CN116836268A - Antibodies against novel coronavirus SARS-CoV-2 and uses thereof - Google Patents

Antibodies against novel coronavirus SARS-CoV-2 and uses thereof Download PDF

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CN116836268A
CN116836268A CN202210291483.XA CN202210291483A CN116836268A CN 116836268 A CN116836268 A CN 116836268A CN 202210291483 A CN202210291483 A CN 202210291483A CN 116836268 A CN116836268 A CN 116836268A
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antibody
amino acid
seq
ser
acid sequence
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胡俊杰
刘志刚
付信磊
万姝南
周晓巍
郝小勃
刘玉兰
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Beijing Wisdomab Biotechnology Co ltd
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Beijing Wisdomab Biotechnology Co ltd
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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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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

Abstract

The application discloses an antibody against novel coronavirus SARS-CoV-2, a nucleic acid molecule encoding the antibody, an expression vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule or vector, a method for preparing and purifying the antibody and the application of the antibody.

Description

Antibodies against novel coronavirus SARS-CoV-2 and uses thereof
Technical Field
The present application relates generally to the fields of genetic engineering and antibody pharmaceuticals; in particular to the antibody field aiming at novel coronavirus SARS-CoV-2 and the application thereof. The application develops a novel antibody aiming at novel coronavirus SARS-CoV-2 and provides the application of the antibody in preventing or treating diseases caused by the novel coronavirus SARS-CoV-2.
Background
The novel coronavirus SARS-CoV-2 is a novel strain of coronavirus found in humans and can cause novel coronavirus pneumonia of acute respiratory infectious disease after infection of humans (Corona Virus Disease 2019, COVID-19) with the coronavirus of the middle east respiratory syndrome (Middle East Respiratory Syndrome Coronavirus, MERS-CoV) and the severe acute respiratory syndrome coronavirus (Severe Acute Respiratory Syndrome Coronavirus, SARS-CoV) belonging to the genus Coronaviridae. The common signs after SARS-CoV-2 infection are fever, dry cough and hypodynamia, few patients are accompanied with symptoms of upper respiratory tract and digestive tract, the serious cases are dyspnea, and the infection can cause pneumonia, acute respiratory distress syndrome, multiple organ failure and even death in the serious cases.
The genome of SARS-CoV-2 is single-stranded positive-stranded RNA, which is about 30kb in length. The virus particles have polymorphism, mainly in the shape of sphere, and rarely in the shape of ellipse or polygon, and have diameters of about 80-120 nm. The surface of the viral envelope is mainly provided with three glycoproteins: spike Protein (S), small Envelope Protein (E, envelope Protein), and Membrane Protein (M, membrane Protein). Like SARS-CoV, the SARS-CoV-2 spike protein contains two functional subunits: the S1 subunit and the S2 subunit, respectively, the S1 subunit promotes viral infection by binding to host cell surface receptors, wherein the Receptor Binding Domain (RBD) at the C-terminus of the S1 subunit is responsible for recognizing the angiotensin converting enzyme ACE2 on the host respiratory tract cell surface; the S2 subunit mediates mainly virus-cell and cell-cell membrane fusion. The S protein plays a critical role in recognizing/binding to host cell surface receptors, mediating fusion of viral envelope with cell membrane and inducing the body to produce neutralizing antibodies.
The novel coronavirus pneumonia has complex clinical symptoms, and the treatment method mainly aims at symptomatic support treatment. The novel coronavirus pneumonia healer blood plasma has better treatment effect due to containing the neutralizing antibody for blocking SARS-CoV-2 spike protein, but has limited sources and limited clinical application. Therefore, the development of specific neutralizing antibodies against SARS-CoV-2 with neutralizing activity can meet the clinical treatment requirements of novel coronavirus pneumonia.
Summary of The Invention
In a first aspect, the present application provides an antibody against novel coronavirus SARS-CoV-2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 24, the LCDR2 amino acid sequence is shown as SEQ ID NO. 25 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 26;
the HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 27, the LCDR2 amino acid sequence is shown as SEQ ID NO. 28 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 29; or (b)
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 30, the LCDR2 amino acid sequence is shown as SEQ ID NO. 31 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 32;
Wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 1, 3 or 5.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 2, 4, 6 or 7.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 1 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7;
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6; or alternatively
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7.
In a second aspect, the application provides an antibody against the novel coronavirus SARS-CoV-2, wherein the amino acid sequence of the heavy chain variable region of said antibody has at least 90% identity to any one of SEQ ID NOS 1, 3 and 5 and the amino acid sequence of the light chain variable region of said antibody has at least 90% identity to any one of SEQ ID NOS 2, 4, 6 and 7.
In some embodiments of the first and second aspects, the antibody is a whole antibody, fab fragment, F (ab') 2 Fragments or single chain Fv fragments (scFv).
In some embodiments of the first and second aspects, the antibody is a monoclonal antibody.
In some embodiments of the first and second aspects, the antibody is a neutralizing antibody.
In some embodiments of the first and second aspects, the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.
In some embodiments of the first and second aspects, the antibody further comprises a light chain constant region selected from the group consisting of kappa subtype or lambda subtype.
In some embodiments of the first and second aspects, the antibody binds to spike protein of novel coronavirus SARS-CoV-2.
In a third aspect, the application provides a nucleic acid molecule encoding an antibody according to the first or second aspect.
In a fourth aspect, the application provides a pharmaceutical composition comprising an antibody according to the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
In a fifth aspect, the application provides the use of an antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect, in the manufacture of a medicament for the prevention or treatment of infection or complications caused by the novel coronavirus SARS-CoV-2.
