Antibodies against coronavirus and uses thereof

Abstract

The present invention relates to the field of molecular virology and immunology, in particular the field of prevention and treatment of Coronavirus infection. Specifically, the present invention relates to antibodies for the treatment of Coronavirus (e.g. SARS-CoV-2 and / or SARS-CoV-1) infection and associated diseases, compositions (e.g. therapeutic and diagnostic agents) comprising said antibodies. Furthermore, the present invention also relates to the use of said antibodies.

Description

Technical Field

[0001] This invention relates to the fields of molecular virology and immunology, particularly to the prevention and treatment of coronavirus infections. Specifically, this invention relates to antibodies for treating coronavirus (e.g., SARS-CoV-2 and / or SARS-CoV-1) infections and related diseases, and compositions comprising said antibodies (e.g., therapeutic agents and diagnostic agents). Furthermore, this invention also relates to the use of said antibodies. Background Technology

[0002] Coronavirus infection can cause respiratory illnesses in humans. Mild coronavirus infections can cause flu-like symptoms, while severe infections can develop into severe viral pneumonia, threatening human life and health. Coronaviruses can infect both humans and animals. If some animal-derived coronaviruses break through the host barrier and infect humans, they can spread rapidly in the population and cause severe illness. For example, severe acute respiratory syndrome (SARS) caused by infection with SARS-CoV (also known as SARS-CoV-1) and COVID-19 caused by infection with SARS-CoV-2 have both had a huge impact and burden on human economic and social development.

[0003] Currently, there are no approved drugs for the prevention or treatment of SARS-CoV-2 infection. Treatment for pneumonia caused by SARS-CoV-2 infection involves only general supportive care, oxygen therapy, and antiviral therapy such as interferon beta-1b, lopinavir / ritonavir, remdesivir, and dexamethasone, with limited clinical effectiveness. Studies have found that recovered COVID-19 patients often develop high levels of SARS-CoV-2 neutralizing antibodies. Research data indicates that treatment with convalescent plasma containing neutralizing antibodies in critically ill patients diagnosed with COVID-19 and concurrent severe respiratory distress syndrome (ARDS) rapidly reduces viral load and effectively improves clinical symptoms. These studies demonstrate the importance of humoral immunity in SARS-CoV-2 and indicate that, in addition to vaccine development, one or more monoclonal antibodies capable of efficiently and specifically neutralizing SARS-CoV-2 should be developed. These antibodies, used alone or in combination, can be applied to the short-term prevention and effective treatment of COVID-19, which is of great significance for the prevention and control of COVID-19 in my country and globally. At the same time, it is foreseeable that with the continuous mutation of the virus and the rapid pace of human development and extensive communication, it is not difficult to predict that newer coronaviruses may emerge continuously. For this reason, the development of neutralizing antibodies (nAbs) against broad-spectrum β-CoV-Bs has become a project of great strategic importance.

[0004] SARS-CoV-1 and SARS-CoV-2 are both β-coronaviruses, belonging to the same branch of the phylogenetic tree, and both are enveloped, single-stranded, positive-sense RNA viruses. Both contain at least three membrane proteins, including a surface spike protein (S), an integrated membrane protein (M), and an integrated membrane protein (E). Like SARS-CoV-1, SARS-CoV-2's receptor mediates viral membrane fusion and cell entry through the specific binding of the receptor-binding domain (RBD) on the S protein to angiotensin-transferase 2 (ACE2) on the host cell, playing a crucial role in viral infection. Therefore, interfering with the binding of the S protein to ACE2 can neutralize viral infection. Thus, the S protein, especially the RBD region, is a major source and recognition region for coronavirus neutralizing antibodies, making the development of neutralizing antibodies targeting the RBD region a preferred approach.

[0005] Therefore, developing drugs such as antibodies that can treat or prevent coronavirus (e.g., SARS-CoV-2) infection is of great significance for controlling related epidemics. Summary of the Invention

[0006] This invention provides monoclonal antibodies capable of neutralizing coronaviruses (e.g., SARS-CoV-2 and / or SARS-CoV-1). These monoclonal antibodies bind to epitopes on the S protein RBD region of coronaviruses and neutralize them, inhibiting the binding of the coronavirus RBD protein to the receptor ACE2, and providing some protection against coronavirus attack in animal models. In particular, the monoclonal antibodies of this invention maintain stable neutralizing efficacy against the currently prevalent mainstream mutant strains of SARS-CoV-2. Therefore, the antibodies of this invention have the potential for prevention and / or treatment of coronavirus (e.g., SARS-CoV-1 and SARS-CoV-2) infection or related diseases, and have significant clinical value.

[0007] The antibody of the present invention

[0008] A first aspect of the present invention provides an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising:

[0009] (a) Heavy chain variable region (VH) containing the following three complementarity-determining regions (CDRs):

[0010] (i) VH CDR1, which consists of the following sequences: SEQ ID NO:7, SEQ ID NO:13 or SEQ ID NO:19, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it.

[0011] (ii) VH CDR2, comprising the following sequences: SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:20, or sequences having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids), and

[0012] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to it;

[0013] And / or,

[0014] (b) Light chain variable regions (VLs) containing the following three complementary determinant regions (CDRs):

[0015] (iv) VL CDR1, which consists of the following sequences: SEQ ID NO:10, SEQ ID NO:16, or SEQ ID NO:22, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the above.

[0016] (v)VL CDR2, which consists of the following sequences: SEQ ID NO:11, SEQ ID NO:17, or SEQ ID NO:23, or sequences having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids), and

[0017] (vi)VL CDR3, which consists of the following sequences: SEQ ID NO:12, SEQ ID NO:18 or SEQ ID NO:24, or sequences having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0018] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0019] In some implementations, the CDRs described above are defined according to the Kabat numbering system.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is derived from monoclonal antibody 10E9, and the antibody or antigen-binding fragment thereof comprises:

[0021] (a) The following three heavy chain CDRs: VH CDR1 with the sequence of SEQ ID NO:7 or a variant thereof, VH CDR2 with the sequence of SEQ ID NO:8 or a variant thereof, and VH CDR3 with the sequence of SEQ ID NO:9 or a variant thereof; and / or the following three light chain CDRs: VL CDR1 with the sequence of SEQ ID NO:10 or a variant thereof, VL CDR2 with the sequence of SEQ ID NO:11 or a variant thereof, and VL CDR3 with the sequence of SEQ ID NO:12 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, e.g., conservative substitutions) compared to the sequence from which it is derived;

[0022] or,

[0023] (b) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:1; and / or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:2; preferably, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

[0024] In some embodiments, the antibody or its antigen-binding fragment comprises a frame region (FR) derived from mouse immunoglobulin.

[0025] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing a sequence as shown in SEQ ID NO:1 or a variant thereof, and a VL containing a sequence as shown in SEQ ID NO:2 or a variant thereof; the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to its derived sequence, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., conserved substitutions). The variant substantially retains the biological function of its derived sequence.

[0026] In some embodiments, the antibody or its antigen-binding fragment possesses at least one of the following characteristics:

[0027] (1) A specific binding RBD to the coronavirus S protein; preferably, the coronavirus is a β coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains;

[0028] (2) The RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)) is bound with an EC50 of less than about 50 ng / mL, for example less than about 40 ng / mL, 30 ng / mL, 20 ng / mL or less, and / or the RBD of the S protein of SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY278554.2)) is bound with an EC50 of less than about 100 ng / mL, for example less than about 90 ng / mL, 80 ng / mL, 70 ng / mL, 65 ng / mL or less; preferably, the EC50 can be determined by ELISA.

[0029] (3) K is less than about 10 nM, for example less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or lower. DThe RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)); preferably, the K D Measured using surface plasmon resonance techniques (e.g., Biacore);

[0030] (4) Neutralize SARS-CoV-2 pseudovirus (e.g., Wuhan-Hu-1 virus strain (GenBank:MN908947)) in vitro at a half-inhibitory concentration (IC50) of less than about 0.1 nM, such as less than about 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM or less, as determined by, for example, the neutralization experiment described in Example 6;

[0031] (5) Neutralize SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank:AY278554.2)) in vitro at a half-inhibitory concentration (IC50) of less than about 0.1 nM, such as less than about 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM or less, as determined by, for example, the neutralization experiment described in Example 6;

[0032] (6) Neutralize SARS-CoV-2 mutant pseudoviruses (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at half-inhibitory concentrations (IC50) of less than about 2 nM, such as less than about 1.5 nM, 1.4 nM, 1.3 nM, 1.2 nM, 1 nM or less, as determined, for example, by the neutralization experiment described in Example 6;

[0033] (7) Neutralizing coronaviruses in a subject (e.g., a human), preventing and / or treating coronavirus infection or disease caused by coronavirus infection; preferably, the coronavirus is a β-coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is derived from monoclonal antibody 30A4-2, and the antibody or antigen-binding fragment thereof comprises:

[0035] (a) The following three heavy chain CDRs: VH CDR1 with the sequence of SEQ ID NO:13 or a variant thereof, VH CDR2 with the sequence of SEQ ID NO:14 or a variant thereof, and VH CDR3 with the sequence of SEQ ID NO:15 or a variant thereof; and / or the following three light chain CDRs: VL CDR1 with the sequence of SEQ ID NO:16 or a variant thereof, VL CDR2 with the sequence of SEQ ID NO:17 or a variant thereof, and VL CDR3 with the sequence of SEQ ID NO:18 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, e.g., conservative substitutions) compared to the sequence from which it is derived;

[0036] or,

[0037] (b) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:3; and / or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:4; preferably, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

[0038] In some embodiments, the antibody or its antigen-binding fragment comprises a frame region (FR) derived from mouse immunoglobulin.

