Antibodies against 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 SARS-CoV-2. In particular, the present invention relates to monoclonal antibodies against SARS-CoV-2, and compositions (e.g., diagnostic and therapeutic agents) comprising said antibodies. 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 SARS-CoV-2 infection and / or a disease caused by said infection (e.g., COVID-19).

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular virology, particularly to the diagnosis, prevention, and treatment of SARS-CoV-2. Specifically, this invention relates to monoclonal antibodies against SARS-CoV-2, 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 SARS-CoV-2 infection and / or diseases caused by said infection (e.g., COVID-19). 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] In this application, the inventors first developed a rabbit-derived antibody with excellent properties, which specifically binds to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein. Building upon this, the inventors further devoted considerable inventive effort to in-depth research and modification of the rabbit-derived antibody, thereby developing a humanized antibody based on it. The antibody of this invention can neutralize SARS-CoV-2, blocking or inhibiting the binding of SARS-CoV-2 to the receptor ACE2. Therefore, the antibody of this invention has the potential for prevention and / or treatment of SARS-CoV-2 infection or diseases caused by SARS-CoV-2 infection, and has significant clinical value.

[0005] The antibody of the present invention

[0006] In one aspect, the present invention provides an antibody or 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 (VH) 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:3, 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:4, 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: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;

[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: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.

[0014] (v)VL CDR2, 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, and

[0015] (vi)VL CDR3, 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.

[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: 3, VH CDR2 with sequence SEQ ID NO: 4, and VH CDR3 with sequence SEQ ID NO: 5; and / or, the following three light chain CDRs as defined by the Kabat numbering system: VL CDR1 with sequence SEQ ID NO: 6, VL CDR2 with sequence SEQ ID NO: 7, and VL CDR3 with sequence SEQ ID NO: 8.

[0018] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0019] (a) Heavy chain variable regions (VH) containing the following three complementary determination regions (CDRs) as defined by the IMGT numbering system:

[0020] (i) VH CDR1, 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.

[0021] (ii) VH CDR2, 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, and

[0022] (iii) VH CDR3, 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;

[0023] And / or,

[0024] (b) Light chain variable regions (VLs) containing the following three complementary determination regions (CDRs) as defined by the IMGT numbering system:

[0025] (iv) VL CDR1, 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.

[0026] (v)VL CDR2, 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) compared to it, and

[0027] (vi)VL CDR3, 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.

[0028] In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

[0029] In some embodiments, the antibody or its antigen-binding fragment comprises: the following three heavy chain CDRs as defined by the IMGT numbering system: VH CDR1 with sequence SEQ ID NO: 9, VH CDR2 with sequence SEQ ID NO: 10, and VH CDR3 with sequence SEQ ID NO: 11; and / or, the following three light chain CDRs as defined by the IMGT numbering system: VL CDR1 with sequence SEQ ID NO: 12, VL CDR2 with sequence SEQ ID NO: 13, and VL CDR3 with sequence SEQ ID NO: 14.

[0030] 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.

[0031] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0032] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0033] (i) The sequence shown in SEQ ID NO: 1;

[0034] (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

[0035] (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;

[0036] and

[0037] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0038] (iv) The sequence shown in SEQ ID NO: 2;

[0039] (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

[0040] (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.

[0041] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0042] 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.

[0043] 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.

[0044] 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 rabbit residues.

[0045] 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 rabbit residues.

[0046] 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 IGHV3-53*04, and heavy chain framework region FR4 derived from the heavy chain germline sequence IGHJ1*01; 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 IGKV1-5*01, and light chain framework region FR4 derived from the light chain germline sequence IGKJ2*02. 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 rabbit residues.

[0047] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0048] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0049] (i) The sequence shown in any one of SEQ ID NOs: 15-18;

[0050] (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 any one of SEQ ID NOs: 15-18; or

[0051] (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 any of the sequences shown in SEQ ID NOs: 15-18;

[0052] and

[0053] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0054] (iv) The sequence shown in SEQ ID NO: 19;

[0055] (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: 19; or

[0056] (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: 19.

[0057] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0058] In some exemplary embodiments, the antibody or its antigen-binding fragment comprises:

[0059] (1) VH containing the sequence shown in SEQ ID NO:15 and VL containing the sequence shown in SEQ ID NO:19;

[0060] (2) VH containing the sequence shown in SEQ ID NO:16 and VL containing the sequence shown in SEQ ID NO:19;

[0061] (3) VH containing the sequence shown in SEQ ID NO:17 and VL containing the sequence shown in SEQ ID NO:19; or

[0062] (4) VH containing the sequence shown in SEQ ID NO:18 and VL containing the sequence shown in SEQ ID NO:19.

[0063] In some embodiments, the antibody or antigen-binding fragment of the present invention may further comprise a constant region sequence or a variant thereof derived from a mammalian (e.g., rabbit or human) immunoglobulin, the variant having one or more amino acid substitutions, deletions or additions compared to the sequence from which it is derived.

[0064] 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,

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 rabbit 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.

[0069] In some embodiments, the light chain constant region is a κ light chain constant region. In some embodiments, the light chain constant region is a rabbit κ light chain constant region. In some embodiments, the light chain constant region is a human κ light chain constant region.

[0070] 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.

[0071] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and sdAb.

[0072] In some embodiments, the antibody is a rabbit-derived antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0073] In some embodiments, the antibody or antigen-binding fragment of the present invention possesses one, two, three, four, five, six, or all seven of the following features:

[0074] (1) The RBD that specifically binds to the S protein of SARS-CoV-2;

[0075] (2) K is less than about 100 nM, for example less than about 50 nM, 40 nM, 30 nM, 20 nM, 10 nM or lower. D The RBD that binds to the S protein of SARS-CoV-2; preferably, the K D Measured using surface plasmon resonance techniques (e.g., Biacore);

[0076] (3) The RBD of the SARS-CoV-2 S protein binds to an EC50 of less than about 100 ng / mL, for example less than about 50 ng / mL, 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 ng / mL or less; preferably, the EC50 can be determined by indirect ELISA.

[0077] (4) 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;

[0078] (5) It does not affect or has little effect on the binding of SARS-CoV-1 to the Ace2 receptor;

[0079] (6) Neutralize SARS-CoV-2 in vitro or in subjects (e.g., humans);

[0080] (7) Prevention and / or treatment of SARS-CoV-2 infection or disease caused by SARS-CoV-2 infection (e.g., COVID-19).

[0081] In this document, the antibodies or antigen-binding fragments thereof of the present invention may include variants that differ from the antibodies or antigen-binding fragments from which they are derived only in the conserved substitution of one or more (e.g., up to 20, 15, 10, or 5 amino acid substitutions) amino acid residues, or have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibodies or antigen-binding fragments from which they are derived, and substantially retain the aforementioned biological functions of the antibodies or antigen-binding fragments from which they are derived.