In a sixth aspect, the present application provides a method of preventing or treating infection or complications caused by the novel coronavirus SARS-CoV-2, comprising administering to a subject in need thereof an antibody according to the first or second aspect, or a pharmaceutical composition according to the fourth aspect.
Drawings
FIG. 1 shows the result of neutralizing activity of a monoclonal antibody against novel coronavirus SARS-CoV-2 against SARS-COV-2 wild-type pseudovirus.
FIG. 2 shows the result of neutralizing activity of anti-novel coronavirus SARS-CoV-2 monoclonal antibody against SARS-COV-2B.1.351 mutant pseudovirus.
FIG. 3 shows the result of neutralization activity of SARS-CoV-2 monoclonal antibody against novel coronavirus against SARS-COV-2B.1.617.2 mutant pseudovirus.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO. 1 shows the amino acid sequence of the heavy chain variable region S4B3VH of the single chain antibody S4B 3.
SEQ ID NO. 2 shows the amino acid sequence of the light chain variable region S4B3VK of the single-chain antibody S4B 3.
SEQ ID NO. 3 shows the amino acid sequence of the humanized heavy chain variable region mutant S4B3VH-h 1.
SEQ ID NO. 4 shows the amino acid sequence of the light chain variable region mutant L1C9 VK.
SEQ ID NO. 5 shows the amino acid sequence of the humanized heavy chain variable region mutant S4B3VH-h 2.
SEQ ID NO. 6 shows the amino acid sequence of the light chain variable region mutant L1B2 VK.
SEQ ID NO. 7 shows the amino acid sequence of the humanized light chain mutant S4B3VK-h 1.
SEQ ID NO. 8 shows the amino acid sequence of the recombinant protein antigen Cov-S-RBD.
SEQ ID NO. 9 shows the amino acid sequence of the recombinant protein antigen Cov-S.
SEQ ID NO. 10 shows the amino acid sequence of the T4 fibritin of the trimer tag.
SEQ ID NO. 11 shows the amino acid sequence of the His tag.
SEQ ID NO. 12 shows the amino acid sequence of the mFc tag.
SEQ ID NO. 13 shows the amino acid sequence of the human (homo sapiens) IgG1 subtype heavy chain constant region.
SEQ ID NO. 14 shows the amino acid sequence of the heavy chain constant region of the mouse (mus musculus) IgG2a subtype.
SEQ ID NO. 15 shows the amino acid sequence of the human (homo sapiens) kappa subtype light chain constant region.
SEQ ID NO. 16 shows the amino acid sequence of the human (homo sapiens) lambda subtype light chain constant region.
SEQ ID NO. 17 shows the amino acid sequence of the mouse (mus musculus) kappa subtype light chain constant region.
SEQ ID NO. 18 shows the amino acid sequence of the light chain constant region of the lambda subtype of mice (mus musculus).
SEQ ID NO. 19 shows the amino acid sequence of the heavy chain variable region of SARS-CoV-2 neutralizing antibody CB 6.
SEQ ID NO. 20 shows the amino acid sequence of the light chain variable region of SARS-CoV-2 neutralizing antibody CB 6.
SEQ ID NOS.21-23 show the amino acid sequences of HCDR1, HCDR2 and HCDR3 of S4B3VH, S4B3VH-h1 and S4B3VH-h 2, respectively.
SEQ ID NOS.24-26 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the light chain variable region mutants L1B2 VK.
SEQ ID NOS.27-29 show the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively, of the light chain variable region mutants L1C9 VK.
SEQ ID NOS.30-32 show the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable regions S4B3VK and S4B4 VK-h 1, respectively.
SEQ ID NO. 33 shows the nucleotide sequence of the primer PmCGR.
SEQ ID NO. 34 shows the nucleotide sequence of the primer PmCKR.
Detailed Description
The inventor of the present application obtains new antibody against novel coronavirus SARS-CoV-2 through antibody engineering technology. In various aspects of the application, novel antibodies against novel coronavirus SARS-CoV-2 are provided, as are nucleic acid molecules encoding the antibodies or antigen binding fragments thereof, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods of making and purifying the antibodies, and medical and biological uses of the antibodies. According to the amino acid sequence of the variable region of the antibody provided by the application, the full-length antibody molecule can be constructed to be used as a medicament for preventing or treating diseases caused by novel coronavirus SARS-CoV-2.
The practice of the present application employs, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
Definition of the definition
The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like. "antibody" as used herein includes not only whole (i.e., full length) antibodies, but also antigen binding fragments thereof (e.g., fab ', F (ab') 2 Fv), variants thereof, fusion proteins comprising an antibody moiety, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.
Typically, an intact or full length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH 3). Each light chain contains a light chain variable region (VL) and a constant region (CL). The full length antibody may be any kind of antibody, such as IgD, igE, igG, igA or IgM (or subclasses thereof described above), but the antibody need not be of any particular class. Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the heavy chain constant domain. In general, immunoglobulins fall into five main categories: igA, igD, igE, igG and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are referred to as α, δ, ε, γ, and μ, respectively. Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
The term "antigen binding fragment or antigen binding portion" as used herein refers to a portion or region of an intact antibody molecule responsible for binding an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3.
It is well known to those skilled in the art that complementarity determining regions (CDRs, typically CDR1, CDR2 and CDR 3) are regions of the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two common ways of defining CDR amino acid sequences for VH or VL, namely Chothia definition and kabat definition. (see, e.g., kabat, "Sequences of Proteins of Immunological Interest", national Institutes of Health, bethesda, md. (1991); A1-Lazikani et al, J.mol. Biol.273:927-948 (1997); and Martin et al, proc. Natl. Acad. Sci. USA86:9268-9272 (1989)). For a given antibody variable region amino acid sequence, the CDR amino acid sequences in VH and VL amino acid sequences may be determined according to Chothia definition or Kabat definition. In an embodiment of the application, the CDR amino acid sequences are defined using Kabat.
For a given antibody variable region amino acid sequence, the mid CDR amino acid sequence of the variable region amino acid sequence can be analyzed in a number of ways, for example, as determined using on-line software Abysis (http:// www.abysis.org /).