[0039] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing a sequence as shown in SEQ ID NO:3 or a variant thereof, and a VL containing a sequence as shown in SEQ ID NO:4 or a variant thereof; the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to its derived sequence, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., conserved substitutions). The variant substantially retains the biological function of its derived sequence.

[0040] In some embodiments, the antibody or its antigen-binding fragment possesses at least one of the following characteristics:

[0041] (1) A specific binding RBD to the coronavirus S protein; preferably, the coronavirus is a β coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains;

[0042] (2) The RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)) is bound with an EC50 of less than about 50 ng / mL, for example less than about 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 ng / mL or less, and / or the RBD of the S protein of SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY278554.2)) is bound with an EC50 of less than about 100 ng / mL, for example less than about 90 ng / mL or less; preferably, the EC50 can be determined by ELISA.

[0043] (3) K is less than about 10 nM, for example less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM or lower. D The RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)); preferably, the K D Measured using surface plasmon resonance techniques (e.g., Biacore);

[0044] (4) Neutralize SARS-CoV-2 pseudovirus (e.g., Wuhan-Hu-1 virus strain (GenBank:MN908947)) in vitro at a half-inhibitory concentration (IC50) of less than about 1 nM, such as less than about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM or less, for example by the neutralization experiment described in Example 6;

[0045] (5) Neutralize SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank:AY278554.2)) in vitro at a half-inhibitory concentration (IC50) of less than about 1 nM, such as less than about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM or less, as determined by, for example, the neutralization experiment described in Example 6;

[0046] (6) Neutralize SARS-CoV-2 mutant pseudoviruses (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at a half-inhibitory concentration (IC50) of less than about 1 nM, such as less than about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM or less, as determined, for example, by the neutralization experiment described in Example 6;

[0047] (7) Neutralizing coronaviruses in a subject (e.g., a human), preventing and / or treating coronavirus infection or disease caused by coronavirus infection; preferably, the coronavirus is a β-coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention is derived from monoclonal antibody 1C5-2, and the antibody or antigen-binding fragment thereof comprises:

[0049] (a) The following three heavy chain CDRs: VH CDR1 with the sequence of SEQ ID NO:19 or a variant thereof, VH CDR2 with the sequence of SEQ ID NO:20 or a variant thereof, and VH CDR3 with the sequence of SEQ ID NO:21 or a variant thereof; and / or the following three light chain CDRs: VL CDR1 with the sequence of SEQ ID NO:22 or a variant thereof, VL CDR2 with the sequence of SEQ ID NO:23 or a variant thereof, and VL CDR3 with the sequence of SEQ ID NO:24 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids, e.g., conservative substitutions) compared to the sequence from which it is derived;

[0050] or,

[0051] (b) Three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO:5; and / or three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO:6; preferably, the three CDRs contained in the VH and / or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

[0052] In some embodiments, the antibody or its antigen-binding fragment comprises a frame region (FR) derived from mouse immunoglobulin.

[0053] In some embodiments, the antibody or its antigen-binding fragment comprises: a VH containing a sequence as shown in SEQ ID NO:5 or a variant thereof, and a VL containing a sequence as shown in SEQ ID NO:6 or a variant thereof; the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to its derived sequence, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., conserved substitutions). The variant substantially retains the biological function of its derived sequence.

[0054] In some embodiments, the antibody or its antigen-binding fragment possesses at least one of the following characteristics:

[0055] (1) A specific binding RBD to the coronavirus S protein; preferably, the coronavirus is a β coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains;

[0056] (2) The RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)) is bound with an EC50 of less than about 50 ng / mL, for example less than about 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 ng / mL, 14 ng / mL, 13 ng / mL, 12 ng / mL or less, and / or the RBD of the S protein of SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY278554.2)) is bound with an EC50 of less than about 500 ng / mL, for example less than about 400 ng / mL, 300 ng / mL, 250 ng / mL or less; preferably, the EC50 can be determined by ELISA.

[0057] (3) K is less than about 10 nM, for example less than about 9 nM, 8 nM, 7 nM, 6 nM, 5 nM or lower. D The RBD of the S protein of SARS-CoV-2 (e.g., Wuhan-Hu-1 strain (GenBank: MN908947)); preferably, the K D Measured using surface plasmon resonance techniques (e.g., Biacore);

[0058] (4) Neutralize SARS-CoV-2 pseudovirus (e.g., Wuhan-Hu-1 virus strain (GenBank:MN908947)) in vitro at a half-inhibitory concentration (IC50) of less than about 1 nM, such as less than about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM or less, for example by the neutralization experiment described in Example 6;

[0059] (5) Neutralize SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank:AY278554.2)) in vitro at a half-inhibitory concentration (IC50) of less than about 1 nM, such as less than about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM or less, as determined by, for example, the neutralization experiment described in Example 6;

[0060] (6) Neutralize SARS-CoV-2 mutant pseudoviruses of concern (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at half-inhibitory concentrations (IC50) of less than about 20 nM, such as less than about 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM or less, for example by the neutralization experiment described in Example 6;

[0061] (7) Neutralizing coronaviruses in a subject (e.g., a human), preventing and / or treating coronavirus infection or disease caused by coronavirus infection; preferably, the coronavirus is a β-coronavirus, such as SARS-CoV-2, SARS-CoV-1 and / or RaTG13; preferably, the SARS-CoV-2 includes mutant strains.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof in any of the above embodiments may be humanized to reduce immunogenicity in humans. Methods for humanizing non-human antibodies are known in the art; for example, methods known in the art can be used to transplant the CDR region of the antibody or antigen-binding fragment of the present invention into a human frame region. In some embodiments, the antibody or antigen-binding fragment thereof in any of the above embodiments may include a frame region (FR) derived from human immunoglobulins.

[0063] In some embodiments, the antibody or its antigen-binding fragment in any of the above embodiments may further comprise a constant region sequence derived from mammalian (e.g., mouse or human) immunoglobulin.

[0064] In some embodiments, the heavy chain of the antibody or its antigen-binding fragment in any of the above embodiments includes a heavy chain constant region derived from mouse or human immunoglobulins (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the light chain of the antibody or its antigen-binding fragment in any of the above embodiments includes a light chain constant region derived from mouse or human immunoglobulins (e.g., κ or λ).

[0065] In some implementations, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0066] In some embodiments, the antigen-binding fragment in any of the above embodiments may be selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0067] In some embodiments, the antibody in any of the above embodiments is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0068] In this document, the antibodies or antigen-binding fragments thereof of the present invention may include variants that differ from the antibodies or antigen-binding fragments from which they are derived only in the conserved substitution of one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibodies or antigen-binding fragments from which they are derived, and substantially retain the aforementioned biological functions of the antibodies or antigen-binding fragments from which they are derived.

[0069] Antibody preparation

[0070] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0071] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0072] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0073] In another aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising isolated nucleic acid molecules as described above. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.

[0074] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment of the present invention, and / or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0075] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of an antibody of the present invention or an antigen-binding fragment thereof, and / or a second nucleotide sequence encoding a light chain of an antibody of the present invention or an antigen-binding fragment thereof; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0076] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid molecules or carriers as described above. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia 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.). In some embodiments, the host cell is a microorganism, such as a prokaryotic cell or a yeast cell.

[0077] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing host cells as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0078] Therapeutic applications

[0079] The antibodies or antigen-binding fragments thereof of the present invention can be used in vitro or in vivo to neutralize coronaviruses, blocking or inhibiting coronavirus infection of cells, thereby achieving the purpose of preventing and / or treating coronavirus infection or related diseases in subjects. In particular, the antibodies or antigen-binding fragments thereof derived from 10E9, 30A4-2 and 1C5-2 provided in the first aspect of the present invention can be used in combination for the prevention and / or treatment of coronavirus infection.

[0080] Therefore, in another aspect, the present invention provides a composition comprising at least two selected from the group consisting of an antibody derived from 10E9 or an antigen-binding fragment thereof, an antibody derived from 30A4-2 or an antigen-binding fragment thereof, and an antibody derived from 1C5-2 or an antigen-binding fragment thereof, as provided in the first aspect of the invention. In some embodiments, the composition comprises an antibody derived from 10E9 or an antigen-binding fragment thereof, an antibody derived from 30A4-2 or an antigen-binding fragment thereof, and an antibody derived from 1C5-2 or an antigen-binding fragment thereof, as provided in the first aspect. In some embodiments, the various antibodies comprised in the composition are provided as separate components or as mixed components. Therefore, the various antibodies comprised in the composition can be administered simultaneously, separately, or sequentially.

[0081] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody described in the first aspect or an antigen-binding fragment thereof or the above-described composition, and a pharmaceutically acceptable carrier and / or excipient.

[0082] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents, such as additional antiviral agents (e.g., interferon, lopinavir, ritonavir, remdesivir, dexamethasone, etc.).