[0082] Antibody preparation

[0083] 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.

[0084] 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.

[0085] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof. In some embodiments, the isolated nucleic acid molecule encodes an antibody of the present invention or an antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof.

[0086] 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.

[0087] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain variable region of an antibody or antigen-binding fragment of the present invention, and / or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment of the present invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0088] In some embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of an antibody of the present invention or an antigen-binding fragment thereof, and / or a second nucleotide sequence encoding a light chain of an antibody of the present invention or an antigen-binding fragment thereof; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

[0089] 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).

[0090] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing host cells as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0091] Pharmaceutical Compositions and Therapeutic Uses

[0092] The antibodies or antigen-binding fragments of the present invention can be used in vitro or in vivo to neutralize SARS-CoV-2, block or inhibit SARS-CoV-2 infection of cells, thereby achieving the purpose of preventing and / or treating SARS-CoV-2 infection or SARS-CoV-2-related diseases in subjects.

[0093] Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0094] 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.).

[0095] In some embodiments, the antibody or antigen-binding fragment 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 of the present invention, along with the additional pharmaceutically active agent, may be administered simultaneously, separately, or sequentially.

[0096] 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.

[0097] 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 of the present invention. This method can be used to neutralize SARS-CoV-2 in vitro or in a subject (e.g., a human).

[0098] 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 of the present invention or a pharmaceutical composition.

[0099] 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).

[0100] 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 an effective amount of an antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, to a subject in need of such treatment.

[0101] 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.

[0102] In another aspect, the present invention relates to the use of the antibody or antigen-binding fragment thereof, or pharmaceutical composition of the present invention, in the preparation of a medicament used for:

[0103] (1) Neutralize SARS-CoV-2 in vitro or in subjects (e.g., humans); and / or

[0104] (2) For the prevention and / or treatment of SARS-CoV-2 infection or SARS-CoV-2-related disease (e.g., COVID-19) in subjects.

[0105] 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).

[0106] The antibodies or antigen-binding fragments thereof of the present invention, or the pharmaceutical compositions thereof, can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The antibodies or antigen-binding fragments thereof or the pharmaceutical compositions thereof of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antibody or antigen-binding fragment of the present invention, and optionally, simultaneously incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] In this paper, the subjects may be mammals, such as humans.

[0111] Conjugate

[0112] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to SARS-CoV-2. Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating the binding of the antibody or antigen-binding fragment to another molecule (e.g., avidin or a multihistidine tag). Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.

[0113] Therefore, in some embodiments, the antibody or its antigen-binding fragment of the present invention carries a detectable marker.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] Reagent kit and detection uses

[0118] The antibody or its antigen-binding fragment of the present invention can specifically bind to the RBD of the S protein of SARS-CoV-2, thereby being used to detect SARS-CoV-2 or its S protein or the RBD of the S protein, and optionally to diagnose whether a subject is infected with SARS-CoV-2 based on the above detection results.

[0119] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or an antigen-binding fragment thereof, or a conjugate of the present invention.

[0120] In some embodiments, the kit comprises the conjugate of the present invention.

[0121] In other embodiments, the kit comprises the antibody or antigen-binding fragment of the present invention. In some embodiments, the antibody or antigen-binding fragment does not contain a detectable label. In some embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment of the present invention; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin.

[0122] In some embodiments, the second antibody is specific to antibodies of the species (e.g., rabbits or humans) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0123] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human or rabbit immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-rabbit IgG antibody or an anti-human IgG antibody.

[0124] 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.

[0125] 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 of the present invention.

[0126] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0127] In some embodiments, the method includes using the conjugate of the present invention.

[0128] In other embodiments, the method includes using the antibody or antigen-binding fragment of the present invention. In some embodiments, the antibody or antigen-binding fragment does not contain a detectable label. In some embodiments, the method further includes using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acrid esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide, or biotin) to detect the antibody or antigen-binding fragment.

[0129] In some embodiments, the second antibody is specific to antibodies of the species (e.g., rabbits or humans) from which the constant region contained in the antibody of the present invention or its antigen-binding fragment originates.

[0130] In some embodiments, the second antibody is an anti-immunoglobulin (e.g., human or rabbit immunoglobulin) antibody, such as an anti-IgG antibody. In some embodiments, the second antibody is an anti-rabbit IgG antibody or an anti-human IgG antibody.

[0131] In some embodiments, the method includes: (1) contacting the sample with an antibody or an antigen-binding fragment of the present invention; and (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of the immune complex. The formation of the immune complex indicates the presence of SARS-CoV-2 or cells infected with SARS-CoV-2.

[0132] 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).

[0133] 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.

[0134] In some implementations, the subject is a mammal, such as a human.

[0135] In another aspect, the use of the antibody or antigen-binding fragment thereof of the present 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.

[0136] In some embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0137] 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.

[0138] 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).

[0139] Terminology Definition

[0140] 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.

[0141] 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: MN908947.3. 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.

[0142] 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.

[0143] 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.

[0144] 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).

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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)).

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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).

[0160] 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., rabbit) 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.).

[0161] In this application, the intended properties of the antibody of the present invention include: (1) specifically binding to the RBD of the S protein of SARS-CoV-2; (2) having a K+ concentration of less than about 100 nM, for example less than about 50 nM, 40 nM, 30 nM, 20 nM, 10 nM or lower. D The RBD that binds to the S protein of SARS-CoV-2; preferably, the K D (2) Detected by surface plasmon resonance technology (e.g., Biacore); (3) Binding to the RBD of the SARS-CoV-2 S protein with an EC50 of less than about 100 ng / mL, for example less than about 50 ng / mL, 40 ng / mL, 30 ng / mL, 20 ng / mL, 15 ng / mL or less; preferably, the EC50 can be determined by indirect ELISA; (4) Blocking or inhibiting the binding of SARS-CoV-2 to the Ace2 receptor, and / or blocking or inhibiting the infection of cells by SARS-CoV-2; (5) Not affecting or substantially not affecting the binding of SARS-CoV-1 to the Ace2 receptor; (6) Neutralizing SARS-CoV-2 in vitro or in a subject (e.g., human); (7) Preventing and / or treating SARS-CoV-2 infection or disease caused by SARS-CoV-2 infection (e.g., COVID-19). The antibodies of the present invention have one or more of the above-described intended properties.

[0162] The chimeric or humanized antibodies of this invention can be prepared based on the sequence of monoclonal antibodies produced by immunized animals (e.g., rabbits). 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.

[0163] 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 rabbit) to the constant region of a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567, 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, U.S. 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.

[0164] To prepare humanized antibodies, the CDR region of an immunized animal (e.g., a rabbit) 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).

[0165] 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).

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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).

[0171] 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.

[0172] 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.

[0173] 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).