Examples of antigen binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having a VL-CL chain and a VH-CH1 chain; (2) F (ab') 2 A fragment, which may be a bivalent fragment having two Fab' fragments linked by a disulfide bridge of the hinge region (i.e., a dimer of Fab); (3) Fv fragments having VL and VH domains of a single arm of an antibody; (4) A single chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; (5) (scFv) 2 It may comprise two VH domains connected by a peptide linker and two VL domains combined with the two VH domains via a disulfide bridge.
The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
The term "neutralizing antibody" as used herein refers to an antibody that is capable of binding to an antigen on the surface of a pathogenic microorganism, thereby preventing the pathogenic microorganism from adhering to a target cell receptor and from invading the cell.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations in a small number of individuals.
In a first aspect, the present application provides an antibody against novel coronavirus SARS-CoV-2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 amino acid sequences and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 amino acid sequences, wherein
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 24, the LCDR2 amino acid sequence is shown as SEQ ID NO. 25 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 26;
the HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 27, the LCDR2 amino acid sequence is shown as SEQ ID NO. 28 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 29; or (b)
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 30, the LCDR2 amino acid sequence is shown as SEQ ID NO. 31 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 32;
wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 1, 3 or 5.
In some embodiments of the first aspect, the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 2, 4, 6 or 7.
In some embodiments of the first aspect, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 1 and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6; or alternatively
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7.
In a second aspect, the application provides an antibody against the novel coronavirus SARS-CoV-2, wherein the amino acid sequence of the heavy chain variable region of said antibody has at least 90% identity to any one of SEQ ID NOS 1, 3 and 5 and the amino acid sequence of the light chain variable region of said antibody has at least 90% identity to any one of SEQ ID NOS 2, 4, 6 and 7.
In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to any of SEQ ID NOs 1, 3 and 5.
In some embodiments of the second aspect, the amino acid sequence of the light chain variable region of the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to any of SEQ ID NOs 2, 4, 6 and 7.
In some embodiments of the second aspect, the amino acid sequence of the heavy chain variable region of the antibody differs from the amino acid sequence set forth in any one of SEQ ID NOs 1, 3 and 5 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions.
In some embodiments of the second aspect, the amino acid sequence of the light chain variable region of the antibody differs from the amino acid sequence set forth in any one of SEQ ID NOs 2, 4, 6 and 7 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and/or additions.
In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs 1, 3 and 5 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining similar functions of the heavy chain variable region of the antibody.
In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOs 1, 3 and 5, the resulting amino acid sequence still retaining similar functions as the heavy chain variable region of the antibody.
In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in any of SEQ ID NOs 1, 3 and 5, provided that the altered amino acid sequence substantially retains similar functions of the heavy chain variable region of the antibody.
In some embodiments of the second aspect, the C-terminal or N-terminal region of the amino acid sequence set forth in any one of SEQ ID NOs 2, 4, 6 and 7 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still retaining similar functions of the light chain variable region of the antibody.
In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal region of the amino acid sequence shown in any one of SEQ ID NOs 2, 4, 6 and 7, the resulting amino acid sequence still retaining similar functions of the light chain variable region of the antibody.
In some embodiments of the second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminus or N-terminus of the amino acid sequence shown in any of SEQ ID NOs 2, 4, 6 and 7, provided that the altered amino acid sequence substantially retains similar function of the light chain variable region of the antibody.
In some embodiments of the first and second aspects, the antibody is a whole antibody, fab fragment, F (ab') 2 Fragments or single chain Fv fragments (scFv).
In some embodiments of the first and second aspects, the antibody is a humanized antibody.
In some embodiments of the first and second aspects, the antibody is a monoclonal antibody.
In some embodiments of the first and second aspects, the antibody is a neutralizing antibody.
In some embodiments of the first and second aspects, the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype.
In some embodiments of the first and second aspects, the heavy chain constant region is of the IgG2 subtype, e.g., the mIgG2a subtype.
In some embodiments of the first and second aspects, the antibody further comprises a light chain constant region selected from the group consisting of kappa subtype or lambda subtype.
In some embodiments of the first and second aspects, the light chain constant region is a kappa subtype.
In some embodiments of the first and second aspects, the antibody binds to spike protein of novel coronavirus SARS-CoV-2.
In a third aspect, the application provides a nucleic acid molecule encoding an antibody according to the first or second aspect.
In some embodiments of the third aspect, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector.
In a fourth aspect, the application provides a pharmaceutical composition comprising an antibody according to the first or second aspect and a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments of the fourth aspect, the pharmaceutical composition is for use in preventing or treating infection or complications caused by the novel coronavirus SARS-CoV-2.
In some embodiments of the fourth aspect, the infection is a pulmonary infection.
In some embodiments of the fourth aspect, the infection causes a novel coronavirus pneumonia.
In some embodiments of the fourth aspect, the complication is fever, cough, shortness of breath, dyspnea, severe acute respiratory syndrome, or renal failure.
In some embodiments of the fourth aspect, the pharmaceutical composition may further comprise one or more of the following: lubricants, such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifying agent; a suspending agent; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and/or flavoring agents, etc.
