Antibodies against sars-cov-1 or sars-cov-2 and uses thereof

Abstract

The present invention relates to the field of immunology and the field of molecular virology, in particular the field of diagnosis, prevention and treatment of coronaviruses, such as SARS-CoV-2 and / or SARS-CoV-1. In particular, the present invention relates to monoclonal antibodies against coronaviruses, as well as compositions comprising said antibodies, e.g. diagnostic and therapeutic agents. Furthermore, the present invention also relates to uses of said antibodies. The antibodies of the present invention can be used for the diagnosis, prevention and / or treatment of a coronavirus infection and / or a disease caused by said infection.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, particularly to the diagnosis, prevention, and treatment of coronaviruses. Specifically, this invention relates to monoclonal antibodies against coronaviruses, and compositions comprising said antibodies (e.g., diagnostic and therapeutic agents). Furthermore, this invention relates to the use of said antibodies. The antibodies of this invention can be used for the diagnosis, prevention, and / or treatment of coronavirus infection and / or diseases caused by said infection. 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 may spread rapidly in the population and cause serious illness.

[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 treatments such as interferon-alpha, lopinavir / ritonavir, and chloroquine phosphate, with limited clinical efficacy. Studies have found that recovered COVID-19 patients typically develop high levels of SARS-CoV-2 neutralizing antibodies. The National Health Commission's Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7) recommends convalescent plasma therapy for patients with rapidly progressing, severe, and critical illness. Research data shows 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, a monoclonal antibody capable of efficiently and specifically neutralizing SARS-CoV-2 should be developed for short-term prevention and effective treatment of COVID-19. This is of great significance for the prevention and control of COVID-19 in my country and globally. Summary of the Invention

[0004] The inventors of this application, through in-depth research and creative labor, obtained murine antibodies capable of specifically and efficiently neutralizing SARS-CoV-2, as well as murine antibodies with broad-spectrum activity against both SARS-CoV-2 and SARS-CoV-1. Building upon this, the inventors further devoted considerable creative effort to in-depth research and modification of these murine antibodies, thereby developing humanized antibodies against them. The humanized antibodies of this invention not only retain functions and properties similar to (or even superior to) their parent murine antibodies, but also possess a high degree of humanization and are less likely to induce immunogenic reactions. Therefore, the antibodies of this invention have the potential for preventing and / or treating coronavirus infection or diseases caused by coronavirus infection, and possess significant clinical value.

[0005] The antibody of the present invention

[0006] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the receptor-binding domain (RBD) of the S protein of SARS-CoV-2, said antibody or antigen-binding fragment comprising:

[0007] (a) Heavy chain variable regions (VHs) containing the following three complementary determination regions (CDRs) as defined by the Kabat numbering system:

[0008] (i) VH CDR1, 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.

[0009] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:6, 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, and

[0010] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:7, 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] And / or,

[0012] (b) Light chain variable regions (VLs) containing the following three complementary determination regions (CDRs) as defined by the Kabat numbering system:

[0013] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:8, 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.

[0014] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:9, 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, and

[0015] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:10, 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.

[0016] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0017] In some embodiments, the antibody or its antigen-binding fragment comprises: the following three heavy chain CDRs as defined by the Kabat numbering system: VH CDR1 with sequence SEQ ID NO: 5, VH CDR2 with sequence SEQ ID NO: 6, and VH CDR3 with sequence SEQ ID NO: 7; and / or, the following three light chain CDRs as defined by the Kabat numbering system: VL CDR1 with sequence SEQ ID NO: 8, VL CDR2 with sequence SEQ ID NO: 9, and VL CDR3 with sequence SEQ ID NO: 10.

[0018] In some embodiments, the antibody or its antigen-binding fragment comprises: 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. In some embodiments, 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.

[0019] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0020] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0021] (i) The sequence shown in SEQ ID NO: 1;

[0022] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 1; or

[0023] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 1;

[0024] and

[0025] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0026] (iv) The sequence shown in SEQ ID NO: 2;

[0027] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 2; or

[0028] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 2.

[0029] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0030] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO:1 and a VL comprising the sequence shown in SEQ ID NO:2.

[0031] In some embodiments, the antibody or antigen-binding fragment of the present invention 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 framework sequence.

[0032] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention may comprise a framework region sequence derived from a human immunoglobulin, wherein the framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to corresponding murine residues.

[0033] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence (i.e., the amino acid sequence encoded by the human heavy chain germline gene), and a light chain framework region sequence derived from a human light chain germline sequence (i.e., the amino acid sequence encoded by the human light chain germline gene), wherein the heavy chain framework region and / or the light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human residues to corresponding murine residues.

[0034] In some embodiments, the VH of the antibody or its antigen-binding fragment comprises: heavy chain framework regions FR1, FR2, and FR3 derived from the heavy chain germline sequence IGHV1-3*01, and heavy chain framework region FR4 derived from the heavy chain germline sequence IGHJ5*02; and the VL of the antibody or its antigen-binding fragment comprises: light chain framework regions FR1, FR2, and FR3 derived from the light chain germline sequence IGKV4-1*01, and light chain framework region FR4 derived from the light chain germline sequence IGKJ2*01. The heavy chain framework regions and / or light chain framework regions optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human residues to corresponding murine residues.

[0035] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0036] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0037] (i) The sequence shown in SEQ ID NO: 17;

[0038] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 17; or

[0039] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 17;

[0040] and

[0041] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0042] (iv) Sequences shown in either SEQ ID NOs 18 or 19;

[0043] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in any of SEQ ID NOs: 18 or 19; or

[0044] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in either SEQ ID NOs: 18 or 19.

[0045] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0046] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises:

[0047] (1) VH containing the sequence shown in SEQ ID NO:17 and VL containing the sequence shown in SEQ ID NO:18; or

[0048] (2) VH containing the sequence shown in SEQ ID NO:17 and VL containing the sequence shown in SEQ ID NO:19.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof described in the first aspect is 36H6 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the monoclonal antibody or its antigen-binding fragment from which they are derived.

[0050] In some embodiments, the antibody or its antigen-binding fragment described in the first aspect has one or more of the following biological functions:

[0051] In some embodiments, the antibody or antigen-binding fragment thereof described in the first aspect possesses one or more of the following features:

[0052] (1) The RBD that specifically binds to the S protein of SARS-CoV-2;

[0053] (2) RBDs that do not bind or barely bind the S protein of SARS-CoV-1;

[0054] (3) Block or inhibit the binding of SARS-CoV-2 to the Ace2 receptor, and / or block or inhibit the infection of cells by SARS-CoV-2;

[0055] (4) It does not affect or has little effect on the binding of SARS-CoV-1 to the Ace2 receptor;

[0056] (5) Neutralize SARS-CoV-2 in vitro or in subjects (e.g., humans);

[0057] (6) Prevention and / or treatment of SARS-CoV-2 infection or disease caused by SARS-CoV-2 infection (e.g., COVID-19).

[0058] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to the receptor-binding domain (RBD) of the S protein of SARS-CoV-2 and SARS-CoV-1, said antibody or antigen-binding fragment comprising:

[0059] (a) Heavy chain variable regions (VHs) containing the following three complementary determination regions (CDRs) as defined by the Kabat numbering system:

[0060] (i) VH CDR1, which consists of the following sequence: SEQ ID NO:11, 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.

[0061] (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:12, 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, and

[0062] (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:13, 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);

[0063] And / or,

[0064] (b) Light chain variable regions (VLs) containing the following three complementary determination regions (CDRs) as defined by the Kabat numbering system:

[0065] (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:14, 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.

[0066] (v)VL CDR2, which consists of the following sequence: SEQ ID NO:15, 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, and

[0067] (vi)VL CDR3, which consists of the following sequence: SEQ ID NO:16, 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.

[0068] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0069] In some embodiments, the antibody or its antigen-binding fragment comprises: the following three heavy chain CDRs as defined by the Kabat numbering system: VH CDR1 with sequence SEQ ID NO: 11, VH CDR2 with sequence SEQ ID NO: 12, and VH CDR3 with sequence SEQ ID NO: 13; and / or, the following three light chain CDRs as defined by the Kabat numbering system: VL CDR1 with sequence SEQ ID NO: 14, VL CDR2 with sequence SEQ ID NO: 15, and VLCDR3 with sequence SEQ ID NO: 16.

[0070] In some embodiments, the antibody or its antigen-binding fragment comprises: 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. In some embodiments, 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.

[0071] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0072] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0073] (i) The sequence shown in SEQ ID NO: 3;

[0074] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 3; or

[0075] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 3;

[0076] and

[0077] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0078] (iv) The sequence shown in SEQ ID NO: 4;

[0079] (v) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO: 4; or

[0080] (vi) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO: 4.

[0081] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0082] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises: a VH comprising the sequence shown in SEQ ID NO:3 and a VL comprising the sequence shown in SEQ ID NO:4.

[0083] In some embodiments, the antibody or antigen-binding fragment of the present invention 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 framework sequence.

[0084] In some embodiments, the humanized antibody or antigen-binding fragment of the present invention may comprise a framework region sequence derived from a human immunoglobulin, wherein the framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) reversion mutations from human residues to corresponding murine residues.

[0085] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence (i.e., the amino acid sequence encoded by the human heavy chain germline gene), and a light chain framework region sequence derived from a human light chain germline sequence (i.e., the amino acid sequence encoded by the human light chain germline gene), wherein the heavy chain framework region and / or the light chain framework region optionally comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) reversion mutations from human residues to corresponding murine residues.