[0174] 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.

[0175] 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.

[0176] 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.

[0177] Beneficial effects of the invention

[0178] The antibody of the present invention can specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, neutralize SARS-CoV-2, and block the binding of SARS-CoV-2 to the Ace2 receptor, thereby blocking SARS-CoV-2 infection of cells. Therefore, the antibody of the present invention has the potential for prevention and / or treatment of SARS-CoV-2 infection or diseases caused by SARS-CoV-2 infection (e.g., COVID-19). Furthermore, the antibody of the present invention can also be used to specifically detect SARS-CoV-2 or its S protein or the RBD of the S protein, which has important clinical value for the diagnosis of SARS-CoV-2 infection.

[0179] 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

[0180] Figure 1 The SDS-PAGE electrophoresis images of the recombinant proteins RBD-mFc, RBD-mGam, and RBD-His are shown.

[0181] Figure 2 The results of ELISA detection of the binding activity of recombinant proteins RBD-mFc and RBD-His in serum of patients in the recovery period of SARS-CoV-2 were shown.

[0182] Figure 3 The results of RBD antibody titer determination in rabbit antiserum are shown.

[0183] Figure 4 The results of Western blot analysis of rabbit antiserum binding activity to RBD-His are shown.

[0184] Figure 5 The results of ELISA assays show the binding activity of rabbit monoclonal antibody 3F4 to RBD-mFc.

[0185] Figure 6 The results of the affinity assay (Biacore) for the rabbit monoclonal antibody 3F4 against RBD-His are shown.

[0186] Figure 7 The results of the assay show the binding activity of rabbit monoclonal antibody 3F4 to the SARS-CoV-2 spike protein S expressed on cells.

[0187] Figure 8The results of the neutralizing activity assay of rabbit monoclonal antibody 3F4 in the SARS-CoV2 VSVpp pseudovirus infection model are shown.

[0188] Figure 9 The results of the cross-blocking ability assay of rabbit monoclonal antibody 3F4 against SARS-CoV-2 and SARS-CoV-1 are shown.

[0189] Figure 10 The results of ELISA assays show the binding activity of the 3F4 humanized antibody to RBD-His.

[0190] Figure 11 The results of neutralizing activity assays of the humanized 3F4 antibody in the SARS-CoV2 VSVpp pseudovirus infection model are shown, where r3F4 is a rabbit monoclonal antibody 3F4.

[0191] Figure 12 The results of the assay for the blocking ability of the humanized antibody r3F4 against SARS-CoV-2 are shown, where r3F4 is a rabbit monoclonal antibody 3F4.

[0192] Sequence information

[0193] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0194] Table 1: Sequence Description

[0195]

[0196]

[0197] Detailed Implementation

[0198] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0199] 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.

[0200] Example 1: Preparation of rabbit-derived monoclonal antibody against SARS-CoV-2 receptor-binding domain RBD protein

[0201] 1.1 Preparation and activity identification of the SARS-CoV-2 receptor-binding domain (RBD) protein:

[0202] The SARS-CoV-2 receptor-binding domain RBD-mFc was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The RBD gene of the SARS-CoV-2 receptor-binding domain RBD-His protein was obtained from the NCBI database (GenBank ID: MN908947.3), with a His tag fused to the C-terminus. Referring to the complete SARS-CoV-2 genome sequence (MN908947.3), the full-length extracellular region sequence of the SARS-CoV-2 spike protein (corresponding to viral Spike gene aa316-aa550) was optimized using human preferred codons to obtain its optimized coding nucleic acid sequence. A signal peptide coding sequence was attached to the N-terminus of the RBD nucleic acid sequence, and the optimized coding nucleic acid sequence of the green fluorescent protein mGamillus (mGam) was attached to the C-terminus. A polyhistidine peptide (6×His or 8×His) for affinity chromatography purification was attached to the C-terminus of the fusion peptide, ultimately yielding the RBD-mGam protein. The coding sequence of SARS-CoV2-RBG was ligated into a plasmid vector suitable for eukaryotic expression. The constructed recombinant plasmid was transfected into ExpiCHO cells (purchased from Thermofisher) or other CHO cells for expression and purification. The same sequence was used to construct and express the RBD-His protein, i.e., a polyhistidine peptide (6×His or 8×His) was ligated to the C-terminus of the RBD sequence. SDS-PAGE electrophoresis results showed that RBD-mFc, RBD-mGam, and RBD-His had high purity. Figure 1 The reactivity of the above proteins with serum from recovered SARS-CoV-2 patients was detected by ELISA, and the results are as follows: Figure 2 As shown, the obtained RBD-mFc, RBD-mGam, and RBD-His proteins are reactive with serum from SARS-CoV-2 convalescent patients.

[0203] 1.2 New Zealand Rabbit:

[0204] Ten-week-old female New Zealand rabbits were purchased from Songlian Experimental Animal Farm in Songjiang District, Shanghai.

[0205] 1.3 Immunization of experimental rabbits:

[0206] The standard in vivo immunization method was used; for detailed procedures, 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:

[0207] Rabbits were injected with 300 μg of SARS-CoV-2 receptor-binding domain (RBD) mFc protein mixed with an equal volume of Freund's complete adjuvant (CFA) to a final volume of 2 mL via multiple injections into the neck and back. On day 8 post-immunization, rabbits were injected with 500 μg of SARS-CoV-2 receptor-binding domain (RBD) mGam protein mixed with an equal volume of Freund's incomplete adjuvant (IFA) to a final volume of 2 mL via multiple injections into the neck and back. On day 11 post-immunization, whole blood was collected via the central auricular artery, serum was separated, and RBD antibody titer was determined. The results showed a serum titer of 10^4 (…). Figure 3 Western blot analysis showed that rabbit antiserum could react with RBD-His antigen. Figure 4 ).

[0208] 1.4 Preparation of rabbit peripheral blood mononuclear cells (PBMCs):

[0209] Rabbit whole blood was diluted 1:1 using serum-free RPMI 1640 medium. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll reagent. 1.5 times the volume of Ficoll solution was added to the bottom of the centrifuge tube, followed by slow addition of rabbit whole blood dilution. The centrifuge was performed at 800g for 30 min at 4°C with slow acceleration and deceleration. After centrifugation, the suspended cells at the interface between Ficoll and RPMI 1640 medium were collected as PBMCs. The PBMCs were collected by a second centrifugation at 1500 rpm for 5 min at 4°C.

[0210] 1.5 Specific B-cell screening for anti-SARS-CoV-2 receptor-binding domain protein RBD:

[0211] Rabbit PBMCs obtained from separation were resuspended in 100 μL of sterile PBS solution. RBD protein was added according to the standard of adding 1 μg of biotin-labeled RBD-mFc protein to 3 ml of rabbit whole blood PBMCs. The mixture was gently pipetted and incubated at 4°C for 30 min. After centrifugation at 1500 rpm for 3 min at 4°C, the supernatant was discarded, and the cells were retained. The cells were resuspended in PBS and washed 2–3 times. 100 μL of the staining system shown below was added to each tube. The cells were then incubated at 4°C in the dark for 30 min.