In some embodiments of the fourth aspect, the pharmaceutical compositions of the present application may be formulated in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules and the like.
In some embodiments of the fourth aspect, the pharmaceutical compositions of the present application may be delivered using any physiologically acceptable mode of administration, including, but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
In some embodiments of the fourth aspect, the pharmaceutical composition for therapeutic use may be formulated for storage in the form of a lyophilized formulation or an aqueous solution by mixing an agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or the like.
In a fifth aspect, the application provides the use of an antibody according to the first or second aspect, a nucleic acid molecule according to the third aspect, or a pharmaceutical composition according to the fourth aspect, in the manufacture of a medicament for the prevention or treatment of infection or complications caused by the novel coronavirus SARS-CoV-2.
In some embodiments of the fifth aspect, the infection is a pulmonary infection.
In some embodiments of the fifth aspect, the infection causes a novel coronavirus pneumonitis.
In some embodiments of the fifth aspect, the complication is fever, cough, shortness of breath, dyspnea, severe acute respiratory syndrome, or renal failure.
In a sixth aspect, the present application provides a method of preventing or treating infection or complications caused by the novel coronavirus SARS-CoV-2, comprising administering to a subject in need thereof an antibody according to the first or second aspect, or a pharmaceutical composition according to the fourth aspect.
In some embodiments of the sixth aspect, the infection is a pulmonary infection.
In some embodiments of the sixth aspect, the infection causes a novel coronavirus pneumonitis.
In some embodiments of the sixth aspect, the complication is fever, cough, shortness of breath, dyspnea, severe acute respiratory syndrome, or renal failure.
In other aspects, the application also provides vectors comprising nucleic acid molecules encoding the antibodies of the application or light or heavy chains thereof, host cells comprising the vectors, and methods of producing the antibodies. In some embodiments, the nucleic acid molecule is operably linked to a regulatory amino acid sequence that can be recognized by a host cell transformed with the vector. In some embodiments, the method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid. In some embodiments, the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
Furthermore, antibodies described herein that are specific for the novel coronavirus SARS-CoV-2 can also be used to detect the presence of the novel coronavirus SARS-CoV-2 in a biological sample. Antibody-based detection methods are well known in the art and include, for example, ELISA, immunoblotting, radioimmunoassay, immunofluorescence, immunoprecipitation, and other related techniques.
It should be understood that the foregoing detailed description is only for the purpose of making apparent to those skilled in the art the contents of the application, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.
Examples
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the application.
Example 1: SARS-CoV-2 antigen and recombinant antibody preparation
1. Preparation of SARS-CoV-2 antigen
In the process of screening anti-SARS-CoV-2 monoclonal antibody, recombinant protein antigens CoV-S-RBD (amino acid sequence shown as SEQ ID NO: 8) and CoV-S (amino acid sequence shown as SEQ ID NO: 9) are needed, and the sources of RBD and S protein antigen genes are shown in Genbank number: YP_009724390, in which a trimeric tag (T4 fibritin, amino acid sequence shown in SEQ ID NO: 10) is added to the C-terminus of the S protein, will favor the formation of a trimer of the S protein. In addition, his tag (His, amino acid sequence is shown as SEQ ID NO: 11) or mFc tag (mFc, amino acid sequence is shown as SEQ ID NO: 12) is added at the C end of the recombinant proteins, which is more beneficial to purification of the recombinant proteins and screening and identification of monoclonal antibodies. Each recombinant protein gene synthesized is cloned into a suitable eukaryotic expression vector (such as pcDNA3.1 of the company of Invitrogen) by using a conventional molecular biology technique, and then the prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F cells of the company of Invitrogen) by using a liposome (such as 293fectin of the company of Invitrogen) or other cationic transfection reagent (such as PEI, etc.), and cultured for 3-4 days under serum-free suspension culture conditions. The culture supernatant is then harvested by centrifugation or the like.
The recombinant protein expressed by His tag fusion is purified in one step by using metal chelating affinity chromatography column (such as HisTrap FF of GE company). The recombinant protein expressed by the mFc fusion is purified by a protein A/G affinity chromatography column (such as MabSelect SURE of GE company, etc.). The recombinant protein preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., GE company Hitrap desaulting, etc.). If necessary, the recombinant protein samples may be subjected to filtration sterilization and then sub-packaged for storage at-20 ℃.
2. Preparation of recombinant antibodies
The nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody are cloned into eukaryotic expression vectors (such as pcDNA3.1 of the company Invitrogen) having nucleotide sequences encoding the heavy chain constant region and the light chain constant region, respectively, by conventional molecular biological means, and the whole antibody is expressed in combination. The heavy chain constant region of the antibody may be of the human IgG1 subtype (amino acid sequence shown as SEQ ID NO: 13) or of the murine IgG2a subtype (amino acid sequence shown as SEQ ID NO: 14), and the light chain constant region may be of the human kappa subtype (amino acid sequence shown as SEQ ID NO: 15), of the human lambda subtype (amino acid sequence shown as SEQ ID NO: 16), of the murine kappa subtype (amino acid sequence shown as SEQ ID NO: 17) or of the murine lambda subtype (amino acid sequence shown as SEQ ID NO: 18). The heavy chain variable region of the SARS-CoV-2 neutralizing antibody CB6 has GenBank number QJU69682.1 (the amino acid sequence is shown as SEQ ID NO: 19), and the light chain variable region has GenBank number QJU69681.1 (the amino acid sequence is shown as SEQ ID NO: 20). The prepared recombinant antibody expression plasmid is transfected into HEK293 cells (e.g., HEK293F cells from Invitrogen) using liposomes (e.g., 293fectin from Invitrogen) or other transfection reagents (e.g., PEI, etc.), and cultured under serum-free suspension culture conditions for 3-5 days. The culture supernatant is then harvested by centrifugation or the like and purified in one step using a ProteinA/G affinity column (e.g., mabSelect SURE, GE, etc.). The recombinant antibody preservation buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (e.g., GE company Hitrap desaulting, etc.). If necessary, the antibody samples may be subjected to filter sterilization and then stored in aliquots at-20 ℃.