[0083] In some embodiments, the antibody or antigen-binding fragment or composition of the present invention, along with the additional pharmaceutically active agent, may be provided as separate components or as a mixed component in the pharmaceutical composition. Therefore, the antibody or antigen-binding fragment or composition of the present invention, along with the additional pharmaceutically active agent, may be administered simultaneously, separately, or sequentially.

[0084] In some exemplary embodiments, the pharmaceutically acceptable carrier and / or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0085] In another aspect, the present invention provides a method for neutralizing coronaviruses in a sample, comprising contacting a sample containing coronaviruses with an antibody or antigen-binding fragment thereof of the present invention, a composition, or a pharmaceutical composition. In some embodiments, the method is performed in vitro. In some embodiments, the method is used for non-diagnostic therapeutic purposes.

[0086] In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, and / or RaTG13. In some embodiments, the coronavirus is SARS-CoV-2 and / or SARS-CoV-1.

[0087] In some embodiments, the SARS-CoV-2 includes a mutant strain. In some embodiments, the S protein of the mutant strain contains a mutation, such as one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In some embodiments, the S protein of the mutant strain contains one or more amino acid substitutions selected from D614G, K417N / T, E484K, N501Y, L452R, and T478K. In some embodiments, the mutant strain is selected from the D614G strain, the Alpha strain (e.g., B.1.1.7), the Beta strain (e.g., B.1.351), the Gamma strain (e.g., P.1), the Delta strain (e.g., B.1.617.2), or any combination thereof.

[0088] In another aspect, the present invention provides a method for neutralizing coronavirus or preventing and / or treating coronavirus infection or coronavirus-related diseases in a subject, comprising: administering an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, composition or pharmaceutical composition to a subject in need of doing so.

[0089] In some embodiments, the composition of the present invention is administered to the subject, wherein the various antibodies contained in the composition may be administered simultaneously, separately, or sequentially.

[0090] In some implementations, the subject is a mammal, such as a human.

[0091] In some embodiments, the antibody or its antigen-binding fragment, composition, or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents such as interferon, lopinavir, ritonavir, remdesivir, dexamethasone, etc.).

[0092] In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, and / or RaTG13. In some embodiments, the coronavirus is SARS-CoV-2 and / or SARS-CoV-1.

[0093] In some embodiments, the SARS-CoV-2 includes a mutant strain. In some embodiments, the S protein of the mutant strain contains a mutation, such as one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In some embodiments, the S protein of the mutant strain contains one or more amino acid substitutions selected from D614G, K417N / T, E484K, N501Y, L452R, and T478K. In some embodiments, the mutant strain is selected from the D614G strain, the Alpha strain (e.g., B.1.1.7), the Beta strain (e.g., B.1.351), the Gamma strain (e.g., P.1), the Delta strain (e.g., B.1.617.2), or any combination thereof.

[0094] In another aspect, the present invention relates to the use of the antibodies or antigen-binding fragments thereof, compositions or pharmaceutical compositions of the present invention in the preparation of medicaments for: (1) neutralizing coronaviruses in vitro or in a subject (e.g., a human); and / or, (2) for the prevention and / or treatment of a subject with coronavirus infection or disease associated with coronavirus infection.

[0095] In some embodiments, the antibody or its antigen-binding fragment, composition, or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents such as interferon, lopinavir, ritonavir, remdesivir, dexamethasone, etc.).

[0096] In some embodiments, the coronavirus is a beta coronavirus. In some embodiments, the coronavirus is SARS-CoV-2, SARS-CoV-1, and / or RaTG13. In some embodiments, the coronavirus is SARS-CoV-2 and / or SARS-CoV-1.

[0097] In some embodiments, the SARS-CoV-2 includes a mutant strain. In some embodiments, the S protein of the mutant strain contains a mutation, such as one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In some embodiments, the S protein of the mutant strain contains one or more amino acid substitutions selected from D614G, K417N / T, E484K, N501Y, L452R, and T478K. In some embodiments, the mutant strain is selected from the D614G strain, the Alpha strain (e.g., B.1.1.7), the Beta strain (e.g., B.1.351), the Gamma strain (e.g., P.1), the Delta strain (e.g., B.1.617.2), or any combination thereof.

[0098] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0099] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention are administered by intravenous injection or bolus.

[0100] The pharmaceutical compositions of the present invention may include an antibody or antigen-binding fragment or composition thereof of the present invention in a "therapeutic effective amount" or a "preventive effective amount". A "preventive effective amount" refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A "therapeutic effective amount" refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of the antibody or antigen-binding fragment of the present invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the method of administration of the drug, and other concurrent treatments, etc.

[0101] In this document, the dosing regimen may be adjusted to obtain the optimal target response (e.g., treatment or prevention). For example, it may be administered as a single dose, multiple times over a period of time, or the dose may be reduced or increased proportionally to the urgency of the treatment situation.

[0102] In this paper, the subjects may be mammals, such as humans.

[0103] Conjugate

[0104] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to the coronavirus S protein. Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0105] Therefore, the present invention also provides a conjugate comprising the antibody of the present invention or an antigen-binding fragment thereof and a detectable label linked thereto.

[0106] In this document, the detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g., 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent materials (e.g., chemiluminescent materials, such as acridine esters, luminol and its derivatives, ruthenium derivatives such as terpyridine ruthenium), magnetic beads (e.g., ), thermal markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0107] In some embodiments, the detectable label is suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label may be selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, or β-galactosidase), chemiluminescent reagents (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent proteins such as FITC, TRITC, or PE), radionuclides, or biotin.

[0108] In some embodiments, the detectable marker described above can be linked to the antibody or its antigen-binding fragment of the present invention via linkers of different lengths to reduce potential steric hindrance.

[0109] Detection Application

[0110] The antibody or antigen-binding fragment of the present invention can specifically bind to the RBD of the coronavirus S protein, thereby being used to detect the coronavirus or its S protein or the RBD of the S protein, and optionally to diagnose whether a subject is infected with the coronavirus based on the above detection results.

[0111] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof, or a conjugate of the present invention.

[0112] In some embodiments, the kit comprises the conjugate of the present invention.

[0113] In other embodiments, the kit comprises the antibody or antigen-binding fragment of the present invention. In some embodiments, the antibody or antigen-binding fragment does not contain a detectable label. In some embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment of the present invention; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0114] In some embodiments, the second antibody is specific to antibodies of the species (e.g., mouse or human) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0115] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human or mouse immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-mouse IgG antibody or an anti-human IgG antibody.

[0116] In some embodiments, the kit of the present invention may further comprise reagents for detecting the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may also comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS, or luminol compounds for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. For example, when the detectable label is a chemiluminescent reagent (e.g., acrid ester compounds), the kit may also comprise a pre-excitation solution and / or an excitation solution for chemiluminescence.

[0117] In another aspect, the present invention provides a method for detecting coronaviruses or their S protein or RBD of the S protein, or the presence or level of coronavirus-infected cells in a sample, comprising using the antibody or antigen-binding fragment of the present invention.

[0118] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0119] In some embodiments, the method includes using the conjugates of the present invention.

[0120] In other embodiments, the method includes using the antibody or antigen-binding fragment of the present invention. In some embodiments, the antibody or antigen-binding fragment does not contain a detectable label. In some embodiments, the method further includes using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment.

[0121] In some embodiments, the second antibody is specific to antibodies of the species (e.g., mouse or human) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0122] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human or mouse immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-mouse IgG antibody or an anti-human IgG antibody.

[0123] In some embodiments, the method includes: (1) contacting the sample with an antibody or an antigen-binding fragment of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of a coronavirus or cells infected with a coronavirus.

[0124] In some embodiments, the method can be used for diagnostic purposes, such as diagnosing whether a subject is infected with a coronavirus based on the presence or level of the coronavirus in a sample. In such embodiments, the sample can be a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0125] In some implementations, the method can be used for non-diagnostic purposes, such as when the sample is not from a subject, for example, a vaccine sample.

[0126] In some implementations, the subject is a mammal, such as a human.

[0127] In another aspect, the use of the antibodies or antigen-binding fragments of the present invention in the preparation of diagnostic reagents for detecting coronaviruses or their S protein or RBD of the S protein, or the presence or level of coronavirus-infected cells in a sample, and / or for diagnosing whether a subject is infected with coronaviruses.

[0128] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0129] In some embodiments, the detection reagent detects coronavirus or its S protein or RBD of the S protein, or the presence or level of coronavirus-infected cells in a sample by the detection method described above, and optionally diagnoses whether a subject is infected with coronavirus based on the detection results.

[0130] In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0131] In some embodiments, the coronavirus described in any of the foregoing aspects is a β-coronavirus. In some embodiments, the coronavirus described in any of the foregoing aspects is SARS-CoV-2, SARS-CoV-1, and / or RaTG13. In some embodiments, the coronavirus described in any of the foregoing aspects is SARS-CoV-2 and / or SARS-CoV-1. In some embodiments, the SARS-CoV-2 includes a mutant strain. In some embodiments, the S protein of the mutant strain contains a mutation, such as one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In some embodiments, the S protein of the mutant strain contains one or more amino acid substitutions selected from D614G, K417N / T, E484K, N501Y, L452R, and T478K. In some embodiments, the mutant strain is selected from the D614G strain, the Alpha strain (e.g., B.1.1.7), the Beta strain (e.g., B.1.351), the Gamma strain (e.g., P.1), the Delta strain (e.g., B.1.617.2), or any combination thereof.