[0212]

[0213] RBD-specific memory B cells were sorted into 96-well plates containing 25 μL of lysis buffer per well, with one cell per well. After completion, 20 μL of lysis buffer containing a single cell was transferred to a PCR plate for reverse transcription, followed by nested PCR to amplify the variable regions of the antibody light and heavy chains.

[0214] The PCR products from the paired heavy and light chains were collected, recovered using a gel extraction kit, and sequenced. The sequencing results were compared with the IMGT database to determine whether the obtained gene was an antibody gene, whether the gene was complete, and whether it could successfully encode the antibody, thus identifying the family to which the antibody gene (V and J regions) belonged. The cloning vectors were digested with enzymes: EcoRI / BamHI for the heavy chain expression plasmid and NheI / SalI for the light chain expression plasmid. The variable region genes of the heavy and light chains were constructed into the corresponding eukaryotic expression vectors pRVRCH (SEQ ID NO:28) and pRVRCL (SEQ ID NO:29) using the Gibson assembly method. pRVRCH contains the nucleic acid sequence encoding the constant region of the rabbit monoclonal antibody heavy chain, and pRVRCL contains the nucleic acid sequence encoding the constant region of the rabbit monoclonal antibody light chain.

[0215] After the expression vector was constructed, HEK293T cells were transiently transfected using liposomes to express a monoclonal antibody against the SARS-CoV-2 receptor-binding domain protein RBD. Twelve hours before transfection, 10^4 cells were seeded into 96-well cell culture plates; tube A: 10 μL of Opti-MEM containing 0.2 μg IgH plasmid and 0.2 μg IgK plasmid; tube B: 10 μL of Opti-MEM containing 0.4 μL Novizan ExFect. 2000 Transfection Reagent; Gently mix tubes A and B separately, let stand at room temperature for 5 min, then add the diluted plasmid to the diluted transfection reagent, gently mix, and incubate at room temperature for 10 min; Add the plasmid-transfection reagent complex to the cells and incubate in a cell culture incubator; After 48 h, collect the cell supernatant, centrifuge at 3000 rpm for 5 min at 4℃, collect the supernatant, and discard the cell debris precipitate. Screen for positive monoclonal antibodies that specifically react with RBD using an indirect ELISA method (see Example 2).

[0216] 1.6 Sequencing of positive monoclonal antibodies against the SARS-CoV-2 receptor-binding domain protein RBD

[0217] A rabbit-derived monoclonal antibody, 3F4, specific to the SARS-CoV-2 receptor-binding domain protein RBD, was obtained using the above method. Sequencing revealed the amino acid sequences of the heavy chain variable region and light chain variable region of 3F4, as shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The CDR sequence was also determined using the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669) and the IMGT numbering system (http: / / www.imgt.org / IMGT_vquest / analysis).

[0218] Table 2: Variable region sequence of 3F4

[0219]

[0220] 1.7 Purification and Preparation of Eukaryotic Expression Antibodies

[0221] To achieve high-level expression of 3F4, suspension cells (293F) in the logarithmic growth phase were prepared and cultured in a cell shaker at 100 rpm, 37°C, and 5% CO2 until the cell density reached 1.5 × 10⁻⁶ cells / year. 6 / mL, cell viability >95%, take 400mL of cells and place them in a new cell culture flask to form one transfection system. Tube A: Add 600μg of light and heavy chain plasmids to 20mL of suspension cell culture medium and vortex to mix; Tube B: Add 1.2mg of PEI transfection reagent to 20mL of suspension cell culture medium and vortex to mix; Add the solution from tube B to tube A, vortex to mix and incubate at room temperature for 15min, then add the mixed liquid to 400mL of cell culture system; place in a cell shaker at 100rpm 37℃ 5% CO2 for antibody expression for 6 days; after culture, collect the cell supernatant and incubate at 4℃ 4000rpm for 10min.

[0222] 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 collected breakthrough and elution peak samples were identified by SDS-PAGE. The purified monoclonal 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.

[0223] Example 2: Reactivity of rabbit monoclonal antibody against SARS-CoV-2 receptor-binding domain protein RBD with SARS-CoV-2 receptor-binding domain protein RBD

[0224] 2.1 Preparation of reaction plates

[0225] The SARS-CoV2-RBD (mFc 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.

[0226] 2.2 ELISA detection of RBD rabbit monoclonal antibody 3F4

[0227] The monoclonal antibody 3F4 obtained in Example 1 was serially diluted 3-fold starting at a concentration of 10 μg / mL using 20 mM PBS buffer, 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 5 times with PBST wash buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 100 μL of HRP-labeled goat anti-rabbit IgG reaction solution was added to each well, and the plate was incubated at 37°C for 30 minutes. After completing the enzyme labeling reaction, the plate was washed 5 times with PBST wash buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 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°C for 15 minutes. After completing the colorimetric reaction, add 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) to each well of the plate and measure the OD450 / 630 values ​​of each well using an ELISA reader.

[0228] The results are as follows Figure 5 As shown, the EC50 value of rabbit monoclonal antibody 3F4 with RBD-mFc is 14.36 ng / mL, indicating good binding activity.

[0229] Example 3: Detection of the affinity constant between rabbit monoclonal antibody against SARS-CoV-2 receptor-binding domain protein RBD and SARS-CoV-2 receptor-binding domain protein RBD

[0230] The binding kinetics of monoclonal antibodies and antigens were analyzed using the Biacore 8K system. All steps were performed in PBS buffer. Monoclonal antibodies diluted to 5 μg / mL were captured using the company's Protein A chip. The SARS-CoV-2 receptor-binding domain protein RBD-His was used as the detection antigen, diluted to five gradients (100 nM, 50 nM, 25 nM, 12.5 nM, and 6.75 nM). The detection procedure was as follows: Capture 60 s, Analyze 300 s, Dissociation 600 s, and Regeneration 60 s. The affinity equilibrium dissociation constant was calculated using the instrument's accompanying data acquisition and analysis software.

[0231] The results are as follows Figure 6 As shown, the equilibrium dissociation constant (K0) of 3F4 for the SARS-CoV-2 receptor-binding domain protein RBD-His is... D The value is 8.79 nM.

[0232] Example 4: Rabbit monoclonal antibody against SARS-CoV-2 receptor-binding domain protein RBD used to detect SARS-CoV-2-S expressed in cells.

[0233] (1) Seed MDCK into a 10cm cell culture plate and transfect when the cell confluence reaches 60-80%.