Example 2: mouse immunity and construction of immune repertoire
BALB/c mice 6-8 weeks old were immunized with the Cov-S-RBD-His and Cov-S-RBD-mFc mixed antigen prepared in example 1 at a dose of 100. Mu.g/mouse, boosted 1 time every 14 days, and the mice were sacrificed 8 weeks after priming and spleen cells were collected. The mouse spleen lymphocytes were isolated using a mouse lymphocyte isolate (daceae, biotechnology Co., ltd., CAT#DKW 33-R0100), and the isolated lymphocytes were subjected to total RNA extraction using a cell total RNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd., CAT#DP430). The extracted total RNA is used as a template, a first strand cDNA synthesis kit (Thermo scientific, CAT#K1621) is used for respectively synthesizing a heavy chain variable region and a light chain variable region of an antibody, a reverse transcription primer adopts a gene specific primer, and a primer pair is respectively positioned in the heavy chain constant region of the antibody and the light chain constant region of the antibody, and specific sequences are PmCGR: TGCATTTGAACTCCTTGCC (SEQ ID NO: 33) and PmCKR: CCATCAATCTTCCACTTGAC (SEQ ID NO: 34) respectively. The synthesized cDNA was immediately stored at-70℃until use. Then, primers were synthesized using cDNA obtained by reverse transcription as a template, and single chain antibody (scFv) genes were constructed by the overlapping extension PCR technique using the primers of reference (Krebber A, bernhauser S, burmester J, et al, available cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system J immunomethods 1997;201 (1): 35-55). Finally, the prepared mouse single chain antibody gene was cloned into vector pADSCFV-S (see example 1 of Chinese patent application No. 201510097117.0, the entire contents of which are incorporated herein by reference), and scFv library was constructed.
Example 3: screening of mouse immune repertoires
1. Screening of mouse immune repertoires
Reference (experimental technical scheme can be seen in Chinese patent application No. 201510097117.0, the whole content of which is incorporated herein by reference), recombinant human Cov-S-RBD-His prepared in example 1 is taken as antigen, and a single-chain antibody S4B3 (the amino acid sequences of the heavy chain variable region S4B3VH and the light chain variable region S4B3VK, respectively, are shown in SEQ ID NO:1 and SEQ ID NO: 2) which specifically binds human Cov-S-RBD-His is finally obtained by three rounds of screening using a solid phase screening strategy (experimental scheme reference phage display: general experimental guideline/(Meclackson, T.) (Megamann (Lowman, H.B.), ma Lan et al. Chemical industry Press, 2008.5, the whole content of which is incorporated herein by reference).
The nucleotide sequences encoding the heavy chain variable region and the light chain variable region of S4B3 are cloned into eukaryotic expression vectors fused with the heavy chain constant region encoding the mouse mIgG2a and the light chain constant region encoding the mouse kappa by using conventional molecular biological means, respectively, so as to prepare the recombinant murine antibody.
2. Affinity analysis of recombinant anti-Cov-S-RBD monoclonal antibodies
The affinity of anti-Cov-S-RBD antibodies was determined by surface plasmon resonance technique using Biacore T200. Amino coupling kit (BR-1000-50) and murine antibody capture reagentReagents and consumables such as cartridge (BR-1008-38), CM5 chip (BR 100012) and 10 XHBS-EP (BR 100669) at pH7.4 were purchased from GE healthcare. Carboxylated CM5 chip surfaces were activated with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1-Ethyl-3- (3-dimethylmineopyl) carbodiimide hydrochloride, EDC) and N-Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) according to the instructions in the kit, and anti-mouse IgG (Fc) antibodies (capture antibodies) were diluted to 25. Mu.g/mL with 10mM sodium acetate (pH 5.0) and then injected at a flow rate of 10. Mu.L/min to achieve a coupling amount of about up to 10000 Response Units (RU). After injection of the capture antibody, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, anti-Cov-S-RBD antibodies were diluted to 2. Mu.g/mL and injected at 10. Mu.L/min, ensuring that about 50RU of the antibody was captured by the anti-murine Fc antibody. The Cov-S-RBD was then subjected to a series of concentration gradients (e.g., 6.17nM, 18.5nM, 55.6nM, 167nM and 500 nM), injected at 30. Mu.L/min from low to high concentration for a binding time of 120S, dissociation time of 600S, and 10mM glycine-HCl (pH 1.7) injected at 10. Mu.L/min for 30S to regenerate the chip surface. Binding rate (K) was calculated by fitting a 1:1 binding model to the binding and dissociation sensorgrams using Biacore T200 evaluation software version 2.0.1 on ) Dissociation rate (K) off ). At a ratio of K off /K on Calculating dissociation equilibrium constant (K) D ). The fitting results are shown in table 1. TABLE 1 affinity constant of recombinant anti-Cov-S-RBD monoclonal antibodies binding to Cov-S-RBD-His
K on K off K D
S4B3-mIgG2a 4.523E+5 1.033E-2 2.284E-8
Example 4: humanization of anti-novel coronavirus SARS-CoV-2 monoclonal antibodies
1. Humanization of S4B3 murine monoclonal antibodies
The murine monoclonal antibody S4B3 was humanized to reduce its immunogenicity. The humanization protocol used classical frame grafting strategies (Tan P, mitchell DA, buss TN, holmes MA, anasetti C, foote J. "Superhumannized" anti-ibodies: reduction of immunogenic potential by complementarity-determining region grafting with human germline sequences: application to an anti-CD28.J Immunol. 2002;169 (2): 1119-1125.). The heavy and light chain variable regions of S4B3 were compared with the human antibody germline gene sequences in the IMGT database, respectively, the appropriate germline gene sequences were selected to provide framework regions 1 to 3 (FR 1+ FR2+ FR 3) of the antibody, and the appropriate J region gene sequences were selected to provide framework region 4 (FR 4). This template may be selected based on a variety of factors, such as: the relative total length of the antibody, the size of the CDRs, the amino acid residues at the junction between the antibody Framework Regions (FR) and the hypervariable regions (CDRs), the homology of the sequence as a whole, and the like. The template selected may be a mixture of sequences or may be a consensus template in order to maintain as much of the appropriate conformation of the parental Complementarity Determining Regions (CDRs) as possible. Meanwhile, in order to avoid protein heterogeneity possibly brought by deamination site NG in an antibody hypervariable region (CDR), mutation design is carried out on a humanized antibody heavy chain variable region. Finally, 2 humanized heavy chain variable region mutants S4B3VH-h1 (the amino acid sequence is shown as SEQ ID NO: 3), S4B3VH-h2 (the amino acid sequence is shown as SEQ ID NO: 5) and a humanized light chain mutant S4B3VK-h1 (the amino acid sequence is shown as SEQ ID NO: 7) are obtained.