[0086] In some embodiments, the antibody or antigen-binding fragment thereof described in the second aspect is 2B4 or its antigen-binding fragment, its chimeric antibody, its humanized antibody, or variants thereof, which substantially retain the biological function of the monoclonal antibody or its antigen-binding fragment from which they are derived.

[0087] In some embodiments, the antibody or its antigen-binding fragment described in the second aspect has one or more of the following biological functions:

[0088] In some embodiments, the antibody or its antigen-binding fragment described in the second aspect possesses one or more of the following features:

[0089] (1) The RBD that specifically binds to the S protein of SARS-CoV-2;

[0090] (2) RBD that specifically binds to the S protein of SARS-CoV-1;

[0091] (3) Block or inhibit the binding of SARS-CoV-2 to the Ace2 receptor, and / or block or inhibit the infection of cells by SARS-CoV-2;

[0092] (4) Block or inhibit the binding of SARS-CoV-1 to the Ace2 receptor, and / or block or inhibit the infection of cells by SARS-CoV-1;

[0093] (5) Neutralize SARS-CoV-2 and / or SARS-CoV-1 in vitro or in subjects (e.g., humans);

[0094] (6) Prevention and / or treatment of SARS-CoV-2 and / or SARS-CoV-1 infection or diseases caused by SARS-CoV-2 and / or SARS-CoV-1 infection (e.g., COVID-19, SARS).

[0095] In some embodiments, the antibody or antigen-binding fragment thereof described in the first or second aspect of the present invention may further comprise a constant region sequence or a variant thereof derived from a mammalian (e.g., mouse or human) immunoglobulin, the variant having one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived.

[0096] In some embodiments, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0097] The light chain of the antibody or its antigen-binding fragment of the present invention comprises a light chain constant region (CL) of human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 conserved substitutions; e.g., 1, 2, 3, 4, or 5 conserved substitutions) compared to the sequence from which it is derived.

[0098] In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conserved substitutions of amino acids compared to its derived sequence. In such embodiments, the variant of the heavy chain constant region (CH) may have the same or substantially the same effector function compared to its derived wild-type sequence.

[0099] In other embodiments, variants of the heavy chain constant region (CH) may contain one or more amino acid mutations to alter one or more of the following properties of the antibody of the present invention: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, altering the antibody's affinity for effector ligands (such as FcR or complement C1q), thereby changing effector function (e.g., reducing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0100] In some embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0101] In some embodiments, the light chain constant region is the κ light chain constant region. In some embodiments, the light chain constant region is the mouse κ light chain constant region. In some embodiments, the light chain constant region is the human κ light chain constant region.

[0102] In some exemplary embodiments, the antibody or antigen-binding fragment of the present invention comprises the heavy chain constant region (CH) shown in SEQ ID NO:20; and / or the light chain constant region (CL) shown in SEQ ID NO:21.

[0103] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0104] In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0105] In this document, the antibody or antigen-binding fragment thereof described in the first or second aspect of the present invention may include variants that differ from the antibody or antigen-binding fragment 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 antibody or antigen-binding fragment from which they are derived, and substantially retain the aforementioned biological functions of the antibody or antigen-binding fragment from which they are derived.

[0106] Antibody preparation

[0107] 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.

[0108] 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.

[0109] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, or a variable region of the heavy chain and / or a variable region of the light chain thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, or a variable region of the heavy chain and / or a variable region of the light chain thereof.

[0110] 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.

[0111] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, and / or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0112] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, and / or a second nucleotide sequence encoding a light chain of an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0113] 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, e.g., mouse cells, human cells, etc.). In some preferred embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHOG44).

[0114] In another aspect, a method is provided for preparing the antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, 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.

[0115] Pharmaceutical Compositions and Therapeutic Uses

[0116] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0117] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents, such as additional antiviral agents (e.g., interferon, lopinavir, ritonavir, chloroquine phosphate, favipiravir, remdesivir, etc.).

[0118] In some embodiments, the antibody or antigen-binding fragment thereof described in the first or second aspect of the present invention, along with the additional pharmaceutically active agent, may be provided as separate components or as a mixture of components in the pharmaceutical composition. Therefore, the antibody or antigen-binding fragment thereof described in the first or second aspect of the present invention, along with the additional pharmaceutically active agent, may be administered simultaneously, separately, or sequentially.

[0119] 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.

[0120] In another aspect, the present invention provides a method for neutralizing SARS-CoV-2, comprising using an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described in the first aspect of the invention. The method can be used to neutralize SARS-CoV-2 in vitro or in a subject (e.g., a human).

[0121] In some embodiments, the method is used to neutralize the virulence of SARS-CoV-2 in a sample. In some embodiments, the method includes contacting a sample containing SARS-CoV-2 with an antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or a pharmaceutical composition containing said antibody or antigen-binding fragment thereof.

[0122] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0123] In another aspect, the present invention provides a method for preventing or treating SARS-CoV-2 infection or disease associated with SARS-CoV-2 virus infection (e.g., COVID-19) in a subject, comprising: administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

[0124] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or its antigen-binding fragment of the present invention can be administered simultaneously, separately, or sequentially with the other pharmaceutically active agent.

[0125] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention, or a pharmaceutical composition thereof, in the preparation of a medicament used for:

[0126] (1) Neutralize SARS-CoV-2 in vitro or in subjects (e.g., humans); and / or

[0127] (2) For the prevention and / or treatment of SARS-CoV-2 infection or SARS-CoV-2-related disease (e.g., COVID-19) in subjects.

[0128] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0129] In another aspect, the present invention provides a method for neutralizing SARS-CoV-2 and / or SARS-CoV-1, comprising using an antibody or antigen-binding fragment thereof or a pharmaceutical composition as described in the second aspect of the invention. The method can be used to neutralize SARS-CoV-2 and / or SARS-CoV-1 in vitro or in a subject (e.g., a human).

[0130] In some embodiments, the method is used to neutralize the virulence of SARS-CoV-2 and / or SARS-CoV-1 in a sample. In some embodiments, the method includes contacting a sample containing SARS-CoV-2 and / or SARS-CoV-1 with an antibody or antigen-binding fragment thereof as described in the second aspect of the invention, or a pharmaceutical composition containing said antibody or antigen-binding fragment thereof.

[0131] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0132] In another aspect, the present invention provides a method for preventing or treating SARS-CoV-2 and / or SARS-CoV-1 infection or diseases associated with SARS-CoV-2 and / or SARS-CoV-1 viral infection (e.g., COVID-19, SARS) in a subject, comprising: administering to a subject in need an effective amount of an antibody or antigen-binding fragment thereof as described in the second aspect of the present invention, or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

[0133] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or its antigen-binding fragment of the present invention can be administered simultaneously, separately, or sequentially with the other pharmaceutically active agent.

[0134] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof described in the second aspect of the invention, or a pharmaceutical composition thereof, in the preparation of a medicament used for:

[0135] (1) Neutralize SARS-CoV-2 and / or SARS-CoV-1 in vitro or in subjects (e.g., humans); and / or

[0136] (2) For the prevention and / or treatment of subjects with SARS-CoV-2 and / or SARS-CoV-1 infection or diseases associated with SARS-CoV-2 and / or SARS-CoV-1 infection (e.g., COVID-19, SARS).

[0137] In some embodiments, the antibody or its antigen-binding fragment is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents).

[0138] The antibodies or antigen-binding fragments thereof described in the first or second aspect of this invention, or the pharmaceutical compositions of this invention, 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 of this 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 injection solutions. For example, sterile injection solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of this invention into a suitable solvent, 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.

[0139] 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.

[0140] The pharmaceutical compositions of the present invention may include an antibody or antigen-binding fragment 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 thereof 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.

[0141] 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.

[0142] In this paper, the subjects may be mammals, such as humans.

[0143] Conjugate

[0144] The antibodies or antigen-binding fragments thereof described in the first or second aspect of this 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 SARS-CoV-2. Therefore, the antibodies or antigen-binding fragments thereof of this invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of this 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 thereof of this 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.

[0145] Therefore, in some embodiments, the antibody or its antigen-binding fragment described in the first or second aspect of the present invention carries a detectable marker.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] Reagent kit and detection uses

[0150] In another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention, or a conjugate of the present invention.

[0151] In some embodiments, the kit comprises the conjugate of the present invention.

[0152] In other embodiments, the kit comprises an antibody or antigen-binding fragment thereof as described in the first or second aspect of the present invention. In some embodiments, the antibody or antigen-binding fragment thereof 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 thereof as described in the first or second aspect 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.

[0153] 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 or antigen-binding fragment described in the first or second aspect of the present invention originates.

[0154] 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.

[0155] 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.

[0156] In another aspect, the present invention provides a method for detecting SARS-CoV-2 or its S protein or RBD of the S protein, or the presence or level of SARS-CoV-2-infected cells in a sample, comprising using the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0157] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0158] In some embodiments, the method includes using the conjugate of the present invention, which comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention.

[0159] In other embodiments, the method includes using the antibody or antigen-binding fragment thereof described in the first aspect of the invention. In some embodiments, the antibody or antigen-binding fragment thereof 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 thereof.

[0160] 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 or antigen-binding fragment described in the first aspect of the invention originates.