[0234] (2) Tube A: Add 30ug of mammalian expression plasmid containing the SARS-CoV-2 spike protein S gene to 2mL Opti-MEM;

[0235] (3) Tube B: Add 60 μL of Novizan ExFect to 2 mL of Opti-MEM. 2000 TransfectionReagent;

[0236] (4) Gently mix tubes A and B separately, let stand at room temperature for 5 minutes, then add the diluted plasmid to the diluted transfection reagent, mix gently, and incubate at room temperature for 10 minutes.

[0237] (5) Add the plasmid-transfection reagent complex to the cells and incubate them in a cell culture incubator;

[0238] (6) 36 hours after transfection, the cells were digested with trypsin and seeded into 96-well cell culture plates at a ratio of 10^4 cells per well and placed in a cell culture incubator for culture.

[0239] (7) After seeding the cells into a 96-well cell culture plate for 12 hours, discard the cell supernatant, add 100 μL of 20 mM PBS to each well and wash once, add 100 μL of 4% paraformaldehyde prepared with 20 mM PBS to each well, fix the cells in the dark for 15 minutes, and add 100 μL of 20 mM PBS to each well and wash three times.

[0240] (8) Add 100 μL of ultrapure water to each well to prepare 3‰ Triton X-100, permeate the cells for 15 min, and wash three times with 100 μL of 20 mM PBS in each well.

[0241] (9) The monoclonal antibody was diluted to 1 μg / mL with 2% BSA prepared with 20 mM PBS and added to a 96-well cell culture plate at 100 μL per well. The plate was incubated at room temperature for 30 min and then washed three times with 100 μL of 20 mM PBS per well.

[0242] (10) Fluorescent secondary antibody Anti-Rabbit IgG (H+L), CF TMThe 647 antibody produced in goat was diluted to 1 μg / mL with 2% BSA prepared in 20 mM PBS. 100 μL of the solution was added to each well of a 96-well cell culture plate and incubated at room temperature for 30 min. Each well was then washed three times with 100 μL of 20 mM PBS.

[0243] (11) The nuclear dye DAPI was diluted with 2% BSA prepared with 20mM PBS at a ratio of 1:2000. 50μL was added to each well and incubated at room temperature for 5min. 100μL of 20mM PBS was added to each well and the cells were washed three times.

[0244] (12) Place the 96-well cell culture plate under a fluorescence microscope to observe and photograph the results.

[0245] The results are as follows Figure 7 As shown, fluorescence is distributed in both the cell membrane and cytoplasm of MDCK cells expressing spike protein S. These results indicate that 3F4 has specific binding activity to SARS-CoV-2 spike protein S expressed on cells, but no non-specific response to negative MDCK cells.

[0246] Example 5: Neutralizing ability of rabbit monoclonal antibody against SARS-CoV-2 receptor-binding domain protein RBD in SARS-CoV-2 VSVpp pseudovirus infection model

[0247] 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: MN908947.3) 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, Millipore, SLHP033RB) to remove cell debris and stored at -80°C for later use. Viral titer was determined by the number of GFP-positive cells infected with BHK21-hACE2 after serially diluted supernatant. The hACE2 gene was integrated into BHK21 cells using the PiggyBac transposon system. A transposon vector containing the hACE2 gene (SBI system biosciences, PB514B-2) and a transposase plasmid were co-transfected into BHK21 cells. Cells stably expressing hACE2, BHK21-hACE2, were obtained by screening using puromycin resistance and red fluorescence.

[0248] Antibody 3F4 was diluted to 2 μg / mL as gradient 1, followed by 3-fold serial dilutions for a total of 6 gradients. The serially diluted antibodies were mixed with diluted SARS-CoV2 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.

[0249] The results are as follows Figure 8 As shown, the IC50 of rabbit monoclonal antibody 3F4 is 1.95 ng / mL, indicating a strong neutralizing effect.

[0250] Example 6: Analysis of the cross-blocking ability of rabbit monoclonal antibodies against the SARS-CoV-2 receptor-binding domain protein RBD

[0251] Based on the complete SARS-CoV2-2 genome sequence (MN908947.3), the full-length extracellular region sequence of the SARS-CoV2-2 spike protein (corresponding to viral Spike gene aa316-aa550) was optimized using human-preferred codons to obtain its optimized coding nucleic acid sequence. A signal peptide coding sequence was ligated to the N-terminus of the RBD nucleic acid sequence, and the optimized coding nucleic acid sequence of the green fluorescent protein mGamillus (mGam) was ligated to the C-terminus. A polyhistidine peptide (6×His or 8×His) for affinity chromatography purification was ligated to the C-terminus of the fusion peptide, ultimately obtaining the RBD fusion fluorescent protein probe RBD-mGam (SARS-CoV2-RBG). The coding sequence of SARS-CoV2-RBG was ligated into a plasmid vector suitable for eukaryotic expression, and the constructed recombinant plasmid was transfected into ExpiCHO cells (purchased from Thermofisher) or other CHO cells for expression and purification.

[0252] Based on the published viral genome sequence of SARS-CoV-1 (AAP13567.1) in Genebank, a fusion fluorescent protein probe, abbreviated as SARS-CoV1-RBG, was constructed by referencing the method for SARS-CoV2-RBG and constructing its RBD with mGamillus. The coding sequence of SARS-CoV1-RBG was ligated into a plasmid vector suitable for eukaryotic expression. The constructed recombinant plasmid was then transfected into ExpiCHO cells (purchased from Thermofisher) or other CHO cells for expression and purification.

[0253] The red fluorescent protein mRuby3 was fused to the C-terminus of the ACE2 gene (NM_021804.1) with a flexible amino acid linker to obtain the sequence hACE2-mRuby3 (abbreviated as hACE2mRb3). This sequence was cloned into the PiggyBac(PB) transposon vector MIHIP-CMVmie constructed in our laboratory to obtain the MIHIP-CMVmie-hACE2mRb3 vector, which can express the hACE2mRb3 protein in cells. The MIHIP-CMVmie-hACE2mRb3 plasmid and the Super PiggyBac Transposase expression plasmid (System Biosciences) were co-transfected into 293T cells using Lipofectamine 3000 transfection reagent (Thermofisher) at a ratio of 4:1. Four hours after transfection, the medium was changed, and after another 24 hours of culture, the cells were passaged into 10cm cell culture dishes and the medium was replaced with one containing 2 μg / mL puromycin (InvivoGen) for pressure selection. The medium was changed every 24 hours to contain cytotoxic antibiotics. After 6-7 days in puromycin-containing medium, surviving cells were confirmed by microscopic examination to be positive for mRuby3 red fluorescent protein, indicating successful integration. The stable cell line was named 293T-ACE2iRb3.