[0132] Terminology Definition

[0133] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures used herein, such as those in virology, biochemistry, nucleic acid chemistry, and immunology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0134] As used in this article, “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)” was formerly known as “novel coronavirus” or “2019-nCoV”. The disease caused by SARS-CoV-2 is called novel coronavirus pneumonia (COVID-19). In this article, the term “SARS-CoV-2” includes various known isolates, including both the original strain (e.g., the first sequenced isolate, GenBank: MN908947.3) and subsequently discovered mutant strains, such as the Variants of Concern (VOC). In some embodiments, the mutant strains include D614G (e.g., GenBank: MN908947), Alpha (B.1.1.7 and Q lineages, e.g., GenBank: MW624725.1), Beta (B.1.351 and descendant lineages, e.g., GenBank: MZ314998.1), Gamma (P.1 and descendant lineages, e.g., GenBank: MZ427312.1), and Delta (B.1.617.2 and AY lineages, e.g., GenBank: OM444216.1). In some embodiments, the term "SARS-CoV-2" includes both isolates whose spike protein does not contain mutations (e.g., compared to the reference strain MN908947.3) and isolates whose spike protein contains mutations (e.g., amino acid substitutions compared to the reference strain MN908947.3, such as D614G, K417N / T, E484K, N501Y, L452R, T478K, or any combination thereof). In some embodiments, the mutant strain is preferably selected from isolates whose spike protein contains mutations (e.g., amino acid substitutions, such as D614G, K417N / T, E484K, N501Y, L452R, T478K, or any combination thereof). In some exemplary embodiments, the mutant strain is selected from the D614G strain, the Alpha strain (e.g., B.1.1.7), the Beta strain (e.g., B.1.351), the Gamma strain (e.g., P.1), and the Delta strain (e.g., B.1.617.2).

[0135] As used in this article, “Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)”, also known as “SARS-CoV-1”, is the pathogen of Severe Acute Respiratory Syndrome (SARS). In this article, the term “SARS-CoV-1” includes various known isolates, such as the CUHK-W1 strain (GenBank:AY278554.2) or GenBank:AAP13567.1. The disease caused by SARS-CoV-1 is called Severe Acute Respiratory Syndrome (SARS).

[0136] Both SARS-CoV-1 and SARS-CoV-2 belong to the genus β-coronavirus and are enveloped, single-stranded, positive-sense RNA viruses. They contain at least three membrane proteins, including a surface spike protein (S), an integrated membrane protein (M), and an integrated membrane protein (E). Both viruses achieve viral membrane fusion and cell entry by specifically binding to angiotensin-converting enzyme 2 (ACE2) on the host cell via the receptor-binding domain (RBD) on the S protein, playing a crucial role in viral infection of cells.

[0137] As used in this article, “RaTG13” is a bat coronavirus, which can be found in, for example, GenBank:MN996532.1.

[0138] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen via at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains, each pair comprising one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The allocation of amino acids in each region or domain can follow the definitions in 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. (1989) Nature 342:878-883.

[0139] As used herein, the term “complementarity-determining region” or “CDR” refers to the amino acid residue in the antibody variable region responsible for antigen binding. Each of the heavy and light chain variable regions contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0140] In this invention, the CDR contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat, Chothia, or IMGT numbering system. In some embodiments, the CDR contained in the antibody or antigen-binding fragment thereof is preferably determined by the Kabat numbering system.

[0141] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0142] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0143] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety”. See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, complementarity-determining region (CDR) fragments, scFv, diabody, single-domain antibody, chimeric antibody, linear antibody, nanobody (technology from Domantis), probody, and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0144] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0145] As used herein, the term “Fd” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0146] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0147] As used herein, the term "Fc" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0148] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, 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 in this invention 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. In some cases, a disulfide bond may also exist between VH and VL of scFv. In some embodiments of the present invention, scFv can form di-scFv, which refers to two or more individual scFvs linked together to form an antibody. In some embodiments of the present invention, scFv can form (scFv)2, which refers to two or more individual scFvs linked together in parallel to form an antibody.

[0149] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another 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)).

[0150] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art as an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also known as nanobodies.

[0151] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0152] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0153] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and whose light chain and / or heavy chain portion is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen in any case (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). In some embodiments, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody are derived from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody are derived from a second antibody (e.g., a human antibody).

[0154] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Typically, at least one or two, but usually all three (heavy and / or light immunoglobulin chains) of the receptor CDR of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDR is referred to as the "donor," and the immunoglobulin providing the framework is referred to as the "receptor." In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the receptor framework can be a naturally occurring human framework, or a sequence having approximately 85%, 90%, 95%, 99%, or higher sequence identity compared to it. Humanized antibodies typically retain the intended properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. Donor antibodies can be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibodies with the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

[0155] In this application, the intended properties of the antibodies of the present invention include: (1) specifically binding to the RBD of the S protein of coronaviruses (e.g., SARS-CoV-2 and / or SARS-CoV-1); (2) neutralizing coronaviruses (e.g., SARS-CoV-2 and / or SARS-CoV-1) in vitro or in a subject (e.g., a human); and (3) preventing and / or treating coronavirus (e.g., SARS-CoV-2 and / or SARS-CoV-1) infection or related diseases. The antibodies of the present invention have one or more of the above-described intended properties.

[0156] The chimeric or humanized antibodies of this invention can be prepared based on the sequence of monoclonal antibodies produced by immunized animals (e.g., mice). The DNA encoding the heavy and light chains can be obtained from targeted hybridomas or specific B cells from immunized animals and engineered using standard molecular biology techniques to contain human immunoglobulin sequences.

[0157] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K0) of that interaction. D () indicates. In this invention, the term "K" is used. D"" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between the two molecules can be determined using methods known in the art, such as surface plasmon resonance (SPR) in a BIACORE instrument.

[0158] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0159] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to 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, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0160] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0161] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0162] The twenty common amino acids mentioned in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0163] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art as being capable of stabilizing the desired activity of the active ingredient in the pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable fluids are selected from water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0164] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., coronavirus infection) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to expected survival (if no treatment was received).

[0165] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has a coronavirus infection or a related disease, or is at risk of having such a disease.

[0166] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., coronavirus infection) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., coronavirus infection); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0167] As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0168] Beneficial effects of the invention

[0169] This invention provides a monoclonal antibody capable of neutralizing coronaviruses, which has at least the following advantages: (1) strong neutralizing ability against SARS-CoV-2 and SARS-CoV-1; (2) stable neutralizing effect against currently prevalent SARS-CoV-2 mutant strains; (3) broad-spectrum ability to cross-bind to the RBDs of SARS-CoV-1, SARS-CoV-2, and RaTG13; and (4) protection against challenge with the SARS-CoV-2 mutant strain B.1.351 in animal models. The antibody of this invention has significant clinical value for the prevention / treatment and diagnosis of coronavirus infection.

[0170] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0171] Figure 1 The ELISA reactivity of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 to the recombinant RBD protein of SARS-CoV-2, SARS-CoV, and RaTG13-CoV viruses was demonstrated.

[0172] Figure 2A The formula for calculating ligand coupling levels in the Biacore affinity assay is shown.

[0173] Figure 2B The results of affinity assays for mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against the recombinant RBD protein of SARS-CoV-2 virus are shown.

[0174] Figure 3 The results of fluorescence detection of SARS-CoV-2 VSVpp and SARS-CoV VSVpp infection in BHK21-hACE2 cells are shown.

[0175] Figure 4 The neutralizing activity assays of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 in SARS-CoV-2 and SARS-CoV VSV pp pseudovirus infection models are shown.

[0176] Figure 5 The neutralizing activity of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against SARS-CoV-2 variants of concern (VOC) was demonstrated.

[0177] Figure 6 The neutralizing activity of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against the Beta (B.1.351) mutant strain of concern was demonstrated.

[0178] Figure 7 The study evaluated the therapeutic effect of a combination of three mouse monoclonal antibodies (10E9, 30A4-2, and 1C5-2) on hamsters inoculated with Beta (B.1.351).

[0179] Figure 8 The study shows the pathological findings of hamster lung tissue after treatment with a combination of three antibodies: mouse monoclonal antibody 10E9, 30A4-2, and 1C5-2.

[0180] Sequence information

[0181] The information relating to the sequence is provided in Table 1 below.

[0182] Table 1: Sequence Description

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] Detailed Implementation

[0190] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0191] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0192] Example 1. Construction of pseudoviruses of SARS-CoV and SARS-CoV-2 based on VSV vector

[0193] 1.1 Construction of plasmids expressing SARS-CoV and SARS-CoV-2 Spike genes

[0194] The spike protein gene of the novel coronavirus Wuhan-Hu-1 strain (GenBank: MN908947) was truncated with 18 amino acids at the C-terminus, and then codon-optimized before being cloned into the eukaryotic expression plasmid vector pCAG to obtain the pCAG-nCoVS-del18 plasmid (Wuhan-Hu-1-spike gene sequence is shown in SEQ ID NO:25, and nCoVS-del18 gene sequence is shown in SEQ ID NO:26); the spike protein gene of the coronavirus SARS-CoV CUHK-W1 strain (GenBank: AY278554.2) was truncated with 18 amino acids at the C-terminus, and then codon-optimized before being cloned into the eukaryotic expression plasmid vector pCAG to obtain the pCAG-SARS-Sdel18 plasmid (SARS CoV CUHK-W1 spike gene sequence is shown in SEQ ID NO:27, and SARS-Sdel18 gene sequence is shown in SEQ ID NO:28).