[0161] 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.

[0162] In some embodiments, the method includes: (1) contacting the sample with the antibody or antigen-binding fragment thereof described in the first aspect of the invention; and (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of the immune complexes. The formation of the immune complexes indicates the presence of SARS-CoV-2 or cells infected with SARS-CoV-2.

[0163] In some embodiments, the method can be used for diagnostic purposes, such as diagnosing whether a subject is infected with SARS-CoV-2 based on the presence or level of SARS-CoV-2 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).

[0164] 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.

[0165] In some implementations, the subject is a mammal, such as a human.

[0166] In another aspect, the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention in the preparation of a kit for detecting SARS-CoV-2 or its S protein or RBD of the S protein, or the presence or level of SARS-CoV-2-infected cells in a sample, and / or for diagnosing whether a subject is infected with SARS-CoV-2.

[0167] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0168] In some embodiments, the kit detects SARS-CoV-2 or its S protein or RBD of the S protein, or the presence or level of SARS-CoV-2-infected cells in a sample using the detection methods described above, and optionally diagnoses whether a subject is infected with SARS-CoV-2 based on the detection results.

[0169] 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).

[0170] In another aspect, the present invention provides a method for detecting the presence or level of a coronavirus or its S protein or RBD of the S protein, or coronavirus-infected cells in a sample, comprising using an antibody or antigen-binding fragment thereof as described in the second aspect of the present invention, wherein the coronavirus is selected from SARS-CoV-2 and / or SARS-CoV-1.

[0171] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0172] In some embodiments, the method includes using the conjugate of the present invention, which comprises the antibody or antigen-binding fragment thereof described in the second aspect of the present invention.

[0173] In other embodiments, the method includes using the antibody or antigen-binding fragment thereof described in the second aspect of the invention. In some embodiments, the antibody or antigen-binding fragment thereof 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 thereof.

[0174] 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 or antigen-binding fragment described in the second aspect of the invention originates.

[0175] 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.

[0176] In some embodiments, the method includes: (1) contacting the sample with the antibody or antigen-binding fragment thereof described in the second aspect of the invention; and (2) detecting the formation of antigen-antibody immune complexes or detecting the amount of the immune complexes. The formation of the immune complexes indicates the presence of the coronavirus or cells infected by the coronavirus.

[0177] In some embodiments, the method can be used for diagnostic purposes, such as diagnosing whether a subject is infected with the coronavirus based on its presence or level 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).

[0178] 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.

[0179] In some implementations, the subject is a mammal, such as a human.

[0180] In another aspect, the use of the antibody or antigen-binding fragment thereof described in the first aspect of the invention in the preparation of a kit for detecting coronavirus or its 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 a coronavirus selected from SARS-CoV-2 and / or SARS-CoV-1.

[0181] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0182] In some embodiments, the kit detects the coronavirus or its S protein or RBD of the S protein, or the presence or level of cells infected with the coronavirus in a sample using the detection methods described above, and optionally diagnoses whether a subject is infected with the coronavirus based on the detection results.

[0183] 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).

[0184] Terminology Definition

[0185] 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.

[0186] As used herein, “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),” formerly known as “novel coronavirus” or “2019-nCov,” belongs to the β-coronavirus genus and is an enveloped, single-stranded, positive-sense RNA virus. The genome sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including a surface spike protein (S), an integrated membrane protein (M), and an membrane protein (E). Like SARS-CoV, the receptor for SARS-CoV-2 specifically binds to angiotensin-converting enzyme 2 (ACE2) on the host cell via the receptor-binding domain (RBD) on the S protein, leading to viral membrane fusion and cellular entry. This receptor plays a crucial role in viral infection of cells.

[0187] As used in this article, the terms “novel coronavirus pneumonia” and “COVID-19” refer to pneumonia caused by SARS-CoV-2 infection. They have the same meaning and can be used interchangeably.

[0188] As used herein, “severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1)” belongs to the β-coronavirus genus and is an enveloped, single-stranded, positive-sense RNA virus. The genome sequence of SARS-CoV-1 is known to those skilled in the art and can be found, for example, in GenBank: AAP13567.1. Pneumonia caused by SARS-CoV-1 is called SARS.

[0189] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of 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 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. 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.

[0190] 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).

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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).

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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)).

[0201] 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.

[0202] 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.

[0203] 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.

[0204] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0205] 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).

[0206] 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.).

[0207] 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.

[0208] To prepare chimeric antibodies, methods known in the art can be used to ligate the variable region of an immunoglobulin from an immunized animal (e.g., a mouse) to the constant region of a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 to Cabilly et al.). For example, DNA encoding VH can be operatively ligated to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is generally preferred to be an IgG1 or IgG4 constant region. For example, DNA encoding VL can be operatively ligated to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant regions are known in the art (see, for example, Kabat, E.A. et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDA Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. Light chain constant regions can be κ or λ constant regions, but κ constant regions are generally preferred.

[0209] To prepare humanized antibodies, the CDR region of an immunized animal (e.g., a mouse) can be transplanted into a human framework sequence using methods known in the art (see Winter’s U.S. Patent No. 5,225,539; Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

[0210] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence in the genome of an organism that encodes an immunoglobulin, which has not undergone a maturation process involving genetic rearrangements and mutations that would lead to the expression of a specific immunoglobulin. In this invention, the term "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding the heavy chain of immunoglobulins, including the V gene (variable), D gene (diversity), J gene (joining), and C gene (constant); similarly, the term "light chain germline gene" refers to a germline antibody gene or gene segment encoding the light chain of immunoglobulins, including the V gene (variable), J gene (joining), and C gene (constant). In this invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also referred to as a "germline sequence." The amino acid sequence encoded by the heavy chain germline gene is called the heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene is called the light chain germline sequence. Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from professional databases (e.g., IMGT, UNSWIg, NCBI, or VBASE2).

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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).

[0216] 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.

[0217] 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.

[0218] As used herein, the term "prevention" refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to methods 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 the expected survival (if no treatment was received).

[0219] As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject (e.g., a human) has SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection (e.g., COVID-19), or is at risk of having such a disease.

[0220] 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., SARS-CoV-2 infection) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., SARS-CoV-2 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.

[0221] 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.

[0222] Beneficial effects of the invention

[0223] This invention provides monoclonal antibodies that neutralize SARS-CoV-2. Specifically, these monoclonal antibodies bind to epitopes on the RBD region of the SARS-CoV-2 S protein and neutralize SARS-CoV-2. The monoclonal antibodies of this invention can inhibit the binding of the SARS-CoV-2 RBD protein to the receptor ACE2. The monoclonal antibodies provided by this invention have the following advantages: (1) Monoclonal antibody 36H6 has strong neutralizing ability, with a half-maximal inhibitory concentration (IC50) of 0.041 nM in the neutralization assay of the SARS-CoV-2 pseudovirus SARS-CoV2-LvPP. (2) Monoclonal antibody 2B4 can cross-bind to the RBD of SARS-CoV-1 and SARS-CoV-2, and can cross-neutralize SARS-CoV-2 and SARS-CoV-1, with half-maximal inhibitory concentrations of 0.739 nM and 0.503 nM, respectively, in the neutralization assays of the pseudovirus SARS-CoV1-LvPP and SARS-CoV2-LvPP, respectively, showing broad-spectrum activity.

[0224] 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

[0225] Figure 1 The image shows an SDS-PAGE electrophoresis diagram of the RBD protein of SARS-CoV-2.

[0226] Figure 2 The monoclonal antibodies 36H6 and 2B4 showed resistance to SARS-CoV-2 ( Figure 2 A) SARS-CoV-1 ( Figure 2 B) and RaTG13-CoV Figure 2 C) ELISA results of RBD protein binding activity.

[0227] Figure 3 The results of the affinity assay (Biacore) for monoclonal antibodies 36H6 and 2B4 against the RBD proteins of SARS-CoV-2, SARS-CoV-1, and RaTG13-CoV are shown.

[0228] Figure 4 The study demonstrated the ability of different doses of SARS-CoV1-LvPP pseudovirus to infect H1299ACE2hR cells.

[0229] Figure 5The ability of different doses of SARS-CoV2-LvPP pseudovirus to infect H1299ACE2hR cells was demonstrated.

[0230] Figure 6 The monoclonal antibodies 2B4 and 36H6 showed resistance to SARS-CoV1-LvPP ( Figure 6 A) and SARS-CoV2-LvPP ( Figure 6 B) Infection neutralization (NAT) dose-effect relationship.

[0231] Figure 7 The results of neutralizing activity assays of monoclonal antibodies 2B4 and 36H6 in a SARS-CoV2 VSVpp pseudovirus infection model are shown.

[0232] Figure 8 The analysis showed the blocking ability of the Fab fragments of monoclonal antibodies 2B4 and 36H6 against SARS-CoV-2 RBD binding to ACE2. Figure 8 A is monoclonal antibody 2B4; Figure 8 B is monoclonal antibody 36H6; Figure 8 C represents an irrelevant control antibody.

[0233] Figure 9 The ELISA results show the binding activity of humanized antibodies 36H6-10 and 36H6-12 to RBD-His.

[0234] Figure 10 The results of neutralizing activity assays for humanized antibodies 36H6-10 and 36H6-12 in a SARS-CoV2VSVpp pseudovirus infection model are presented.