[0254] 293T-ACE2iRb3 cells were seeded at a density of 15,000 cells / well on a black glass plate and cultured for 12-24 hours until they adhered. The SARS-CoV2-RBG probe was diluted to an appropriate concentration (20-30 nM) and mixed with antibody dilutions of different fractions, creating 10 serial dilutions of 50 nM each. 50 μL of culture medium was removed from the original cell culture plate, and 50 μL of the prepared mixture was added to the cell culture plate. The plate was incubated at 37°C for 60 minutes. Imaging analysis was performed directly using the Opera Phenix confocal high-content system without prior washing. Imaging fluorescence channels included Ex488 / Em510 (green fluorescent protein detection channel, probe signal), Ex561 / Em592 (red fluorescent protein detection channel, ACE2), and Ex641 / Em670 (near-infrared fluorescent protein iRFP670 imaging channel, cell nucleus). At least 25 fields of view (confocal mode) were captured using a 20x or 40x immersion lens. After completion, the data was uploaded to Columbus image management and analysis software for quantitative image analysis. Analysis parameters included: number of nuclear iRFP670 positive cells (N, >1000), mean intensity of red fluorescence signal (ACE2-mRuby3, for intercellular pore differences) on the cell membrane, and mean intensity and SD of green fluorescence signal in the cytoplasm (reflecting the amount of protein probe bound and taken up by the cell).

[0255] The results are as follows Figure 9 As shown, antibody 3F4 can block the binding of the SARS-CoV-2 receptor-binding domain RBD protein to the Ace2 receptor, but does not block the binding of the SARS-CoV-1 receptor-binding domain RBD protein to the Ace2 receptor.

[0256] Example 7: Preparation of 3F4 humanized antibody

[0257] 7.1 Humanization Design of Monoclonal Rabbit Antibody 3F4

[0258] Humanization of rabbit monoclonal antibody 3F4 was carried out according to the Combined CDRs method (Zhang YF, Ho M. Humanization of rabbit monoclonal antibodies via grafting combined Kabat / IMGT / Paratome complementarity-determining regions: Rationale and examples. MAbs. 2017;9(3):419–429. doi:10.1080 / 19420862.2017.1289302).

[0259] First, the complementarity-determining region (CDR) of the rabbit antibody 3F4 was determined using the Kabat method (Kabat EA; Wu TT, Perry HM, Gottesman KS, Coelier K. Sequences of proteins of immunological interest, US Department of Health and Human Services, PHS, NIH, Bethesda, 1991) and the IMGT method. The specific sequence is shown in Table 2 above.

[0260] The CDR regions determined by the IMGT method and the Kabat method were combined using the Combined CDRs method, and the final CDR regions used for CDR transplantation are shown in Table 3.

[0261] Table 3: Combined CDRs of rabbit monoclonal antibody 3F4

[0262]

[0263]

[0264] By searching the IMGT gene database, the variable region sequence of the human germline gene with the highest homology to the FR region of the rabbit antibody 3F4 was first identified. Homology analysis determined that the IGHV3-53*04 and IGKV1-5*01 germline gene sequences were used as templates for the heavy and light chains of the humanized antibody, respectively. Since the germline gene does not contain the FR4 region required for modification, the FR4 region needed to be aligned separately. Ultimately, the IGHJ1*01 and IGKJ2*02 germline gene sequences were selected as templates for the heavy and light chain FR4 modifications of the humanized antibody, respectively. The CDR regions of the heavy and light chains of the rabbit antibody 3F4 were transplanted into the FR frames of the human templates vH and vK, respectively. Sequence alignment of the FR regions of the rabbit antibody and the germline gene was performed, and selective reversion mutations were performed on the differentially expressed amino acids. Finally, four humanized heavy chains and one humanized light chain were designed, resulting in four humanized antibodies. The variable region sequences are shown in the table below.

[0265] Table 4: Variable Region Sequence of Humanized Antibodies

[0266]

[0267] 7.2 Construction of Recombinant Plasmid for Antibody Expression

[0268] The light and heavy chain variable region genes of the 3F4 humanized antibody were obtained using the splitting overlapping extension-PCR (SOE-PCR) method. The heavy and light chain variable region genes were separated into oligonucleotide sequences of approximately 80 bp each, with an overlap of approximately 20 bp between fragments. 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 was constructed into the PTT5-H vector (with AgeI / SalI restriction sites) containing the coding sequence of the heavy chain constant region (SEQ ID NO:20), and the light chain variable region fragment was constructed into the PTT5-K vector (with AgeI / BsiWI restriction sites) containing the coding sequence of the light chain constant region (SEQ ID NO:21). The recombinant vectors were transformed into DH5α competent cells (Shenzhen Kangti), and after 12 h of growth on ampicillin-resistant LB plates, single colonies were picked and sent for sequencing (Shanghai Sangon Biotech). The recombinant plasmids that were correctly sequenced were extracted in large quantities using the endotoxin-free plasmid extraction kit (TianGen, DP117).

[0269] 7.3 Eukaryotic expression and purification of antibodies

[0270] 293F cells were transiently transfected with two plasmids to express the humanized antibody 3F4. 293F cells with a viability higher than 95% were prepared and transfected at a rate of 4 × 10⁻⁶ cells / cell. 6 200 ml of the culture medium was seeded into a 1 L cell culture flask. 0.5 mg of each of the light and heavy chain plasmids were mixed, and 1 mg of the mixed light and heavy chain plasmids was combined with 2 mg of PEI. The mixture was vigorously shaken for 8 seconds and then allowed to stand for 8 minutes. The mixture was then added to 200 ml of cell culture medium. 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 and centrifuged at 10,000 rpm for 30 minutes. The supernatant was then collected and further purified as described in Example 1.7.

[0271] Example 8: ELISA binding activity of 3F4 humanized antibody against SARS-CoV-2

[0272] 8.1 Preparation of reaction plates

[0273] 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.

[0274] 8.2 ELISA detection of 3F4 humanized antibodies

[0275] The 3F4 humanized antibody obtained in Example 8 was serially diluted with 20 mM PBS buffer, 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 wash 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 incubated at 37°C for 30 minutes. After completing the enzyme labeling reaction, the plate was washed five times with PBST wash buffer (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), and 50 μL of TMB chromogenic reagent (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well. The plate was incubated at 37°C for 15 minutes. After completing the colorimetric reaction, 50 μL of stop solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) was added to each well of the ELISA plate, and the OD450 / 630 values ​​of each well were measured using an ELISA reader. The reactivity of the 3F4 humanized antibody with SARS-CoV2-RBD was determined based on the readings after the reaction. Results are as follows: Figure 10 As shown, the 3F4 humanized antibody exhibits good binding activity against RBD-His.