[0195] 1.2 VSV-based pseudovirus packaging of SARS-CoV and SARS-CoV-2

[0196] Using the VSV reverse genetics operating system, this study successfully constructed a recombinant VSV: VSVdG-EGFP-G. VSVdG-EGFP-G is a recombinant viral form in which the G gene in its genome is replaced by the fluorescent reporter gene EGFP. It can be used to generate rVSV pseudoviruses containing heterologous envelope proteins. These heterologous proteins can be viral proteins with high biosecurity requirements. Because the replication ability of the VSVdG-EGFP-G pseudovirus is limited to a single generation, it can be used in BSL-2 laboratories to study viruses required by BSL-3 / BSL-4 laboratories, such as SARS-CoV and SARS-CoV-2. Vero-E6 cells were transfected with the pCAG-nCoVS-del18 plasmid. Twenty-four hours later, they were infected with the virus VSVdG-EGFP-G. One hour after infection, the cell supernatant was replaced with 5% FBS DMEM containing anti-VSV-G rat serum to block infection by residual VSVdG-EGFP-G. Twenty-four hours later, the cell supernatant was harvested to obtain a pseudovirus based on the VSV vector carrying SARS-CoV-2-Spike-del18, which was aliquoted and frozen at -80°C for later use. Using the same packaging method, a pseudovirus based on the pCAG-SARS-Sdel18 plasmid carrying SARS-CoV-2-Spike-del18 was obtained, aliquoted, and frozen at -80°C for later use.

[0197] Example 2. Screening for monoclonal antibodies that simultaneously neutralize SARS-CoV and SARS-CoV-2

[0198] 2.1 Mouse Immunization

[0199] 2.1.1 Immunogen Preparation: The immunogen was the VSV vector-based pseudovirus carrying SARS-CoV-2-Spike-del18 and the pseudovirus carrying SARS-CoV-Spike-del18 obtained in Example 1. The immunization dose was 5 × 10⁻⁶. 6 PFU / dose is mixed with an equal volume of Freund's adjuvant and thoroughly mixed in an injection emulsifier to form a water-in-oil emulsion. Freund's complete adjuvant is used for primary immunization, and Freund's incomplete adjuvant is used for subsequent booster immunizations.

[0200] 2.1.2 Mouse Immunization: Six- to eight-week-old female BALB / c mice were immunized bilaterally by multiple subcutaneous injections in the groin using the prepared immunogens described above. The injection volume was 300 μL per mouse per injection. The immunization procedure adopted a sequential immunization approach. Immunizations at weeks 1, 3, and 5 were based on pseudoviruses carrying SARS-CoV-Spike-del18 via VSV vectors. Immunizations at weeks 2, 4, and 6 were based on pseudoviruses carrying SARS-CoV-2-Spike-del18 via VSV vectors. Approximately 200 μL of orbital venous blood was collected before each immunization for titer determination. The neutralizing titers of serum against SARS-CoV and SARS-CoV-2 pseudoviruses were detected using a pseudovirus neutralization method. Fusion experiments were performed at week 7.

[0201] 2.2 Antibody Screening

[0202] 2.2.1 Preparation before fusion: Resuscitate mouse myeloma cells (SP2 / 0) and culture them in RPMI 1640 medium containing 10% fetal bovine serum to the logarithmic growth phase in preparation for fusion.

[0203] 2.2.2 Preparation and Screening of Fusion Hybridomas

[0204] Mouse spleen cells were prepared into a cell suspension and fused with mouse myeloma cells SP2 / 0 to obtain hybridoma cells. Feeder cells were then prepared and co-cultured with the hybridoma cells. During hybridoma cell culture, a large number of unfused myeloma cells and mouse spleen cells die in RPMI 1640-HAT selection medium, and a very small number of hybridoma cells do not survive. These feeder cells are required to survive. In our laboratory, mouse peritoneal macrophages and young mouse thymocytes were used as feeder cells.

[0205] 2.2.2.1 Preparation of mouse peritoneal macrophages: (i) A 6-week-old BALB / c mouse was euthanized by cervical indwelling, disinfected by immersion in 75% alcohol for 3-5 minutes, and then placed in a laminar flow hood with the mouse's abdomen facing upward. The mouse's abdominal skin was lifted with forceps, a small incision was made, and the mouse's outer skin was cut open vertically at the incision site using two hemostatic forceps to expose the mouse's peritoneum; (ii) The peritoneum was lifted with sterile forceps, and 5 mL of RPMI1640 culture medium was injected into the mouse's peritoneal cavity with a syringe. The mouse was then shaken to mix the culture medium in the peritoneal cavity, and then the culture medium in the peritoneal cavity was carefully aspirated with a syringe; (iii) The culture medium containing macrophages was added to RPMI1640-HAT selection medium containing 20% ​​fetal bovine serum and mixed with the fusion cells.

[0206] 2.2.2.2 Preparation of mouse thymocytes: (i) A 3-week-old BALB / c mouse was euthanized by cervical indwelling, immersed in 75% alcohol for 3-5 minutes for disinfection, and then placed in a laminar flow hood with the mouse's abdomen facing upward. The skin of the mouse's chest cavity was lifted with forceps, a small incision was made, and the mouse's outer skin was cut open vertically with two hemostatic forceps to expose the endothelium of the mouse's chest. (ii) The chest cavity was clamped with another sterile forceps and the chest cavity was cut open with scissors. (iii) The milky white thymus in the chest cavity was removed with clean sterile forceps, ground in a 70μm cell sieve to obtain thymus feeder cells, and the thymocytes were added to RPMI1640-HAT selection medium containing 20% ​​fetal bovine serum and mixed with the fusion cells.

[0207] 2.2.2.3 Preparation of mouse myeloma cells: SP2 / 0 cells grown to the logarithmic growth phase were selected for fusion. Before fusion, myeloma cells were transferred from the culture flask to a centrifuge tube and washed once with RPMI-1640 medium (1500 rpm × 5 min). The cells were resuspended in RPMI-1640 medium and counted.

[0208] 2.2.2.4 Preparation of spleen cells from immunized mice: (i) Take BALB / c mice that have undergone booster immunization as described in 3.1.3, collect whole blood and serum from the mice. (ii) Then, euthanize the mice by cervical inclination, immerse them in 75% alcohol for 3-5 min for disinfection, and then place them in a laminar flow hood with the mice in a right lateral decubitus position. (iii) Open the abdominal cavity of the mice with sterile forceps and surgical scissors, cut out the spleen, cut the spleen into small pieces, grind them in a 70 μm cell sieve to obtain spleen cells. (iv) Place the spleen cells in a 50 mL centrifuge tube, remove the adipose tissue with a glass pipette (curved tube), then add RPMI-1640 medium to 30 mL, centrifuge at 1500 rpm for 5 min, and repeat 3 times. (v) Resuspend the spleen cells in RPMI-1640 medium and count them.

[0209] 2.2.2.5 Preparation of hybridomas by PEG fusion: (i) Before fusion, incubate 1 mL of PEG1450 (purchased from SIGMA) and 40 mL of RPMI1640 medium at 37°C; (ii) Mix the prepared myeloma cells and spleen cells in a 50 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, and gently tap the bottom of the tube to loosen the cells into a paste; (iii) Slowly add the incubated PEG to the cells, shaking the cells while adding, then mix the cells, and terminate the fusion with incubated RPMI1640 medium after 1 min; (iv) Centrifuge at 1500 rpm for 5 min, add the cells to RPMI1640-HAT selection medium containing feeder cells and 20% fetal bovine serum, then add 200 μL to each well of a 96-well plate, and add 5% PEG. (v) After 5 days of culture, remove the 20% fetal bovine serum RPMI1640-HAT medium and replace it with RPMI1640-HT medium containing 10% fetal bovine serum. Continue culturing for another 5 days and then collect the cell supernatant for detection.

[0210] 2.2.2.6 Hybridoma Screening: Pseudovirus Neutralization Screening. Using the SARS-CoV and SARS-CoV-2 pseudoviruses based on the VSV vector in Example 1, 50 μL of the supernatant from the fusion cell culture was used for pseudovirus neutralization experiments. Positive clone wells that could simultaneously inhibit SARS-CoV and SARS-CoV-2 pseudovirus infection were selected and cloned to screen for monoclonal hybridoma cells.

[0211] 2.3 Production of mouse monoclonal antibody ascites

[0212] Five BALB / c mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin oil. The mice were ready for use 3 days after sensitization. Hybridoma cells in logarithmic growth phase were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in PBS to a concentration of 1-2 x 10^6 cells / mL. 0.5 mL of these cells was injected intraperitoneally into each mouse. After 7 days, the mice's abdomens were significantly distended. The mice were then euthanized by cervical insemination, and the peritoneal cavity was carefully opened, and all ascites fluid was aspirated.

[0213] 2.4 Purification of monoclonal antibody ascites

[0214] After high-speed centrifugation of the monoclonal antibody ascites fluid, the supernatant was collected and an equal volume of saturated ammonium sulfate solution was added. The mixture was allowed to precipitate on ice for 30 min, followed by centrifugation at 25,000 rpm for 10 min. The precipitate was dissolved in 0.2 M disodium hydrogen phosphate dodecahydrate buffer and then purified using a Protein A affinity chromatography column (purchased from GE Healthcare, USA) to obtain purified mouse monoclonal antibodies. Three antibody strains were ultimately obtained: 10E9 (type IgG2a), 30A4-2 (type IgG1), and 1C5-2 (type IgG1).