Based on the S4B3VH-h1 heavy chain variable region, the phage display system based on the double vector utilizes light chain replacement (specific operation can be referred to in example 4.3 of Chinese patent 201510097117.0) to carry out further in vitro affinity maturation on the antibody S4B3VH-h1/S4B3VK-h1, so as to obtain two high affinity light chain variable region mutants L1C9VK (the amino acid sequence is shown as SEQ ID NO: 4) and L1B2VK (the amino acid sequence is shown as SEQ ID NO: 6).
The nucleotide sequences of the humanized heavy chain variable region and the light chain variable region of S4B3 are cloned to eukaryotic expression vectors fused with the nucleotide sequences of the mouse mIgG2a heavy chain constant region and the mouse kappa light chain constant region respectively by using a conventional molecular biological means, so as to prepare the recombinant murine antibody.
2. Affinity assay for humanized anti-novel coronavirus SARS-CoV-2 monoclonal antibody
With reference to example 3.2, the affinity of humanized anti-novel coronavirus SARS-CoV-2 monoclonal antibody to CoV-S-His was detected with Biacore T200 and the fitting results are shown in Table 2.
TABLE 2 affinity constant of humanized anti-novel coronavirus SARS-CoV-2 monoclonal antibody binding to CoV-S-His
K on K off K D
S4B3VH-h1+L1C9VK-mIgG2a 2.129E+4 1.065E-4 5.002E-9
S4B3VH-h2+L1C9VK-mIgG2a 1.92E+4 1.105E-4 5.756E-9
S4B3VH-h1+L1B2VK-mIgG2a 2.11E+4 6.035E-5 2.86E-9
S4B3VH-h2+L1B2VK-mIgG2a 2.051E+4 6.245E-5 3.044E-9
S4B3VH-h1+S4B3VK-h1-mIgG2a 1.42E+4 2.056E-4 1.448E-8
S4B3VH-h2+S4B3VK-h1-mIgG2a 1.168E+4 2.786E-4 2.386E-8
Example 5: neutralizing Activity of monoclonal antibodies against novel coronavirus SARS-CoV-2
And transferring ACE2 into HEK-293T cells to construct an ACE2 over-expression cell strain HEK-293T-ACE 2. The SARS-CoV-2 pseudovirus expresses new coronavirus spike protein on the surface of replication defective virus to form chimeric virus particles, which combine with HEK-293T-ACE2 cell surface over-expressed ACE2 to simulate the infection process of new coronavirus to target cells through spike protein combined ACE2. The assay detects the level of infection of the virus by means of a luciferase reporter gene.
HEK-293T-ACE2 cells were resuspended to 5X 10 with maintenance medium (DMEM medium with 5% serum) 5 And (5) using the solution in a standby mode. The anti-novel coronavirus SARS-CoV-2 monoclonal antibodies (S4B 3-mIgG2a, S4B3VH-h1+L1C9VK-mIgG2a, S4B3 VH-h1+L1B2VK-mIgG 2a and CB 6) were diluted with maintenance medium, and in the neutralization experiments of wild-type pseudovirus (FIG. 1) and B.1.617.2 mutant pseudovirus (FIG. 3), the initial concentration of each anti-novel coronavirus SARS-CoV-2 monoclonal antibody was 600nM, 3-fold gradient dilution was performed for 10 concentration points; the concentration of the negative control isotype antibody (IgG 2 a) was 600nM single-point concentration, without gradient dilution. In the neutralization experiment of the pseudovirus of the B.1.351 mutant strain (figure 2), the initial concentration of the anti-novel coronavirus SARS-CoV-2 monoclonal antibody is 60nM, and the monoclonal antibody is diluted in a 3-time gradient, and the concentration is 10 concentration points in total; the concentration of the negative control isotype antibody (IgG 2 a) was 60nM single-point concentration, and no gradient dilution was performed. mu.L of antibody was mixed with 10. Mu.L of SARS-CoV-2 pseudovirus (SARS-CoV-2 pseudovirus liquid (WT, wild-type pseudovirus) -LUC: RM02858; SARS-CoV-2 pseudovirus liquid (B.1.351, south Africa strain) -LUC: RM02860 or SARS-CoV-2 pseudovirus liquid (B.1.617.2, india strain) -LUC: RM02913; available from Wobotake Biotechnology Co., ltd.) in 96-well cell plates, incubated at 37℃for 1 hour, followed by adding prepared 100. Mu.L of HEK-293T-ACE2 cell suspension and incubating in an incubator for 48 hours. After centrifugation, the cell supernatant was aspirated, and 20. Mu.L of cell lysate was added for 5min to complete lysis, and 20. Mu.L of cell lysate was transferred to a 96-well white microplate and an automatic loading program was set up with a Spectgmax i3X microplate reader (Molecular devices) with 50. Mu.L of luciferase substrate (Luciferase Assay System, E1500, promega) per well for 10s reading. Experimental results were treated with GraphPad Prism. The results showed that the cell control group not infected with pseudovirus had no fluorescence, the isotype antibody (IgG 2 a) group as the negative control had no inhibitory effect on the fluorescence generated by pseudovirus infection, while the fluorescence values of each anti-novel coronavirus SARS-CoV-2 monoclonal antibody group were decreased with increasing antibody concentration, demonstrating that the anti-novel coronavirus SARS-CoV-2 monoclonal antibody pair SARS-COV-2 wild-type pseudovirus (figure 1), B.1.351 mutant pseudovirus (figure 2) and B.1.617.2 mutant pseudovirus (figure 3) all have obvious neutralization activity, and can neutralize EC of 3 strains of viruses 50 The values are similar.
Sequence listing
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<120> antibodies against novel coronaviruses SARS-CoV-2 and uses thereof
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370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
530 535 540
Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr
545 550 555 560
Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr
565 570 575
Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr
580 585 590
Ser Asn Gln Val Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val
595 600 605
Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr
610 615 620
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
625 630 635 640
Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala
645 650 655
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
660 665 670
Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly
675 680 685
Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr
690 695 700
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
705 710 715 720
Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu
725 730 735
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
740 745 750
Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu
755 760 765
Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp
770 775 780
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro
785 790 795 800
Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu
805 810 815
Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile
820 825 830
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
835 840 845
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