[0276] Example 9: Determination of the neutralizing activity of 3F4 humanized antibody in a SARS-CoV2 VSVpp pseudovirus infection model

[0277] The neutralization method for 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: MN908947.3) 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. Forty-eight hours after transfection, VSVdG-EGFP-G (Addgene, 31842) was... 1 The virus was inoculated into cells expressing the SARS-CoV-2 Sde18 truncated protein and incubated for 1 hour. The VSVdG-EGFP-G virus in the supernatant was then removed, and anti-VSV-G rat serum was added to block infection by residual VSVdG-EGFP-G. Progeny viruses carrying the SARS-CoV-2 Sde18 truncated protein were used to obtain 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, Millipore, SLHP033RB) to remove cell debris and stored at -80°C for later use. Viral titer was determined by the number of GFP-positive cells infected with BHK21-hACE2 after serially diluted supernatant. The hACE2 gene was integrated into BHK21 cells using the PiggyBac transposon system. A transposon vector containing the hACE2 gene (SBI system biosciences, PB514B-2) and a transposase plasmid were co-transfected into BHK21 cells. Cells stably expressing hACE2, BHK21-hACE2, were obtained by screening using puromycin resistance and red fluorescence.

[0278] The 3F4 humanized antibody was diluted to 13.33 nM as a gradient, followed by 3-fold gradients for a total of 10 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. Figure 11 As shown, the 3F4 humanized antibody can block pseudovirus infection of cells.

[0279] Example 10: Analysis of the ability of 3F4 humanized antibody to block the binding of SARS-CoV-2 S trimeric protein to ACE2

[0280] Based on the complete SARS-CoV2-2 genome sequence (MN908947.3), the full-length extracellular region sequence of the SARS-CoV2-2 spike protein (corresponding to viral Spike genes aa16-aa1207, abbreviated as SARS-CoV2-Secd) was optimized using human partial codons to obtain its optimized coding nucleic acid sequence. A signal peptide coding sequence was attached to the N-terminus of the SARS-CoV2-Secd nucleic acid sequence, and a trimer domain coding sequence (abbreviated as TFD) was attached to the C-terminus. Further, a flexible linker amino acid linker coding sequence, green fluorescent protein mGamillus (abbreviated as mGam), and a polyhistidine polypeptide (6×His or 8×His) for easy affinity chromatography purification were attached, ultimately yielding the spike protein extracellular region trimer fusion fluorescent protein probe Strimer-mGam (abbreviated as SARS-CoV2-STG).

[0281] The red fluorescent protein mRuby3 was fused to the C-terminus of the ACE2 gene (NM_021804.1) with a flexible amino acid linker to obtain the sequence hACE2-mRuby3 (abbreviated as hACE2mRb3). This sequence was cloned into the PiggyBac(PB) transposon vector MIHIP-CMVmie constructed in our laboratory to obtain the MIHIP-CMVmie-hACE2mRb3 vector, which can express the hACE2mRb3 protein in cells. The MIHIP-CMVmie-hACE2mRb3 plasmid and the Super PiggyBac Transposase expression plasmid (System Biosciences) were co-transfected into 293T cells using Lipofectamine 3000 transfection reagent (Thermofisher) at a ratio of 4:1. Four hours after transfection, the medium was changed, and after another 24 hours of culture, the cells were passaged into 10cm cell culture dishes and the medium was replaced with one containing 2 μg / mL puromycin (InvivoGen) for pressure selection. The medium was changed every 24 hours to contain cytotoxic antibiotics. After 6-7 days in puromycin-containing medium, surviving cells were confirmed by microscopic examination to be positive for mRuby3 red fluorescent protein, indicating successful integration. The stable cell line was named 293T-ACE2iRb3.

[0282] 293T-ACE2iRb3 cells were seeded at a density of 15,000 cells / well on a black glass plate and cultured for 12-24 hours until they adhered. The SARS-CoV2-STG probe was diluted to an appropriate concentration (2-4 nM) and mixed with antibody dilutions of different fractions, creating 10 serial dilutions of 50 nM each. 50 μL of culture medium was removed from the original cell culture plate, and 50 μL of the prepared mixture was added to the cell culture plate. The plate was incubated at 37°C for 60 minutes. Imaging analysis was performed directly using the Opera Phenix confocal high-content system without prior washing. Imaging fluorescence channels included Ex488 / Em510 (green fluorescent protein detection channel, probe signal), Ex561 / Em592 (red fluorescent protein detection channel, ACE2), and Ex641 / Em670 (near-infrared fluorescent protein iRFP670 imaging channel, cell nucleus). At least 25 fields of view (confocal mode) were captured using a 20x or 40x immersion lens. After completion, the data was uploaded to Columbus image management and analysis software for quantitative image analysis. Analysis parameters included: number of nuclear iRFP670 positive cells (N, >1000), mean intensity of red fluorescence signal (ACE2-mRuby3, for intercellular pore differences) on the cell membrane, and mean intensity and SD of green fluorescence signal in the cytoplasm (reflecting the amount of protein probe bound and taken up by the cell). The difference between the average intensity of the green fluorescence signal in the cytoplasm of different test wells and that in the positive control well / the average fluorescence intensity of the positive control well × 100% (inhibition rate). Figure 12 As shown, the 3F4 humanized antibody can block the binding of the SARS-CoV-2 receptor-binding domain RBD protein to the Ace2 receptor.