[0235] Sequence information

[0236] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0237] Table 1: Sequence Description

[0238]

[0239] Detailed Implementation

[0240] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0241] 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.

[0242] Example 1: Synthesis of the anti-SARS-CoV-2 receptor binding domain RBD gene and construction of the expression vector

[0243] Referring to the complete SARS-CoV2-2 genome sequence (MN908947.3), the nucleotide sequence encoding the SARS-CoV2-2 receptor-binding domain RBD protein (corresponding to amino acids 316-550 of the S protein) was optimized according to human codon preferences. Furthermore, a signal peptide coding sequence (the guide sequence of human B2M) was ligated to the N-terminus of the optimized nucleic acid sequence, and a polyhistidine polypeptide (6×His) for affinity chromatography purification was ligated to the C-terminus. This protein was named RBD-His, and its nucleotide sequence is shown in SEQ ID NO:22, and its amino acid sequence is shown in SEQ ID NO:23. Using NEBuilder HiFi DNA AssemblyMaster Mix (NEB), the above nucleotide sequence was ligated into the expression vector EIRBsMie-C18hA2dtSCT (with AgeI / BglII restriction sites). DNA sequencing confirmed that the sequence was completely consistent with the design, finally obtaining the SARS-CoV-2 RBDhis expression vector EIRBsMie-RBDhis.

[0244] Example 2: Expression and purification of SARS-CoV-2 RBD antigen

[0245] 1. Expression of SARS-CoV-2 RBD antigen

[0246] With 3x10 6 The density of ExpiCHO cells was determined by adding an appropriate amount of ExpiCHO culture medium. TM Expression Medium (Thermo Scientific) was cultured in Erlenmeyer flasks and placed in a constant temperature shaker at 37°C, 8% CO2, and appropriate rotation speed for 24 hours, until the cell density reached 6 x 10⁻⁶ cells / mL. 6 The density.

[0247] Use ExpiFectamine according to the kit instructions. TM The CHO Transfection Kit (Thermo Scientific) was used to transfect the vector EIRBsMie-RBDhis obtained in Example 1 into ExpiCHO cells. After culturing under the same conditions for 17-24 hours, feed and enhancer provided in the kit were added, and the cells were transferred to a constant temperature shaker at 32°C, 5% CO2, and appropriate rotation speed for another 6 days.

[0248] 2. Purification of SARS-CoV-2 RBD antigen

[0249] After 6 days of culture, the ExpiCHO-expressing cell suspension was collected, centrifuged at 12,000 rpm at room temperature for 30 min, the supernatant was collected and dialyzed in PBS, and then filtered through a 0.22 μm filter membrane.

[0250] The supernatant sample dialyzed into PBS was purified by medium-pressure Ni-Excel chromatography (GE medium), with 30 mM imidazole used to remove impurities, and 250 mM imidazole used to elute the target protein. The target protein was identified as the SARS-CoV-2 RBD antigen, and was named SARS-CoV-2RBD. SDS-PAGE electrophoresis results showed that the purity of SARS-CoV-2 RBD was over 90%. Figure 1 The obtained target protein was dialyzed into PBS buffer and stored at -20°C.

[0251] Example 3: Obtaining murine monoclonal antibodies

[0252] 1. Mouse immunization

[0253] The standard in vivo immunization method was used; for details, please refer to Ed Harlow et al., “Antibodies A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988. A brief summary of the procedure is as follows:

[0254] The SARS-CoV-2 RBD protein obtained in Example 2 was mixed with an equal volume of Freund's adjuvant and emulsified. Six- to eight-week-old female BALB / c mice were immunized bilaterally via multiple subcutaneous injections in the groin, with a booster immunization two weeks after the initial immunization. Serum antibody titers were measured using indirect ELISA, and fusion experiments were performed four weeks later.

[0255] Seventy-two hours before the fusion of mouse spleen cells with mouse myeloma cells (SP2 / 0), a final booster immunization was performed on the spleen. The antigen used in this immunization was an unadjuvanted antigen diluted to 1 mg / mL. 50 μL of protein was injected longitudinally along the spleen. Simultaneously, mouse myeloma cells (SP2 / 0) were resuscitated and cultured in RPMI 1640 medium containing 10% fetal bovine serum to the logarithmic growth phase, in preparation for fusion.

[0256] 2. Preparation and screening of hybridoma cells

[0257] Mice were given a spleen booster 72 hours later. The spleen was used to prepare a cell suspension, which was then fused with mouse myeloma cells SP2 / 0 to obtain hybridoma cells. Feeder cells were prepared and co-cultured with the hybridoma cells. In our laboratory, mouse peritoneal macrophages and thymocytes from young mice were used as feeder cells.

[0258] Hybridoma cells were screened by indirect ELISA. 50 ng / well of SARS-CoV-2 RBD protein from Example 2 was coated and blocked. 60 μL of fusion cell culture supernatant was added, and the cells were reacted at 37°C for 1 hour. Horseradish peroxidase-labeled goat anti-mouse secondary antibody (GAM-HRP) was then added, and the cells were reacted at 37°C for 30 min before color development. Positive clones were then selected.

[0259] Five BALB / c mice were used, and ascites was obtained by intraperitoneal injection. The ascites was centrifuged at high speed, and the supernatant was collected. An equal volume of saturated ammonium sulfate solution was added, and the mixture was precipitated on ice for 30 min. Then, it was centrifuged 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). Two purified mouse monoclonal antibodies were obtained (Table 2), named 36H6 (also known as m36H6) and 2B4 (also known as m2B4), respectively.

[0260] Table 2. Monoclonal antibody information

[0261]

[0262] Example 4: Obtaining the sequence of a murine monoclonal antibody

[0263] RNA was extracted from 36H6 and 2B4 hybridoma cells cultured to the logarithmic growth phase using the Trizol method (Invitrogen). The extracted RNA precipitate was dissolved in 50 μl of DEPC water. Next, the heavy chain variable region and light chain variable region were subjected to reverse transcription PCR, and the PCR products were recovered and sequenced. The sequences were then aligned using BLAST to determine the heavy chain and light chain variable region sequences of the antibody. The final amino acid sequences of the variable regions of 36H6 and 2B4 cells are shown in Tables 1 and 2.

[0264] Furthermore, the CDR sequences of mouse monoclonal antibodies 36H6 and 2B4 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 final determined amino acid sequences of the CDRs of the variable regions of 36H6 and 2B4 are shown in Tables 1 and 3.

[0265] Table 3. Sequences of antibodies 36H6 and 2B4

[0266] Table 3: Monoclonal Antibody Variable Region Sequence

[0267]

[0268] Example 5: ELISA binding activity of monoclonal antibodies 2B4 and 36H6 against SARS-CoV-2, SARS-CoV-1, and RaTG13

[0269] Referring to the viral genome sequences of SARS-CoV-1 (AAP13567.1) and RaTG13 (MN996532.1) published in Genebank, respectively, RBD expression vectors for these two coronaviruses were constructed using the method described in Example 1, and recombinant proteins were prepared using the method described in Example 2. The recombinant protein derived from the RBD of SARS-CoV-1 was named SARS-CoV1-RBD, and the recombinant protein derived from the RBD of RaTG13-CoV was named RaTG13-RBD.

[0270] The SARS-CoV2-RBD, SARS-CoV1-RBD, and RaTG13-RBD proteins obtained in Example 2 and above were diluted with 50 mM CB 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), and 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. The plates were then 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.

[0271] The mouse monoclonal antibodies 36H6 and 2B4 obtained in Example 3 were serially diluted with PBS solution containing 20% ​​newborn calf serum to concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, and then subjected to ELISA detection according to the following steps:

[0272] (1) Sample reaction: Take an enzyme-labeled plate that has been coated with SARS-CoV2-RBD, SARS-CoV1-RBG and RaTG13-RBG proteins respectively, add 100 μL of diluted sample to each well, and incubate at 37℃ for 30 minutes.

[0273] (2) Enzyme-labeled reaction: After completing the sample reaction steps, wash the enzyme-labeled plate 5 times with PBST washing solution (20mM PB7.4, 150mM NaCl, 0.1% Tween 20), add 100μL of horseradish peroxidase (HRP) labeled goat anti-mouse IgG (GAM) reaction solution to each well, and incubate at 37℃ for 30 minutes.

[0274] (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.

[0275] (4) Termination of reaction and measurement of readings: After completing the colorimetric reaction, add 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) to each well of the ELISA plate and measure the OD450 / 630 value of each well using an ELISA reader. Reactivity determination of mouse monoclonal antibodies 36H6 and 2B4 with SARS-CoV2-RBD, SARS-CoV1-RBG, and RaTG13-RBG: Determination is based on the readings after the reaction. If the detected value / background value is greater than 5, it is considered positive.

[0276] Results Analysis: The results are as follows Figure 2As shown, 36H6 exhibited strong binding activity against SARS-CoV2-RBD at concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, but no binding activity against SARS-CoV1-RBD and RaTG13-RBD. 2B4 also showed strong binding activity against SARS-CoV2-RBD and SARS-CoV1-RBD at concentrations of 10 μg / mL, 1 μg / mL, 0.1 μg / mL, and 0.01 μg / mL, but no binding activity against RaTG13-RBD. These results indicate that 36H6 can specifically recognize the SARS-CoV-2 RBD, while 2B4 can cross-recognize both SARS-CoV-2 and SARS-CoV-1 RBDs, demonstrating a certain degree of broad-spectrum binding.