850 855 860
Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala
865 870 875 880
Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly
885 890 895
Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala
900 905 910
Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser
915 920 925
Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala
930 935 940
Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala
945 950 955 960
Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu
965 970 975
Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu
980 985 990
Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala
995 1000 1005
Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln
1010 1015 1020
Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe
1025 1030 1035 1040
Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1045 1050 1055
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp
1060 1065 1070
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr
1075 1080 1085
His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr
1090 1095 1100
Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile
1105 1110 1115 1120
Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe
1125 1130 1135
Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val
1140 1145 1150
Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln
1155 1160 1165
Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser
1170 1175 1180
Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp
1185 1190 1195 1200
Pro
<210> 10
<211> 27
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys
1 5 10 15
Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
20 25
<210> 11
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
His His His His His His
1 5
<210> 12
<211> 232
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
1 5 10 15
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
20 25 30
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
35 40 45
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
50 55 60
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
65 70 75 80
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
85 90 95
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
100 105 110
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
115 120 125
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
130 135 140
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
145 150 155 160
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
165 170 175
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
180 185 190
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
195 200 205
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
210 215 220
Ser Phe Ser Arg Thr Pro Gly Lys
225 230
<210> 13
<211> 330
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 13
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 14
<211> 330
<212> PRT
<213> mice (Mus musculus)
<400> 14
Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly
1 5 10 15
Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
100 105 110
Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
115 120 125
Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
130 135 140
Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp
145 150 155 160
Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg
165 170 175
Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
180 185 190
His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn
195 200 205
Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly
210 215 220
Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu
225 230 235 240
Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met
245 250 255
Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu
260 265 270
Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe
275 280 285
Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
290 295 300
Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr
305 310 315 320
Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
325 330
<210> 15
<211> 107
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 15
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 16
<211> 106
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 16
Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp
20 25 30
Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro
35 40 45
Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys
65 70 75 80
Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95
Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
100 105
<210> 17
<211> 107
<212> PRT
<213> mice (Mus musculus)
<400> 17
Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
1 5 10 15
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
20 25 30
Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
35 40 45
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
65 70 75 80
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser
85 90 95
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 18
<211> 106
<212> PRT
<213> mice (Mus musculus)
<400> 18
Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp
20 25 30
Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro
35 40 45
Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu
65 70 75 80
Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val
85 90 95
Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser
100 105
<210> 19
<211> 449
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Leu Phe Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys
210 215 220
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445
Lys
<210> 20
<211> 217
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro
85 90 95
Glu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys Ser
210 215
<210> 21
<211> 10
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gly Tyr Ile Phe Asn Ser Tyr Ser Ile His
1 5 10
<210> 22
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Asn Ser Gln Arg Phe Lys
1 5 10 15
Gly
<210> 23
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Asp Gly Tyr Asp Asp Ser Tyr Gly Leu Asp Tyr
1 5 10
<210> 24
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Arg Ala Ser Gln Asn Ile His Asn Tyr Leu Asp
1 5 10
<210> 25
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Ser Ala Ser Ser Arg Glu Thr
1 5
<210> 26
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Gln Gln Phe Gln Gln Ile
1 5
<210> 27
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Arg Ala Ser Gln Asn Ile Gly Gly
1 5
<210> 28
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Gln Ala Ser Thr Leu Glu Ser
1 5
<210> 29
<211> 6
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Gln Gln Tyr Ser Gly Phe
1 5
<210> 30
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala
1 5 10
<210> 31
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Ser Ala Ser Tyr Arg Tyr Ser
1 5
<210> 32
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Gln Gln Tyr Asn Ser Tyr Pro Leu Thr
1 5
<210> 33
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
tgcatttgaa ctccttgcc 19
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ccatcaatct tccacttgac 20