[0215] Example 3. Amplification and sequencing of the light and heavy chain variable region genes of monoclonal antibodies 10E9, 30A4-2, and 1C5-2

[0216] Hybridoma RNA preparation: Hybridoma cells (10E9, 30A4-2, and 1C5-2) cultured to the logarithmic growth phase were transferred to 15 mL centrifuge tubes. Cells were collected by centrifugation at 1500 rpm for 3 min, resuspended in 200 μl sterile PBS (pH 7.45), and transferred to a new RNase-free 1.5 mL centrifuge tube. 800 μl of Trizol solution (Invitrogen) was added, and the tubes were vigorously vortexed for 30 seconds and then incubated at 4°C for 10 min. The cells were then frozen at -80°C and sent to a sequencing company for sequencing.

[0217] Sequencing revealed the amino acid sequences of the heavy and light chain variable regions of 10E9, 30A4-2, and 1C5-2. The VH and VL sequences of 10E9 are shown in SEQ ID NO:1 and 2, respectively; the VH and VL sequences of 30A4-2 are shown in SEQ ID NO:3 and 4, respectively; and the VH and VL sequences of 1C5-2 are shown in SEQ ID NO:5 and 6, respectively.

[0218] Furthermore, the CDR sequences of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 were determined using the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669). The amino acid sequences of the CDRs for the heavy chain variable region and light chain variable region of 10E9 are shown in SEQ ID NO:7-12, respectively; the amino acid sequences of the CDRs for the heavy chain variable region and light chain variable region of 30A4-2 are shown in SEQ ID NO:13-18, respectively; and the amino acid sequences of the CDRs for the heavy chain variable region and light chain variable region of 1C5-2 are shown in SEQ ID NO:19-24, respectively.

[0219] Example 4. ELISA binding activity of monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against SARS-CoV-2, SARS-CoV-1, and RaTG13.

[0220] 4.1 Expression and purification of RBD proteins of SARS-CoV, SARS-CoV-2 and RaTG13

[0221] Referring to the viral genome sequences already published in Genebank, namely SARS-CoV-2Wuhan-Hu-1 (GenBank: MN908947), SARS-CoV (AAP13567.1), and RaTG13 (MN996532.1), we constructed RBD protein expression clones for the three coronaviruses and prepared recombinant proteins. The recombinant protein of SARS-CoV-2 was named SARS-CoV-2-RBD, the recombinant protein of SARS-CoV was named SARS-CoV-RBD, and the recombinant protein of RaTG13-CoV was named RaTG13-RBD.

[0222] 4.2 Preparation of reaction plates

[0223] The obtained SARS-CoV-2-RBD, SARS-CoV-RBD, and RaTG13-RBD proteins were diluted with 50 mM MCB buffer (NaHCO3 / Na2CO3 buffer, final concentration 50 mM, pH 9.6) at pH 9.6 to a final concentration of 2 μg / mL. 100 μL of coating buffer was added to each well of a 96-well microplate, and the plates were coated at 2–8°C for 16–24 hours, followed by coating at 37°C for 2 hours. The plates were washed once with PBST washing buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20). Then, 200 μL of blocking buffer (20 mM Na2HPO4 / NaH2PO4 buffer solution at pH 7.4 containing 20% ​​fetal bovine serum and 1% casein) was added to each well, and the plates were blocked at 37°C for 2 hours. The blocking buffer was discarded. After drying, the plates were stored in aluminum foil bags at 2–8°C for later use.

[0224] 4.3 ELISA detection of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2

[0225] The mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 obtained in Example 3 were diluted to 10 μg / mL with PBS containing 20% ​​newborn calf serum, and then diluted 3-fold or 5-fold in eight different ways. ELISA detection was performed according to the following steps:

[0226] (1) Sample reaction: Take the enzyme-labeled plates that have been coated with SARS-CoV-2-RBD, SARS-CoV-RBD and RaTG13-RBD proteins respectively, add 100 μL of diluted sample to each well, and incubate at 37℃ for 30 minutes.

[0227] (2) Enzyme labeling reaction: After completing the sample reaction steps, wash the enzyme label plate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween 20), add 100μL of HRP-labeled goat anti-mouse IgG (GAM) reaction solution to each well, and incubate at 37℃ for 30 minutes.

[0228] (3) Colorimetric reaction: After completing the enzyme labeling reaction steps, wash the microplate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween 20), add 50μL of TMB colorimetric reagent (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) to each well, and incubate at 37℃ for 15 minutes.

[0229] (4) Termination of reaction and measurement of readings: After the colorimetric reaction is completed, add 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) to each well of the plate after the reaction is completed, and measure the OD450 / 630 value of each well on the microplate reader.

[0230] Reactivity of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 with SARS-CoV-2-RBD, SARS-CoV-RBD, and RaTG13-RBD was determined based on the post-reaction readings. A positive result was considered achieved if the ratio of the detected value to the background value was greater than 5.

[0231] (5) Results Analysis: ELISA reactivity results are as follows Figure 1 As shown, mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 all exhibited strong binding activity against SARS-CoV-2-RBD and SARS-CoV-RBD. Figure 1 -A, 1-B); among them, 10E9 and 1C5-2 also have some reactivity to RaTG13-RBD ( Figure 1 -C), possessing certain spectroscopic properties, and the corresponding reactivity EC50 is summarized as follows: Figure 1 -D is shown.

[0232] Example 5. Biacore affinity assay of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 for SARS-CoV-2 RBD recombinant protein.

[0233] This study used surface plasmon resonance (SPR) technology to detect the affinity of three monoclonal antibodies against SARS-CoV and SARS-CoV-2 (10E9, 30A4-2, and 1C5-2) obtained in Example 2 for the RBD antigen of SARS-CoV-2. The detection method used in this example was the capture method, employing a Protein G chip (GE) to capture the mouse monoclonal antibodies.

[0234] 5.1 Determination of Ligand Response Value (RU)

[0235] The formula for calculating ligand coupling level is as follows: Figure 2A As shown, based on the molecular weight of the mouse monoclonal antibody and the molecular weights of the three RBD analytes, the ligand response value is approximately 1000 RU.

[0236] The three antibodies, 10E9, 30A4-2, and 1C5-2, were diluted with PBS to appropriate concentrations: 50 μg / mL for 30A4-2, 40 μg / mL for 30A4-2, and 40 μg / mL for 1C5-2, to stabilize the response value of their binding to the Protein G chip at around 1000 RU.

[0237] 5.2 Affinity Testing

[0238] The SARS-CoV-2 RBD antigen was serially diluted 2-fold from an initial concentration of 1000 nM downwards (a total of 10 dilution gradients were tested, with the highest test concentration being 1000 nM and the lowest being 1.6 nM). Antigen-antibody affinity was detected using a Biacore 8000 surface plasmon resonance (SPR) analyzer (GE). Results are as follows: Figure 2B As shown.

[0239] 5.3 Results Analysis

[0240] Three antibodies, 10E9, 30A4-2, and 1C5-2, exhibited good affinity for the SARS-CoV-2 viral RBD antigen. Among them, antibody 10E9 showed the highest affinity for the SARS-CoV-2 RBD antigen, with a dissociation constant Kd = 0.99 nM. The dissociation constants for 30A4-2 and 1C5-2 against the SARS-CoV-2 viral RBD antigen were Kd = 2.79 nM and Kd = 4.69 nM, respectively. This indicates that the mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 possess good cross-binding activity against the RBD of both coronaviruses.

[0241] Example 6. Neutralization experiments of monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against SARS-CoV and SARS-CoV-2 pseudoviruses.

[0242] 6.1 Construction of a pseudovirus neutralization system based on SARS-CoV VSVpp and SARS-CoV2 VSVpp

[0243] To construct a VSV pseudovirus carrying the SARS-CoV-2 spike protein, the spike gene of the Wuhan-Hu-1 strain (sequence source: GenBank: MN908947) was codon-optimized for expression in human cells. An 18-amino acid truncated C-terminus of the SARS-CoV-2 spike gene was cloned into the eukaryotic expression vector pCAG, obtaining pCAG-nCoVSde18. The plasmid pCAG-nCoVSde18 was transfected into Vero-E6 cells. Forty-eight hours after transfection, VSVdG-EGFP-G (Addgene, 31842) virus was inoculated into cells expressing the truncated SARS-CoV-2 Sde18 protein and incubated for one hour. The VSVdG-EGFP-G virus in the supernatant was then removed, and anti-VSV-G rat serum was added to block infection by residual VSVdG-EGFP-G. The progeny viruses carried the truncated SARS-CoV-2 Sde18 protein, yielding the pseudovirus VSV-SARS-CoV-2VSVpp. The SARS-CoV-1 pseudovirus SARS-CoV VSVpp was constructed in the same manner. Twenty-four hours after VSVdG-EGFP-G infection, cell supernatants were collected, centrifuged, and filtered (0.45 μm pore size, Millipore, SLHP033RB) to remove cell debris and stored at -80°C for later use. Viral titers were determined by the number of GFP-positive cells infected with serially diluted supernatants in BHK21-hACE2. The hACE2 gene was integrated into BHK21 cells using the PiggyBac transposon system. A transposon vector containing the hACE2 gene (SBI system biosciences, PB514B-2) and a transposase plasmid were co-transfected into BHK21 cells. Cells stably expressing hACE2, BHK21-hACE2, were obtained by selection using puromycin resistance and red fluorescence. Fluorescence images of BHK21-hACE2 cells infected with pseudoviruses SARS-CoV-2VSVpp and SARS-CoV VSVpp are shown below. Figure 3 As shown in Figure A.

