg/ml、小于4μg/ml、小于3μg/ml、小于2μg/ml、小于1μg/ml、小于0.5μg/ml、小于0.25μg/ml、小于0.2μg/ml、小于0.1μg/ml、小于0.05μg/ml、或小于0.001μg/ml的IC 50中和新型冠状病毒(SARS-CoV-2)。
  43. 一种药物组合物，其包含如权利要求1至42之一所述的抗原结合单元和药学上可接受的赋形剂。
  44. 一种分离的核酸，其编码如权利要求1至42之一所述的抗原结合单元。
  45. 一种载体，其包含编码如权利要求1至42之一所述的抗原结合单元的核酸序列。
  46. 一种宿主细胞，其表达如权利要求1至42之一所述的抗原结合单元。
  47. 一种宿主细胞，其包含编码如权利要求1至42之一所述的抗原结合单元的核酸。
  48. 一种产生如权利要求1至42之一所述的抗原结合单元的方法，其包括：在适合表达所述抗原结合单元的条件下培养权利要求46或 47的宿主细胞；以及分离由所述宿主细胞表达的所述抗原结合单元。
  49. 一种缀合物，其包含如权利要求1至42之一所述的抗原结合单元，其中所述抗原结合单元与化学功能部分缀合。
  50. 如权利要求49所述的缀合物，其中所述化学功能部分选自射性同位素、酶、荧光化合物、化学发光化合物、生物发光化合物底物辅因子和抑制剂。
  51. 一种预防和/或治疗有需要的患者的SARS-CoV-2感染的方法，包括向所述患者给予如权利要求1至42之一所述的抗原结合单元的方法或者编码所述抗原结合单元的核酸。
  52. 一种用于检测SARS-CoV-2的试剂盒，其包含如权利要求1至42之一所述的抗原结合单元。
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