Claims (10)

1. An antibody against novel coronavirus SARS-CoV-2 comprising a heavy chain variable region comprising the amino acid sequences of HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3, wherein
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 24, the LCDR2 amino acid sequence is shown as SEQ ID NO. 25 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 26;
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 27, the LCDR2 amino acid sequence is shown as SEQ ID NO. 28 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 29; or (b)
The HCDR1 amino acid sequence is shown as SEQ ID NO. 21, the HCDR2 amino acid sequence is shown as SEQ ID NO. 22, the HCDR3 amino acid sequence is shown as SEQ ID NO. 23, the LCDR1 amino acid sequence is shown as SEQ ID NO. 30, the LCDR2 amino acid sequence is shown as SEQ ID NO. 31 and the LCDR3 amino acid sequence is shown as SEQ ID NO. 32;
wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.
2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 1, 3 or 5.
3. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID No. 2, 4, 6 or 7.
4. The antibody of any one of claims 1-3, wherein
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 4;
the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 6; or alternatively
The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7.
5. An antibody against the novel coronavirus SARS-CoV-2, wherein the amino acid sequence of the heavy chain variable region of said antibody has at least 90% identity to any one of SEQ ID nos. 1, 3 and 5 and the amino acid sequence of the light chain variable region of said antibody has at least 90% identity to any one of SEQ ID nos. 2, 4, 6 and 7.
6. The antibody of any one of claims 1-5, wherein
The antibody is a whole antibody, a Fab fragment, F (ab') 2 Fragments or single chain Fv fragments (scFv), preferably the antibody is a humanized antibody; and/or
The antibody is a monoclonal antibody; and/or
The antibody is a neutralizing antibody; and/or
The antibody further comprises a heavy chain constant region selected from the IgG1 subtype, the IgG2 subtype, or the IgG4 subtype, preferably the heavy chain constant region is of the IgG2 subtype; and/or
The antibody further comprises a light chain constant region selected from the kappa subtype or the lambda subtype, preferably the light chain constant region is of the kappa subtype.
7. The antibody of any one of claims 1-6, wherein the antibody binds to spike protein of novel coronavirus SARS-CoV-2.
8. A nucleic acid molecule encoding the antibody of any one of claims 1-7.
9. A pharmaceutical composition comprising the antibody of any one of claims 1-7 and a pharmaceutically acceptable excipient, diluent or carrier.
10. Use of an antibody according to any one of claims 1-7, a nucleic acid molecule according to claim 8 or a pharmaceutical composition according to claim 9 in the manufacture of a medicament for the prevention or treatment of infection or complications caused by the novel coronavirus SARS-CoV-2, preferably a pulmonary infection, e.g. causing a novel coronavirus pneumonia; and/or the complication is fever, cough, shortness of breath, dyspnea, severe acute respiratory syndrome or renal failure.
CN202210291483.XA 2022-03-23 2022-03-23 Antibodies against novel coronavirus SARS-CoV-2 and uses thereof Pending CN116836268A (en)

Priority Applications (1)

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Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210291483.XA CN116836268A (en) 2022-03-23 2022-03-23 Antibodies against novel coronavirus SARS-CoV-2 and uses thereof

Publications (1)

Publication Number Publication Date
CN116836268A true CN116836268A (en) 2023-10-03

Family

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Family Applications (1)

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Country Status (1)

Country Link
CN (1) CN116836268A (en)

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