[0244] Results analysis: such as Figure 3As shown in Figure B, when SARS-CoV-2 VSVpp and SARS-CoV VSVpp infected BHK21-hACE2 cells, the number of GFP-positive cells decreased with increasing infection dilution. Within the range of 1000-20000 GFP-positive cells, after pseudovirus infection, the number of positive cells showed a good correlation with the infection dilution (SARS-CoV-2 VSVpp: R). 2 =0.98, SARS-CoV VSVpp:R 2 =0.98). Based on the formula: Viral titer = Number of positive cells × Dilution factor / Virus volume, the corresponding viral titer was calculated as: SARS-CoV-2VSVpp = 2.97 × 10⁻⁶. 7 PFU / mL; SARS-CoV VSVpp=2.73×10 7 PFU / mL.

[0245] 6.2 Neutralization experiments on pseudoviruses of SARS-CoV VSVpp and SARS-CoV2 VSVpp

[0246] Antibodies 10E9, 30A4-2, and 1C5-2 were diluted to 666.7 nM as gradient 1, followed by 3-fold gradients down to a total of 11 gradients. The serially diluted antibodies were mixed with diluted VSVpp virus (MOI = 0.05) and incubated at 37°C for 1 h. All samples and viruses were diluted with 10% FBS-DMEM. 80 μL of the mixture was added to pre-coated BHK21-hACE2 cells. After 12 hours of incubation, fluorescence imaging of infected cells was performed using a disc-based confocal high-content imaging system (Opera Phenix or Operetta CLS, purchased from PerkinElmer). The number of green fluorescent positive cells was then quantitatively analyzed using Columbus image management and analysis software. The percentage reduction in the number of GFP-positive cells in the antibody-treated group compared to the untreated control wells was calculated, and the inhibition rate was calculated. The IC50 of the antibody was calculated using nonlinear regression analysis.

[0247] The results are as follows Figure 4As shown, 10E9, 30A4-2, and 1C5-2 all exhibited strong neutralizing activity against SARS-CoV-2 VSVpp and SARS-CoV VSVpp, with 10E9 showing superior neutralizing activity, achieving a neutralizing IC50 of 0.03 nM against SARS-CoV VSVpp and 0.04 nM against SARS-CoV VSVpp. Monoclonal antibody 30A4-2 showed neutralizing IC50 values ​​of 0.11 nM against SARS-CoV and 0.29 nM against SARS-CoV-2 pseudovirus, while monoclonal antibody 1C5-2 showed neutralizing IC50 values ​​of 0.34 nM against SARS-CoV and 0.37 nM against SARS-CoV-2 pseudovirus, respectively.

[0248] 6.3 Neutralization experiment against pseudoviruses of Variants of Concern (VOC), the currently prevalent novel coronavirus SARS-CoV-2.

[0249] To test the neutralizing effect of the monoclonal antibody produced in this patent against the currently prevalent SARS-CoV-2 variants of concern (VOC), this study, following the pseudovirus production method in Example 6.1.1, cloned the spike gene of the SARS-CoV-2 VOC variant into the eukaryotic expression vector pCAG to obtain the corresponding plasmid. This plasmid was then used to replace plasmid pCAG-nCoVSde18 to produce the corresponding pseudovirus, containing D614G (sequence source: GenBank: MN908947), Alpha (B.1.1.7 and Q lineages, GenBank: MW624725.1), Beta (B.1.351 and descendant lineages, GenBank: MZ314998.1), Gamma (P.1 and descendant lineages, GenBank: MZ427312.1), and Delta (B.1.617.2 and AY). Five pseudoviruses (Lineages, GenBank: OM444216.1) were used. 10E9, 30A4-2, and 1C5-2 were diluted to 66.67 nM as gradient 1, followed by 3-fold gradients down to a total of 11 gradients. The serially diluted antibodies were mixed with diluted VSVpp virus (MOI = 0.05) and incubated at 37°C for 1 h. All samples and viruses were diluted with 10% FBS-DMEM. 80 μL of the mixture was added to pre-coated BHK21-hACE2 cells. After 12 hours of incubation, fluorescence imaging of infected cells was performed using a disc-based confocal high-content imaging system (Opera Phenix or Operatta CLS, PerkinElmer). The number of green fluorescent positive cells was then quantitatively analyzed using Columbus image management and analysis software. The percentage reduction in the number of GFP-positive cells in the antibody-treated group compared to the untreated control wells was calculated, and the inhibition rate was calculated. The IC50 of the antibody was calculated using nonlinear regression analysis.

[0250] Test results as follows Figure 5 As shown in Table 2, 10E9, 30A4-2, 1C5-2, and the combination of the three antibodies have a stable and strong neutralizing effect against the currently prevalent SARS-CoV-2 variants of concern (VOC). The neutralizing effect is not affected by viral mutations. The corresponding IC50 values ​​for the neutralizing effect are summarized in the table below.

[0251] Table 2: Summary of neutralizing IC50 of 10E9, 30A4-2 and 1C5-2 against SARS-CoV-2 variants of concern (VOC)

[0252]

[0253] Example 7. Neutralizing effect of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against the SARS-CoV-2 beta (B.1.351) mutant strain.

[0254] To further evaluate the neutralizing efficacy of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2 against SARS-CoV-2, this example tested the neutralizing efficacy of the antibodies against the SARS-CoV-2 mutant strain Beta (B.1.351) using the following method:

[0255] 10E9, 30A4-2, and 1C5-2 were diluted to 6666.67 nM as gradient 1, followed by 5-fold serial dilutions for a total of 5 gradients. The serially diluted antibodies were mixed with the SARS-CoV-2 beta (B.1.351) mutant strain and incubated at 37°C for 1 h. All samples and viruses were diluted with 10% FBS-DMEM, and the mixture was added to pre-coated Vero cells. Viral titer was characterized by the number of viral plaques formed. The percentage reduction in plaque count in the antibody-treated group compared to the untreated control wells was calculated, and the inhibition rate was calculated. The IC50 of the antibody was calculated using nonlinear regression analysis.

[0256] Results Analysis: The results are as follows Figure 6 As shown, 10E9, 30A4-2, and 1C5-2 all exhibited strong neutralizing activity against the novel coronavirus Beta (B.1.351) mutant strain. Among them, 10E9 showed the best neutralizing activity, with a neutralizing IC50 of 30.2 nM against the novel coronavirus Beta (B.1.351) mutant strain. The neutralizing IC50s of monoclonal antibodies 30A4-2 and 1C5-2 were 103.4 nM and 132.9 nM, respectively.

[0257] Example 8. Evaluation of the therapeutic and protective efficacy of combined use of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 against novel coronavirus in a hamster model.

[0258] To evaluate the therapeutic effects of mouse monoclonal antibodies 10E9, 30A4-2, and 1C5-2, this example uses intranasal challenge with 1×10⁻⁶ antibodies in hamsters. 4Twenty-four hours after administering PFU to the novel coronavirus strain Beta (B.1.351), the hamsters were treated with a combination of three antibodies: 10E9, 30A4-2, and 1C5-2. 1 ml of the antibodies were injected intraperitoneally at a dose of 35 mg / kg. The hamsters were continuously monitored for signs of lung inflammation, viral load in the respiratory tract, and histopathological characteristics after antibody treatment.

[0259] The results are as follows Figure 7 As shown. Figure 7 As shown in Figure A, out of 18 hamsters, 12 hamsters were vaccinated with 1×10⁻⁶ dredges. 4 Twenty-four hours after PFU administration of the novel coronavirus strain Beta (B.1.351), six hamsters received a triple antibody combined with a single injection at a dose of 35 mg / kg / dose. Another six hamsters received no treatment after inoculation and served as the untreated group. The remaining six hamsters received no treatment and served as the blank control group. Data on body weight were collected... Figure 7 B) It can be seen that in the blank control group, hamsters grew normally without any treatment, and their weight gradually increased over time. On the fifth day, the average weight of the hamsters increased by 9.43%. In contrast, the weight of the untreated hamsters continued to decrease over time after the challenge, and the average weight of the hamsters decreased by 13.76% on the fifth day after the challenge. The weight of the hamsters in the triple antibody combined injection treatment group only decreased slightly over time, and the average weight of the hamsters decreased by only 2.28% on the fifth day after the challenge, which was significantly lower than that of the untreated group (day 2: p = 0.0022; day 3: p = 0.0043; day 4: p = 0.0022; day 5: p = 0.0043). At the same time, all hamsters in the untreated group showed obvious symptoms of weakness, ruffled hair, hunchback, or abdominal breathing, which were not observed in the antibody treatment group and the blank control group. From the perspective of hamster survival rate ( Figure 7 C) In the untreated group, one hamster died on day 4 and two died on day 5, with a 5-day survival rate of 50%. In contrast, no hamsters died in the antibody treatment group and the blank control group, with a survival rate of 100%. This result indicates that the combined treatment of three antibodies, namely hamster monoclonal antibodies 10E9, 30A4-2 and 1C5-2, has a significant therapeutic effect on hamsters inoculated with Beta (B.1.351).