[0277] Example 6: Detection of affinity constants of monoclonal antibodies 2B4 and 36H6 for the receptor-binding domain (RBD) proteins of SARS-CoV-2, SARS-CoV-1, and RaTG13

[0278] This study used surface plasmon resonance (SPR) technology to detect the affinity of monoclonal antibodies 2B4 and 36H6 for the RBD proteins of SARS-CoV-2, SARS-CoV-1, and RaTG13, respectively. The detection method used in this example was the capture method, employing a Protein G chip (GE) to capture the mouse monoclonal antibodies. Based on the molecular weight of the mouse monoclonal antibodies and the molecular weights of the three RBD analytes, the ligand response value was calculated to be approximately 1000 RU using the ligand conjugation level calculation formula. The two antibodies, 2B4 and 36H6, were diluted with PBS to appropriate concentrations: 50 μg / mL for 2B4 and 40 μg / mL for 36H6, to stabilize their binding response value with the Protein G chip at approximately 1000 RU. The RBD proteins of SARS-CoV-2, SARS-CoV-1, and RaTG13 were sequentially diluted 2-fold from an initial concentration of 200 nM (a total of 11 dilution gradients were tested, with the highest test concentration being 200 nM and the lowest test concentration being 0.19 nM). The antigen-antibody affinity was detected using a Biacore 8000 surface plasmon resonance analyzer (GE).

[0279] The results are as follows Figure 3 As shown, 2B4 exhibits the highest affinity for the SARS-CoV-2 RBD protein, with a low equilibrium dissociation constant (K). D The value is 3.05 nM. Figure 3 A), 2B4 also has a certain affinity for the RBD proteins of SARS-CoV-1 and RaTG13, with an equilibrium dissociation constant (K). D) is 34.1 nM ( Figure 3 B) and 67nM ( Figure 3 C). 36H6 has a good affinity only for the RBD protein of SARS-CoV-2, and the equilibrium dissociation constant (K) D The value is 5.84 nM. Figure 3 D). This indicates that monoclonal antibody 36H6 is a specific antibody against the RBD of SARS-CoV-2, while 2B4 has certain cross-binding activity against the RBDs of the other two coronaviruses.

[0280] Example 7: Neutralization experiments of monoclonal antibodies 2B4 and 36H6 against SARS-CoV-1 and SARS-CoV-2 pseudoviruses

[0281] 1. Preparation of pseudovirus packaging cells

[0282] Resuscitate 293T cells and culture them in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase, then incubate at 7 × 10⁻⁶ cells / year. 6 293T cells were seeded at a density of 10cm on a cell culture plate and cultured in a 5% CO2 cell culture incubator at 37°C for 12 hours. Transfection was performed when the cells reached 95-99% confluence.

[0283] Use Lipofectamine according to the kit instructions. TM 3000 (Thermo Fisher Scientific) co-transfected 293T cells with plasmid psPAX2 (lentiviral packaging plasmid), pLvEF1αmNGNL (lentiviral shuttle plasmid carrying a green fluorescent reporter gene), and pCMV-SARS1-S (expressing the full-length Spike protein of SARS-CoV-1) to package SARS-CoV-1 pseudovirus (abbreviated as SARS-CoV1-LvPP); and co-transfected 293T cells with plasmid psPAX2, pLvEF1αmNGNL, and EIRBsMie-SARS2-SFL plasmid to package SARS-CoV-2 pseudovirus (abbreviated as SARS-CoV2-LvPP). Six hours after transfection, the packaging medium was removed and replaced. Twenty-four hours after transfection, the pseudovirus was collected for the first time, and all cell supernatant was collected and stored at 4°C. The supernatant was then replaced with preheated fresh medium and cultured at 37°C in a 5% CO2 cell incubator for 24 hours. Forty-eight hours after transfection, pseudoviruses were collected a second time, and all cell supernatant was mixed with the supernatant collected in the first instance. The mixture was centrifuged at 2000 rpm for 30 minutes, and the supernatant was filtered through a 0.45 μm filter. Viral titers were determined using the H1299 cell line overexpressing human ACE2 (H1299ACE2hR), aliquoted, and stored at -80°C. The methods for determining the infection titers of the two pseudoviruses are as follows:

[0284] (1) H1299ACE2hR cells in the logarithmic growth phase were subjected to 1.2 × 10⁻⁶ HCl. 4 The cells were seeded at a density of 100 μL / well in 96-well plates and cultured overnight in a 37°C, 5% CO2 cell culture incubator.

[0285] (2) 30 μL of supernatant from two pseudoviruses, SARS-CoV1-LvPP and SARS-CoV2-LvPP, was added to each well to infect cells. Different viral titers were infected by serial 3-fold dilutions, with a total of 5 dilution gradients: original (30 μL), 3-fold dilution (10 μL), 9-fold dilution (3.3 μL), 27-fold dilution (1.1 μL), and 81-fold dilution (0.37 μL).

[0286] (3) Thirty-six to 48 hours after infecting H1299ACE2hR cells, fluorescence imaging of the infected cells was performed using a disc-based confocal high-content imaging system (Opera Phenix or Operetta CLS, purchased from PerkinElmer) (20x immersion lens, 25 fields of view). After completion, the obtained fluorescence images were quantitatively analyzed using Columbus image management and analysis software to detect the number of mNeonGreen positive cells. Results of H1299ACE2hR cell infection with two serially diluted pseudoviruses are shown below. Figure 4 and Figure 5 As shown in the figure. This result demonstrates that the obtained pseudoviruses SARS-CoV1-LvPP and SARS-CoV2-LvPP can efficiently infect H1299-hAce2 cells, and the number and percentage of green fluorescent positive cells are linearly correlated with the viral load.

[0287] (4) Through linear regression analysis, combined with the number of cells that could be "lit up" after infection with different doses of pseudovirus, the initial infection titer of SARS-CoV1-LvPP could be calculated to be 1.4 × 10⁻⁶ cells / well. 5 TU / mL (original pseudovirus concentration was 4.67 × 10⁻⁶) 5 TU / mL), while the first-well infection of SARS-CoV2-LvPP pseudovirus was 3.03 × 10⁻⁶. 5 TU / mL (original pseudovirus concentration was 1.01 × 10⁻⁶) 6 TU / mL).

[0288] 2. Neutralizing ability of monoclonal antibodies 2B4 and 36H6 against pseudoviruses of SARS-CoV-1 and SARS-CoV-2

[0289] To test whether the two monoclonal antibodies developed in this invention can neutralize SARS-CoV-1 and SARS-CoV-2 in vitro, we used two pseudoviruses, SARS-CoV1-LvPP and SARS-CoV2-LvPP, on H1299ACE2hR cells. The specific testing method is as follows:

[0290] (1) H1299ACE2hR cells in the logarithmic growth phase were subjected to 1.2 × 10⁻⁶ HCl. 4 The cells were seeded at a density of 100 μL / well in 96-well plates and cultured overnight in a 37°C, 5% CO2 cell culture incubator.

[0291] (2) The two antibodies were prepared into different concentrations using DMEM medium containing 10% fetal bovine serum, with the highest concentration being 1000 nM, and were serially diluted 2 times.

[0292] (3) Dilute 60 μL of antibody at different concentrations in a 1.0 × 10⁻⁶ solution. 5 Equal amounts of SARS-CoV1-LvPP (TU / mL) and SARS-CoV1-LvPP were mixed and incubated at 37°C for 1 hour to allow the antibody to fully bind to the pseudovirus.

[0293] (4) Take 100 μL of the virus antibody incubation solution obtained in the previous step and add it to the H1299ACE2hR cells that have been plated. Incubate the cells at 37°C and 5% CO2 for infection.

[0294] (5) 36-48 hours after infection, fluorescence imaging of infected cells was performed using a high-content imaging system based on rotating confocal imaging (Opera Phenix or Operatta CLS, purchased from PerkinElmer) (20x immersion lens, 25 fields of view). After completion, the obtained fluorescence images were quantitatively analyzed using Columbus image management and analysis software to calculate the number of green fluorescent protein (mNeonGreen) positive cells in each cell well.

[0295] (6) Compare the average number of positive cells in the control wells without antibody to calculate the infection inhibition rate per well. The calculation formula is as follows: (Number of green fluorescent positive cells in positive control wells - Number of green fluorescent positive cells in test wells) / Number of green fluorescent positive cells in positive control wells × 100%. After calculating the inhibition rates of the two antibodies under different dosage conditions, the inhibition curves were plotted using GraphpadPrism 8 software, and the half-maximum inhibitory concentration (IC50) was calculated using a 4-parameter curve fitting model.

[0296] The results are as follows Figure 6As shown, both monoclonal antibodies 2B4 and 36H6 exhibit strong neutralizing activity against SARS-CoV-2, with 36H6 showing stronger activity than 2B4, achieving an IC50 of 0.041 nM. However, this antibody showed no neutralizing effect against SARS-CoV-1. Monoclonal antibody 2B4 demonstrated strong neutralizing activity against both SARS-CoV-1 and SARS-CoV-2, with IC50 values ​​of 0.739 nM and 0.503 nM against the two viruses, respectively. These results indicate that 36H6 is a highly effective neutralizing antibody specifically targeting SARS-CoV-2, while 2B4 is a general neutralizing antibody against both SARS-CoV-1 and SARS-CoV-2.

[0297] Example 8: Neutralizing ability of monoclonal antibodies 2B4 and 36H6 against SARS-CoV2 VSVpp pseudovirus

[0298] The neutralization method for the SARS-CoV2-VSVpp pseudovirus was performed according to the method described in the reference (doi:https: / / doi.org / 10.1101 / 2020.04.08.026948). The main experimental procedure is briefly described below: To construct a VSV pseudovirus carrying the SARS-CoV-2 spike protein, the SARS-CoV-2 spike gene (sequence source: GenBank: MN908947) with an 18-amino acid truncated C-terminus 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, the 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 was then removed from the supernatant, and anti-VSV-G rat serum was added to block infection by residual VSVdG-EGFP-G. The progeny viruses carried the SARS-CoV-2 Sde18 truncated protein, yielding the pseudovirus VSV-SARS-CoV2 VSVpp. Twenty-four hours after VSVdG-EGFP-G infection, the cell supernatant was collected, centrifuged, and filtered (0.45 μm pore size, Millipore, SLHP033RB) to remove cell debris and stored at -80°C for later use. The serially diluted supernatant was used to infect the BHK21 cell line BHK21-hACE2, which overexpresses human ACE2. Viral titer was determined by the number of GFP-positive cells after infection.

[0299] Antibodies 36H6 and 2B4 were diluted to 106.4 nM as gradients, followed by 16 gradients of 1-2-fold decrease. The serially diluted antibodies were mixed with diluted SARS-CoV-2 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 the 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 determined.

[0300] The IC50 of the antibodies was calculated using nonlinear regression analysis. The inhibitory results of antibodies m36H6 and m2B4 against SARS-CoV-2 VSVpp virus are as follows: Figure 7 As shown, the IC50 values ​​are 0.021 nM and 0.893 nM, respectively.

[0301] Example 9: Test of the ability of monoclonal antibodies 2B4 and 36H6 to block SARS-CoV-2 RBD binding to receptor ACE2

[0302] To evaluate whether monoclonal antibodies 2B4 and 36H6 could block the binding between the SARS-CoV-2 spike protein and the ACE2 receptor in vitro, we used a Biacore 8000 (GE) analyzer. The methods are as follows:

[0303] (1) Papain (purchased from Sigma) was used to digest 2B4, 36H6, and the irrelevant control antibody (controlmAb). After digestion, the samples were chromatographically analyzed using MabSelect SuRe. Successfully digested Fab fragments could not bind to the MabSelect SuRe medium, while undigested residual antibody and Fc fragments were captured and removed by the MabSelect SuRe medium. The MabSelect SuRe chromatography permeated the samples, dialyzed to 20 mM PB7.4, and then concentrated using a Millipore 10 kDa concentrator. The concentrated samples were then used for later use.

[0304] (2) Dilute the C-terminal ACE2 recombinant protein (ACE2-Ig) fused with the Fc fragment of the IgG antibody to an appropriate concentration and capture ACE2-Ig using a Protein A chip (GE).

[0305] (3) The SARS-CoV-2RBDhis antigen prepared in Example 2 was diluted to 20mM PB7.4, with a concentration of 200nM. It was then mixed with different concentrations of 2B4, 36H6, and control antibody Fab (800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM) in equal volumes and incubated for 60 minutes.

[0306] (4) Perform Biacore tests on the series of protein samples prepared in step (3) to obtain their respective binding and dissociation kinetic data, and plot them as follows: Figure 8 As shown.

[0307] The results are as follows Figure 8 As shown, both Fab fragments of 2B4 and 36H6 can directly block the binding of SARS-CoV-2 RBD to ACE2. The 36H6 Fab fragment exhibits stronger blocking activity, showing a significant blocking effect at a 1:2 molar ratio with RBD, and almost complete blocking at a 1:1 or higher molar ratio. Similarly, the Fab fragment of the 2B4 antibody also shows a significant blocking effect at a 1:1 molar ratio with RBD, and complete blocking at higher molar ratios. In contrast, the Fab fragment of the control antibody shows no significant blocking effect even at high concentrations. These results demonstrate that the 2B4 and 36H6 antibodies can directly block the binding of SARS-CoV-2 RBD to ACE2, which may explain their high neutralizing activity.

[0308] Example 10: Preparation of 36H6 humanized antibody

[0309] The IMGT gene database was searched to identify the human germline variable region sequence with the highest homology to the FR region of the murine antibody 36H6. Homology analysis determined the IGHV1-3*01 and IGKV4-1*01 germline gene sequences as templates for the heavy and light chains of the humanized antibody, respectively. Since the germline gene does not contain the required FR4 region for modification, the FR4 region needed to be aligned separately. The IGHJ5*02 and IGKJ2*01 germline gene sequences were ultimately selected as templates for the heavy and light chain FR4 modifications of the humanized antibody. The heavy and light chain CDR regions of the murine antibody 36H6 were transplanted into the FR frames of the human templates vH and vK, respectively. Sequence alignment of the FR regions of the murine antibody and the germline gene was performed, and selective reversion mutations were performed on the differentially expressed amino acids. Finally, one humanized heavy chain and two humanized light chains were designed, resulting in two humanized antibodies. The variable region sequences are shown in the table below.

[0310]

[0311] The light and heavy chain variable region genes of the 36H6 humanized antibody were obtained using the splitting overlapping extension-PCR (SOE-PCR) method. The amplification products were analyzed by agarose gel electrophoresis, and the PCR products were purified using a DNA purification and recovery kit (TianGen, DP118-02). Using the Gibson assembly method, the heavy chain variable region fragment of the mouse antibody was constructed into a PTT5-H vector (with AgeI / SalI restriction sites) containing the coding sequence of the human heavy chain constant region (SEQ ID NO:20), and the light chain variable region fragment was constructed into a PTT5-K vector (with AgeI / BsiWI restriction sites) containing the coding sequence of the human light chain constant region (SEQ ID NO:21). The recombinant vectors were transformed into DH5α competent cells (Shenzhen Kangti), and after 12 hours of growth on ampicillin-resistant LB plates, single colonies were picked and sent for sequencing (Shanghai Sangon Biotech). The correctly sequenced recombinant plasmids were extracted in large quantities using an endotoxin-free plasmid extraction kit (TianGen, DP117). After expression and purification, humanized antibodies h36H6-10 and h36H6-12 were obtained.

[0312] Example 11: Expression and purification of humanized antibodies h36H6-10 and h36H6-12

[0313] 11.1 Eukaryotic expression of 36H6 humanized antibody

[0314] Humanized antibodies against h36H6-10 and h36H6-12 were expressed by transient transfection of Expi-293F cells with dual plasmids. Expi-293F cells with a viability higher than 95% were prepared and cultured at a density of 4 × 10⁻⁶ cells / cells. 6 200 ml of the recombinant plasmid was seeded into 1 L cell culture flasks. 0.5 mg of PTT5-36H6-H3 recombinant plasmid and 0.5 mg of PTT5-36H6-K1 recombinant plasmid were used as the light and heavy chain plasmids for expressing h36H6-10; 1 mg of the mixed light and heavy chain plasmids was mixed with 2 mg of PEI, vigorously vortexed for 8 seconds, and allowed to stand for 8 minutes. The mixture was then added to 200 ml of cell culture. After 4 hours, 200 ml of Freestyle medium was added, and the cells were incubated at 37°C for 7 days using a 5% CO2 incubator. The cell supernatant was collected, centrifuged at 10,000 rpm for 30 minutes, and then purified.

[0315] 11.2 Purification and preparation of 36H6 humanized antibody:

[0316] Filter the cell supernatant using a 0.22 μm filter. Turn on the AKTA instrument and rinse tubing A and tubing B with solution A (200 mM disodium hydrogen phosphate dodecahydrate) and solution B (100 mM citric acid monohydrate), respectively. Mount the protein A column. Equilibrate the protein A column with solution A at a flow rate of 8 mL / min for at least 15 min. Once the UV value, pH value, and conductivity detected by the instrument have stabilized, proceed to the next step. Load the sample at a flow rate of 6-10 mL / min. The UV value will subsequently rise; this peak is the breakthrough peak. Continue washing the column with solution A, and collect the breakthrough peak sample for analysis. Once the pH value no longer changes, inject solution B at a flow rate of 6-10 mL / min. The pH value will subsequently decrease, and the UV value will rise; this peak is the elution peak. The antibody is mainly present in the elution peak; collect the elution peak sample for analysis. Equilibrate the column with solution A, then fill the tubing and protein A column with 20% ethanol. Remove the column and store at 4°C. The purified antibody was dialyzed overnight with 20 mM PBS buffer, and the concentration was determined by UV spectrophotometry or BCA. The antibody was then aliquoted into 1.5 mL tubes and stored at -20°C for later use.

[0317] Example 12: ELISA binding activity of humanized antibodies h36H6-10 and h36H6-12 against SARS-CoV-2

[0318] 12.1 Preparation of reaction plates

[0319] The SARS-CoV2-RBD (His tag) protein was diluted to a final concentration of 2 μg / mL with 50 mM CB buffer (NaHCO3 / Na2CO3 buffer, final concentration 50 mM, pH 9.6) at pH 9.6. 100 μL of coating buffer was added to each well of a 96-well microplate, and the plate was coated at 2–8°C for 16–24 hours, followed by coating at 37°C for 2 hours. The plate was 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 plate was incubated at 37°C for 2 hours. The blocking buffer was discarded. After drying, the plate was stored in aluminum foil bags at 2–8°C for later use.