[0260] Furthermore, on the fifth day after challenge, hamsters were sacrificed to assess viral load and histopathological characteristics in the respiratory tract, and SARS-CoV-2 viral RNA was quantified using quantitative real-time PCR. The detection results of the ORF-ab1 gene and the N gene are as follows: Figure 7 D and Figure 7As shown in Figure E. The results showed that, compared with the untreated group, the viral RNA level in the lung tissue of hamsters treated with antibodies was significantly reduced, decreasing by 2.80 log10 (ORF-ab1 copies / ml) and 1.57 log10 (N copies / ml); in the trachea and nasal turbinate tissues, hamsters treated with antibodies showed a more significant reduction in viral RNA compared with the untreated group, with a decrease of 1.30 log10 (ORF-ab1 copies / ml) and 1.31 log10 (N copies / ml) in the nasal turbinate tissue and a decrease of 1.90 log10 (ORF-ab1 copies / ml) and 1.83 log10 (N copies / ml) in the trachea tissue.

[0261] The pathological findings of hamster lung tissue after combined treatment with 10E9, 30A4-2, and 1C5-2 triple antibodies are as follows: Figure 8 As shown in the gross images of the lungs, the untreated lungs exhibited multifocal diffuse congestion and consolidation, while the lung tissue lesions were reduced in the antibody treatment group. Figure 8 A). Histopathological examination revealed 100% lung consolidation, alveolar destruction, and diffuse inflammation in the untreated group, while the antibody-treated group showed only mild inflammation in limited lung areas and minimal pneumonia pathology in most lung regions. The pathological severity score of hamsters in the antibody-treated group was higher than that of untreated hamsters (mean score 2.83 vs. 0.88), but significantly lower than that of the untreated group (mean score 10.68). Figure 8 B, p = 0.0022). These results indicate that the combination therapy of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 has a significant therapeutic effect on hamsters inoculated with Beta (B.1.351).

[0262] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the RBD of the coronavirus S protein, comprising: (a) The following three heavy chain CDRs: VH CDR1 with sequence SEQ ID NO: 7, VHCDR2 with sequence SEQ ID NO: 8, and VH CDR3 with sequence SEQ ID NO: 9; and the following three light chain CDRs: VLCDR1 with sequence SEQ ID NO: 10, VL CDR2 with sequence GVT, and VL CDR3 with sequence SEQ ID NO: 12; or, (b) The three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 1; and the three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 2; wherein the three CDRs contained in the VH and the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system; in, The coronavirus is selected from SARS-CoV-2 or its mutant strains, SARS-CoV-1 and / or RaTG13, and the mutant strain is selected from D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain or any combination thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: a VH containing the sequence shown in SEQ ID NO: 1 or a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity compared to the sequence shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising a constant region derived from mouse or human immunoglobulin.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein, The heavy chain of the antibody or its antigen-binding fragment includes a heavy chain constant region derived from mouse or human immunoglobulin, and the light chain of the antibody or its antigen-binding fragment includes a light chain constant region derived from mouse or human immunoglobulin.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein, The heavy chain constant region is the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

6. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv; and / or the antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

7. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-6, or the variable region of the heavy chain and the variable region of the light chain thereof.

8. A vector comprising the nucleic acid molecule of claim 7.

9. The vector of claim 8, wherein the vector is a cloning vector or an expression vector.

10. A host cell comprising the nucleic acid molecule of claim 7 or the vector of claim 8 or 9.

11. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-6, comprising culturing the host cell of claim 10 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

12. A composition comprising: (i) The antibody or antigen-binding fragment thereof as claimed in claim 1; (ii) An antibody or an antigen-binding fragment thereof, comprising: (a) three heavy chain CDRs: VH CDR1 of SEQ ID NO: 13, VH CDR2 of SEQ ID NO: 14, and VH CDR3 of SEQ ID NO: 15; and three light chain CDRs: VL CDR1 of SEQ ID NO: 16, VL CDR2 of SEQ ID NO: 17, and VL CDR of SEQ ID NO: 18; or (b) three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 3; and three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 4; wherein the three CDRs contained in the VH and the three CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering system; and (iii) An antibody or an antigen-binding fragment thereof comprising: (a) three heavy chain CDRs: VH CDR1 of SEQ ID NO: 19, VH CDR2 of SEQ ID NO: 20, and VH CDR3 of SEQ ID NO: 21; and three light chain CDRs: VL CDR1 of SEQ ID NO: 22, VL CDR2 of SEQ ID NO: 23, and VL CDR of SEQ ID NO: 24; or (b) three CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO: 5; and three CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO: 6; wherein the three CDRs contained in the VH and the three CDRs contained in the VL are defined by the Kabat, IMGT, or Chothia numbering system; in, The antibodies or antigen-binding fragments described in (ii) and (iii) specifically bind to the RBD of the coronavirus S protein, wherein the coronavirus is selected from SARS-CoV-2 or its mutant strains, SARS-CoV-1 and / or RaTG13, and the mutant strains are selected from D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain or any combination thereof.

13. The composition of claim 12, wherein: (i) The antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO: 1 or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2 or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2; (ii) The antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO: 3 or a sequence having at least 90% sequence identity with said sequence; and a VL comprising the sequence shown in SEQ ID NO: 4 or a sequence having at least 90% sequence identity with said sequence; and (iii) The antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO: 5 or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 6 or a sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO:

6.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-6 or a composition as described in claim 12 or 13, and a pharmaceutically acceptable carrier and / or excipient.

15. The pharmaceutical composition of claim 14, wherein, The pharmaceutical composition also contains additional antiviral agents.

16. The pharmaceutical composition of claim 15, wherein, The additional antiviral agents are selected from interferon, lopinavir, ritonavir, remdesivir, or dexamethasone.

17. A method for neutralizing coronaviruses in samples for non-diagnostic and non-therapeutic purposes, comprising contacting a sample containing coronaviruses in vitro with an antibody or antigen-binding fragment thereof as described in any one of claims 1-6 or a composition as described in claim 12 or 13; wherein, The coronavirus is selected from SARS-CoV-2 or its mutant strains, SARS-CoV-1 and / or RaTG13, and the mutant strain is selected from D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain or any combination thereof.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-6, the composition according to claim 12 or 13, or the pharmaceutical composition according to any one of claims 14-16 for the preparation of a medicament for neutralizing coronavirus in a subject, or for preventing and / or treating coronavirus infection or disease associated with coronavirus infection; wherein, The coronavirus is selected from SARS-CoV-2 or its mutant strains, SARS-CoV-1 and / or RaTG13, and the mutant strain is selected from D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain or any combination thereof.

19. The use as described in claim 18, wherein, The subjects were mammals.

20. The use as described in claim 19, wherein, The mammal in question is selected from humans.

21. The use as described in claim 18, wherein, The antibody or its antigen-binding fragment, composition or pharmaceutical composition may be used alone or in combination with other antiviral agents.

22. The use as claimed in claim 21, wherein, The additional antiviral agents are selected from interferon, lopinavir, ritonavir, remdesivir, or dexamethasone.

23. A conjugate comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-6 and a detectable label attached to said antibody or antigen-binding fragment thereof.

24. The conjugate of claim 23, wherein, The detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

25. The conjugate of claim 24, wherein, The enzyme is selected from horseradish peroxidase or alkaline phosphatase.

26. The conjugate of claim 24, wherein, The chemiluminescent reagent is selected from acridine esters, luminol and its derivatives, or ruthenium derivatives.

27. The conjugate of claim 24, wherein, The fluorescent dye is selected from fluorescein or fluorescent protein.

28. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-6 or the conjugate according to any one of claims 23-27 in the preparation of a diagnostic reagent, said diagnostic reagent being used to detect the presence or level of coronavirus in a sample, and / or to diagnose whether a subject is infected with coronavirus; wherein, The coronavirus is selected from SARS-CoV-2 or its mutant strains, SARS-CoV-1 and / or RaTG13, and the mutant strain is selected from D614G strain, Alpha strain, Beta strain, Gamma strain, Delta strain or any combination thereof.

29. The use as described in claim 28, wherein, The test reagent detects the presence or level of coronavirus in a sample through immunological detection, and optionally diagnoses whether a subject is infected with coronavirus based on the test results.

30. The use as described in claim 29, wherein, The immunological assays are selected from enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay, or radioimmunoassay.

31. The use as described in claim 28, wherein, The samples are blood samples, excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

32. The use as described in claim 31, wherein, The blood sample is selected from whole blood, plasma, or serum.

33. The use as described in claim 28, wherein, The subjects were mammals.

34. The use as described in claim 33, wherein, The mammal in question is a human.

35. The method of claim 17 or the use of claim 18 or 28, wherein, The Alpha strain was selected from B.1.1.

7.

36. The method of claim 17 or the use of claim 18 or 28, wherein, The Beta strain was selected from B.1.

351.

37. The method of claim 17 or the use of claim 18 or 28, wherein, The Gamma strain was selected from P.

1.

38. The method of claim 17 or the use of claim 18 or 28, wherein, The Delta strain was selected from B.1.617.2.

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