[0320] 12.2 ELISA detection of 36H6 humanized antibody

[0321] The humanized antibodies h36H6-10 and h36H6-12 obtained in Example 11 were serially diluted with SD-1 solution starting at a concentration of 10 μg / mL, for a total of 7 dilutions. 100 μL of the diluted sample was added to each well of an ELISA plate coated with SARS-CoV2-RBD protein, and the plate was incubated at 37°C for 60 minutes. The plate was then washed five times with PBST washing buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 100 μL of HRP-labeled goat anti-human IgG reaction solution was added to each well. The plate was then incubated at 37°C for 30 minutes. After completing the enzyme-labeled reaction steps, the ELISA plate was washed five times with PBST washing buffer (20mM PB7.4, 150mM NaCl, 0.1% Tween 20). 50 μL of TMB chromogenic reagent (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well, and the plate was incubated at 37℃ for 15 minutes. After the chromogenic reaction, 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well, and the OD450 / 630 values ​​of each well were measured using an ELISA reader. Reactivity determination of 36H6 humanized antibody with SARS-CoV2-RBD: The determination was based on the readings after the reaction. If the detected value / background value was greater than 5, the result was considered positive.

[0322] The results are as follows Figure 9 As shown, both humanized antibodies against SARS-CoV-2-RBD exhibited strong binding activity at concentrations of 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, and 0.12 μg / mL. These results indicate that the humanized h36H6-10 and h36H6-12 strains can maintain strong binding activity against SARS-CoV-2-RBD.

[0323] Example 13: Determination of neutralizing activity of h36H6-10 and h36H6-12 humanized antibodies in a SARS-CoV2 VSVpp pseudovirus infection model

[0324] The neutralizing activities of humanized antibodies h36H6-10 and h36H6-12, chimeric antibody 36H6-Cab (whose heavy chain and light chain respectively contain the heavy chain constant region shown in SEQ ID NO:21 and the light chain constant region shown in SEQ ID NO:22), and parental mouse monoclonal antibody 36H6 (mAb-36H6) were determined in a pseudovirus infection model using the detection method described in Example 8.

[0325] The antibody was diluted to 1.333 nM as a gradient, followed by 3-fold gradients for a total of 6 gradients. The serially diluted antibodies were mixed with diluted SARS-CoV-2 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 the infected cells was performed using a disc-based confocal high-content imaging system (Opera Phenix or Operetta CLS, purchased from PerkinElmer). The obtained fluorescence images were then quantitatively analyzed using Columbus image management and analysis software to detect the number of green fluorescent positive cells. 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.

[0326] The results are as follows Figure 10 As shown, both humanized 36H6 antibodies exhibited strong neutralizing activity against SARS-CoV-2. The IC50 of the murine monoclonal antibody 36H6 was 0.017 nM (essentially equivalent to the result measured in Example 8), while the IC50 of the humanized antibody h36H6-10 was 0.026 nM. Furthermore, h36H6-12 was even stronger than h36H6-10, with an IC50 of 0.016 nM. These results indicate that the neutralizing activity of both humanized 36H6 antibodies is no weaker than that of the parental murine monoclonal antibody 36H6.

[0327] 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. SEQUENCE LISTING <110> Yangshengtang Co., Ltd.; Xiamen University <120> Antibodies against SARS-CoV-1 or SARS-CoV-2 and their uses <130> IDC210043 <150> 202010369877.3 <151> 2020-04-30 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> artificial <220> <223> 36H6 VH <400> 1 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Tyr Tyr Val Ser Ser Tyr Gly Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 2 <211> 113 <212> PRT <213> artificial <220> <223> 36H6 VL <400> 2 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Light <210> 3 <211> 119 <212> PRT <213> artificial <220> <223> 2B4 VH <400> 3 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Thr Val Glu Trp Phe Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Gln 115 <210> 4 <211> 113 <212> PRT <213> artificial <220> <223> 2B4 VL <400> 4 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30 Tyr Asn Gln Glu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 5 <211> 8 <212> PRT <213> artificial <220> <223> 36H6 CDR‑H1 <400> 5 Gly Tyr Thr Phe Thr Glu Tyr Thr 1 5 <210> 6 <211> 8 <212> PRT <213> artificial <220> <223> 36H6 CDR‑H2 <400> 6 Ile Asn Pro Asn Asn Gly Asp Thr 1 5 <210> 7 <211> 13 <212> PRT <213> artificial <220> <223> 36H6 CDR‑H3 <400> 7 Ala Arg Glu Gly Asp Tyr Tyr Val Ser Ser Tyr Gly Tyr 1 5 10 <210> 8 <211> 12 <212> PRT <213> artificial <220> <223> 36H6 CDR‑L1 <400> 8 Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr 1 5 10 <210> 9 <211> 3 <212> PRT <213> artificial <220> <223> 36H6 CDR‑L2 <400> 9 Trp Ala Ser 1 <210> 10 <211> 9 <212> PRT <213> artificial <220> <223> 36H6 CDR‑L3 <400> 10 Gln Gln Tyr Tyr Ser Phe Pro Leu Thr 1 5 <210> 11 <211> 8 <212> PRT <213> artificial <220> <223> 2B4 CDR‑H1 <400> 11 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 12 <211> 8 <212> PRT <213> artificial <220> <223> 2B4 CDR‑H2 <400> 12 Ile Asp Pro Tyr Asp Ser Glu Thr 1 5 <210> 13 <211> 12 <212> PRT <213> artificial <220> <223> 2B4 CDR‑H3 <400> 13 Ala Arg Trp Gly Thr Val Glu Trp Phe Phe Asp Tyr 1 5 10 <210> 14 <211> 12 <212> PRT <213> artificial <220> <223> 2B4 CDR‑L1 <400> 14 Gln Ser Leu Leu Asn Ser Tyr Asn Gln Glu Asn Tyr 1 5 10 <210> 15 <211> 3 <212> PRT <213> artificial <220> <223> 2B4 CDR‑L1 <400> 15 Phe Ala Ser 1 <210> 16 <211> 9 <212> PRT <213> artificial <220> <223> 2B4 CDR‑L1 <400> 16 Gln Gln His Tyr Ser Thr Pro Phe Thr 1 5 <210> 17 <211> 120 <212> PRT <213> artificial <220> <223> h36H6‑10 / h36H6‑12 VH <400> 17 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ser Pro Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile Asn Pro Asn Asn Gly Asp Thr Ile Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Tyr Tyr Val Ser Ser Tyr Gly Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 113 <212> PRT <213> artificial <220> <223> h36H6‑10 VL <400> 18 Asp Ile Val Met Ser Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Light <210> 19 <211> 113 <212> PRT <213> artificial <220> <223> h36H6‑12 VL <400> 19 Asp Ile Val Met Ser Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Light <210> 20 <211> 330 <212> PRT <213> artificial <220> <223> Human IgG1 heavy chain constant region <400> 20 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110<e Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 21 <211> 107 <212> PRT <213> artificial <220> <223> Human κ light chain constant region <400> 21 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Glin 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 22 <211> 792 <212> PRT <213> artificial <220> <223> Nucleotide sequence encoding the RBD-His protein <400> 22 Ala Thr Gly Gly Cys Cys Cys Gly Cys Ala Gly Cys Gly Thr Gly Ala 1 5 10 15 Cys Cys Cys Thr Gly Gly Thr Gly Thr Thr Cys Cys Thr Gly Gly Thr 20 25 30 Gly Cys Thr Gly Gly Thr Gly Ala Gly Cys Cys Thr Gly Ala Cys Cys 35 40 45 Gly Gly Thr Cys Thr Gly Thr Ala Cys Gly Cys Cys Ala Gly Cys Ala 50 55 60 Ala Cys Thr Thr Cys Cys Gly Cys Gly Thr Gly Cys Ala Gly Cys Cys 65 70 75 80 Cys Ala Cys Cys Gly Ala Gly Ala Gly Cys Ala Thr Cys Gly Thr Gly 85 90 95 Cys Gly Cys Thr Thr Cys Cys Cys Cys Ala Ala Cys Ala Thr Cys Ala 100 105 110 Cys Cys Ala Ala Cys Cys Thr Gly Thr Gly Cys Cys Cys Cys Thr Thr 115 120 125 Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Thr Thr Cys Ala Ala Cys 130 135 140 Gly Cys Cys Ala Cys Cys Cys Gly Cys Thr Thr Cys Gly Cys Cys Ala 145 150 155 160 Gly Cys Gly Thr Gly Thr Ala Cys Gly Cys Cys Thr Gly Gly Ala Ala 165 170 175 Cys Cys Gly Cys Ala Ala Gly Cys Gly Cys Ala Thr Cys Ala Gly Cys 180 185 190 Ala Ala Cys Thr Gly Cys Gly Thr Gly Gly Cys Cys Gly Ala Cys Thr 195 200 205 Ala Cys Ala Gly Cys Gly Thr Gly Cys Thr Gly Thr Ala Cys Ala Ala 210 215 220 Cys Ala Gly Cys Gly Cys Cys Ala Gly Cys Thr Thr Cys Ala Gly Cys 225 230 235 240 Ala Cys Cys Thr Thr Cys Ala Ala Gly Thr Gly Cys Thr Ala Cys Gly 245 250 255 Gly Cys Gly Thr Gly Ala Gly Cys Cys Cys Cys Ala Cys Cys Ala Ala 260 265 270 Gly Cys Thr Gly Ala Ala Cys Gly Ala Cys Cys Thr Gly Thr Gly Cys 275 280 285 Thr Thr Cys Ala Cys Cys Ala Ala Cys Gly Thr Gly Thr Ala Cys Gly 290 295 300 Cys Cys Gly Ala Cys Ala Gly Cys Thr Thr Cys Gly Thr Gly Ala Thr 305 310 315 320 Cys Cys Gly Cys Gly Gly Cys Gly Ala Cys Gly Ala Gly Gly Thr Gly 325 330 335 Cys Gly Cys Cys Ala Gly Ala Thr Cys Gly Cys Cys Cys Cys Cys Gly 340 345 350 Gly Cys Cys Ala Gly Ala Cys Cys Gly Gly Cys Ala Ala Gly Ala Thr 355 360 365 Cys Gly Cys Cys Gly Ala Cys Thr Ala Cys Ala Ala Cys Thr Ala Cys 370 375 380 Ala Ala Gly Cys Thr Gly Cys Cys Cys Gly Ala Cys Gly Ala Cys Thr 385 390 395 400 Thr Cys Ala Cys Cys Gly Gly Cys Thr Gly Cys Gly Thr Gly Ala Thr 405 410 415 Cys Gly Cys Cys Thr Gly Gly Ala Ala Cys Ala Gly Cys Ala Ala Cys 420 425 430 Ala Ala Cys Cys Thr Gly Gly Ala Cys Ala Gly Cys Ala Ala Gly Gly 435 440 445 Thr Gly Gly Gly Cys Gly Gly Cys Ala Ala Cys Thr Ala Cys Ala Ala 450 455 460 Cys Thr Ala Cys Cys Thr Gly Thr Ala Cys Cys Gly Cys Cys Thr Gly 465 470 475 480 Thr Thr Cys Cys Gly Cys Ala Ala Gly Ala Gly Cys Ala Ala Cys Cys 485 490 495 Thr Gly Ala Ala Gly Cys Cys Cys Thr Thr Cys Gly Ala Gly Cys Gly 500 505 510 Cys Gly Ala Cys Ala Thr Cys Ala Gly Cys Ala Cys Cys Gly Ala Gly 515 520 525 Ala Thr Ala Thr Ala Cys Cys Ala Gly Gly Cys Cys Gly Gly Cys Ala 530 535 540 Gly Cys Ala Cys Cys Cys Cys Cys Thr Gly Cys Ala Ala Cys Gly Gly 545 550 555 560 Cys Gly Thr Gly Gly Ala Gly Gly Gly Cys Thr Thr Cys Ala Ala Cys 565 570 575 Thr Gly Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys Cys Thr Gly Cys 580 585 590 Ala Gly Ala Gly Cys Thr Ala Cys Gly Gly Cys Thr Thr Cys Cys Ala 595 600 605 Gly Cys Cys Cys Ala Cys Cys Ala Ala Cys Gly Gly Cys Gly Thr Gly 610 615 620 Gly Gly Cys Thr Ala Cys Cys Ala Gly Cys Cys Cys Thr Ala Cys Cys 625 630 635 640 Gly Cys Gly Thr Gly Gly Thr Gly Gly Thr Gly Cys Thr Gly Ala Gly 645 650 655 Cys Thr Thr Cys Gly Ala Gly Cys Thr Gly Cys Thr Gly Cys Ala Cys 660 665 670 Gly Cys Cys Cys Cys Cys Gly Cys Cys Ala Cys Cys Gly Thr Gly Thr 675 680 685 Gly Cys Gly Gly Cys Cys Cys Cys Ala Ala Gly Ala Ala Gly Ala Gly 690 695 700 Cys Ala Cys Cys Ala Ala Cys Cys Thr Gly Gly Thr Gly Ala Ala Gly 705 710 715 720 Ala Ala Cys Ala Ala Gly Thr Gly Cys Gly Thr Gly Ala Ala Cys Thr 725 730 735 Thr Cys Ala Ala Cys Thr Thr Cys Ala Ala Cys Gly Gly Cys Cys Thr 740 745 750 Gly Ala Cys Cys Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Gly Ala 755 760 765 Thr Cys Thr Gly Gly Thr Cys Ala Cys Cys Ala Cys Cys Ala Cys Cys 770 775 780 Ala Cys Cys Ala Cys Cys Ala Cys 785 790 <210> 23 <211> 264 <212> PRT <213> artificial <220> <223> Amino acid sequence of RBD-His protein <400>  23 Met Ala Arg Ser Val Thr Leu Val Phe Leu Val Leu Val Ser Leu Thr 1 5 10 15 Gly Leu Tyr Ala Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys 225 230 235 240 Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Arg 245 250 255 Ser Gly His His His His His His 260

Claims

1. An antibody or an antigen-binding fragment thereof specifically binding to the receptor-binding region of the S protein of SARS-CoV-2, said antibody or antigen-binding fragment comprising: (a) A heavy chain variable region (VH) containing the following three complementary determinant regions: VH CDR1 of SEQ ID NO: 5, VH CDR2 of SEQ ID NO: 6, and VH CDR3 of SEQ ID NO: 7; and (b) Light chain variable regions (VLs) containing the following three complementary determination regions: the following three light chain CDRs: VL CDR1 with sequence SEQ ID NO: 8, VL CDR2 with sequence SEQ ID NO: 9, and VL CDR3 with sequence SEQ ID NO:

10.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising: a VH comprising the sequence shown in SEQ ID NO: 1 and a VL comprising the sequence shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from human immunoglobulin.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein, The VH of the antibody or its antigen-binding fragment comprises: heavy chain framework regions FR1, FR2, and FR3 derived from the heavy chain germline sequence IGHV1-3*01, and heavy chain framework region FR4 derived from the heavy chain germline sequence IGHJ5*02; and, The VL of the antibody or its antigen-binding fragment comprises: light chain framework regions FR1, FR2 and FR3 derived from the light chain germline sequence IGKV4-1*01, and light chain framework region FR4 derived from the light chain germline sequence IGKJ2*01.

6. The antibody or antigen-binding fragment thereof according to claim 5, comprising: (1) VH containing the sequence shown in SEQ ID NO: 17 and VL containing the sequence shown in SEQ ID NO: 18; or (2) VH containing the sequence shown in SEQ ID NO: 17 and VL containing the sequence shown in SEQ ID NO:

19.

7. The antibody or antigen-binding fragment thereof of claim 1, further comprising a constant region derived from human immunoglobulin.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin, and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the light chain constant region is the κ light chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 8, wherein the heavy chain constant region is the IgG1, IgG2, IgG3 or IgG4 heavy chain constant region.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO:

20.

12. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO:

21.

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

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein, The antigen-binding fragment is a humanized antibody.

15. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16.

18. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-14, comprising culturing a host cell according to claim 17 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.

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

20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition further comprises an additional pharmaceutically active agent.

21. A method for neutralizing the virulence of SARS-CoV-2 in a sample for non-disease diagnostic purposes, comprising contacting a sample containing SARS-CoV-2 with an antibody or antigen-binding fragment thereof as described in any one of claims 1-14.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14 for the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample, or for preventing and / or treating SARS-CoV-2 infection or disease associated with SARS-CoV-2 infection in a subject.

23. The use as described in claim 22, wherein the disease associated with SARS-CoV-2 infection is COVID-19.

24. The use as described in claim 22, wherein the subject is a mammal.

25. The use as described in claim 22, wherein the antibody or its antigen-binding fragment is used alone or in combination with another pharmaceutically active agent.

26. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-14, and a detectable label linked to said antibody or antigen-binding fragment thereof.

27. The conjugate of claim 26, wherein the detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

28. The conjugate of claim 27, wherein the detectable marker is selected from horseradish peroxidase, alkaline phosphatase, acridine esters, luminol, fluorescein, or fluorescent protein.

29. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-14 or a conjugate as claimed in any one of claims 26-28.

30. The kit of claim 29, wherein the kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1-14, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

31. The kit of claim 30, wherein the second antibody further comprises a detectable marker selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

32. The kit of claim 31, wherein the detectable marker is selected from horseradish peroxidase, alkaline phosphatase, acridine esters, luminol, fluorescein, or fluorescent protein.

33. A method for detecting the presence or level of SARS-CoV-2 in a sample for non-disease diagnostic purposes, comprising using an antibody or antigen-binding fragment thereof as described in any one of claims 1-14 or a conjugate as described in any one of claims 26-28.

34. The method of claim 33, wherein the method is an enzyme immunoassay, a chemiluminescent immunoassay, a fluorescence immunoassay, or a radioimmunoassay.

35. The method of claim 33, wherein the method comprises: Use the antibody or antigen-binding fragment thereof according to any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof contains a detectable label.

36. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-14 in the preparation of a kit for detecting the presence or level of SARS-CoV-2 in a sample, and / or for diagnosing whether a subject is infected with SARS-CoV-2.

37. The use according to claim 36, wherein the kit comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-14, wherein the antibody or antigen-binding fragment thereof comprises a detectable label.

38. The use as claimed in claim 36, wherein the sample is a blood sample, excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

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