[0283] 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-2 and their uses <130> IDC210054 <150> 202010365125.X <151> 2020-04-30 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> artificial <220> <223> 3F4 VH <400> 1 Gln Ser Val Lys Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Trp 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Lys Ile Thr 65 70 75 80 Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Thr Ser 85 90 95 Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 110 <212> PRT <213> artificial <220> <223> 3F4 VL <400> 2 Asp Pro Met Leu Thr Gln Thr Ala Ser Ser Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Asp Asn 20 25 30 Asn Trp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Leu 65 70 75 80 Glu Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Asn Ile Phe Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Leu 100 105 110 <210> 3 <211> 5 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑H1 <400> 3 Ser Tyr Trp Met Ser 1 5 <210> 4 <211> 16 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑H2 <400> 4 Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑H3 <400> 5 Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp Leu 1 5 10 15 <210> 6 <211> 13 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑L1 <400> 6 Gln Ser Ser Gln Ser Val Tyr Asp Asn Asn Trp Leu Gly 1 5 10 <210> 7 <211> 7 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑L2 <400> 7 Ser Ala Ser Thr Leu Ala Ser 1 5 <210> 8 <211> 10 <212> PRT <213> artificial <220> <223> Kabat 3F4 CDR‑L3 <400> 8 Ala Gly Gly Tyr Ser Gly Asn Ile Phe Ala 1 5 10 <210> 9 <211> 8 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑H1 <400> 9 Gly Phe Ser Leu Ser Ser Tyr Trp 1 5 <210> 10 <211> 7 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑H2 <400> 10 Ile Phe Thr Gly Gly Ser Thr 1 5 <210> 11 <211> 18 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑H3 <400> 11 Ala Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu 1 5 10 15 Asp Leu <210> 12 <211> 8 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑L1 <400> 12 Gln Ser Val Tyr Asp Asn Asn Trp 1 5 <210> 13 <211> 3 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑L2 <400> 13 Ser Ala Ser 1 <210> 14 <211> 10 <212> PRT <213> artificial <220> <223> IMGT 3F4 CDR‑L3 <400> 14 Ala Gly Gly Tyr Ser Gly Asn Ile Phe Ala 1 5 10 <210> 15 <211> 124 <212> PRT <213> artificial <220> <223> h3F4‑1‑VH <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp 100 105 110 Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 124 <212> PRT <213> artificial <220> <223> h3F4‑2‑VH <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp 100 105 110 Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 124 <212> PRT <213> artificial <220> <223> h3F4‑3‑VH <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala 85 90 95 Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp 100 105 110 Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 124 <212> PRT <213> artificial <220> <223> h3F4‑4‑VH <400> 18 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg His Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu Asp 100 105 110 Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 110 <212> PRT <213> artificial <220> <223> h3F4‑1 / h3F4‑2 / h3F4‑3 / h3F4‑4‑VL <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Ser Val Tyr Asp Asn 20 25 30 Asn Trp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Ser Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Asn Ile Phe Ala Phe Gly Gln Gly Thr Lys Leu Glu Ile Leu 100 105 110 <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 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 Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 22 <211> 10 <212> PRT <213> artificial <220> <223> Combined CDR‑H1 <400> 22 Gly Phe Ser Leu Ser Ser Tyr Trp Met Ser 1 5 10 <210> 23 <211> 16 <212> PRT <213> artificial <220> <223> Combined CDR‑H2 <400> 23 Ile Ile Phe Thr Gly Gly Ser Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 1 5 10 15 <210> 24 <211> 18 <212> PRT <213> artificial <220> <223> Combined CDR‑H3 <400> 24 Ala Arg Thr Ser Tyr Tyr Asp Val Ser Gly Trp Gly Val Gly Arg Leu 1 5 10 15 Asp Leu <210> 25 <211> 13 <212> PRT <213> artificial <220> <223> Combined CDR‑L1 <400> 25 Gln Ser Ser Gln Ser Val Tyr Asp Asn Asn Trp Leu Gly 1 5 10 <210> 26 <211> 7 <212> PRT <213> artificial <220> <223> Combined CDR‑L2 <400> 26 Ser Ala Ser Thr Leu Ala Ser 1 5 <210> 27 <211> 10 <212> PRT <213> artificial <220> <223> Combined CDR‑L3 <400> 27 Ala Gly Gly Tyr Ser Gly Asn Ile Phe Ala 1 5 10 <210> 28 <211> 1047 <212> DNA <213> artificial <220> <223> Heavy chain expression plasmid pRVRCH <400> 28 gccgccacca tggaatggag ctgggtcttt ctcttcttcc tgtcagtaac tacaggtgaa 60 ttctccactc ggggcaagga tcctaaggct ccatcagtct tcccactggc cccctgctgc 120 ggggacacac ccagctccac ggtgaccctg ggctgcctgg tcaaagggta cctcccggag 180 ccagtgaccg tgacctggaa ctcgggcacc ctcaccaatg gggtacgcac cttcccgtcc 240 gtccggcagt cctcaggcct ctactcgctg agcagcgtgg tgagcgtgac ctcaagcagc 300 cagcccgtca cctgcaacgt ggcccaccca gccaccaaca ccaaagtgga caagaccgtt 360 gcgccctcga catgcagcaa gcccacgtgc ccaccccctg aactcctggg gggaccgtct 420 gtcttcatct tccccccaaa acccaaggac accctcatga tctcacgcac ccccgaggtc 480 acatgcgtgg tggtggacgt gagccaggat gaccccgagg tgcagttcac atggtacata 540 aacaacgagc aggtgcgcac cgcccggccg ccgctacggg agcagcagtt caacagcacg 600 atccgcgtgg tcagcaccct ccccatcgcg caccaggact ggctgagggg caaggagttc 660 aagtgcaaag tccacaacaa ggcactcccg gcccccatcg agaaaaccat ctccaaagcc 720 agagggcagc ccctggagcc gaaggtctac accatgggcc ctccccggga ggagctgagc 780 agcaggtcgg tcagcctgac ctgcatgatc aacggcttct acccttccga catctcggtg 840 gagtgggaga agaacgggaa ggcagaggac aactacaaga ccacgccggc cgtgctggac 900 agcgacggct cctacttcct ctacagcaag ctctcagtgc ccacgagtga gtggcagcgg 960 ggcgacgtct tcacctgctc cgtgatgcac gaggccttgc acaaccacta cacgcagaag 1020 tccatctccc gctctccggg taaatga 1047 <210> 29 <211> 403 <212> DNA <213> artificial <220> <223> Light chain expression plasmid pRVRCL <400> 29 gccgccacca tgagtgtgcc cactcaggtc ctggggttgc tgctgctgtg gcttacagat 60 gccagatgct agcttcctgt cagggtgatg tcgaccagtt gcacctactg tcctcatctt 120 cccaccagct gctgatcagg tggcaactgg aacagtcacc atcgtgtgtg tggcgaataa atactttccc gatgtcaccg tcacctggga ggtggatggc accacccaaa caactggcat 240 300. cgagaacagt aaaacaccgc agaattctgc agattgtacc tacaacctca gcagcactct gacactgacc agcacacagt acaacagcca caaagagtac acgtgcaagg tgacccaggg cacgacctca gtcgtccaga gcttcaatag gggtgactgt tag

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to the receptor-binding region (RBD) of the S protein of SARS-CoV-2, said antibody or antigen-binding fragment comprising: a VH as shown in SEQ ID NO: 16 and a VL as shown in SEQ ID NO:

19.

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

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from IgG1, IgG2, IgG3 or IgG4, and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from κ or λ.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) The heavy chain constant region (CH) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the wild-type sequence from which it is derived; and / or (b) The light chain constant region (CL) of human immunoglobulin or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions or additions or any combination thereof compared to the wild-type sequence from which it is derived.

5. 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.

6. 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.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein, The antigen-binding fragment is selected from Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, and diabody.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment has one or more of the following characteristics: (a) The RBD that specifically binds to the S protein of SARS-CoV-2; (b) Blocking or inhibiting the binding of SARS-CoV-2 to the Ace2 receptor, and / or blocking or inhibiting SARS-CoV-2 infection of cells; (c) It does not affect the binding of SARS-CoV-1 to the Ace2 receptor; (d) Neutralize SARS-CoV-2 in vitro or in subjects; (e) Prevention and / or treatment of SARS-CoV-2 infection or diseases associated with SARS-CoV-2 infection.

9. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-8.

10. A vector comprising the nucleic acid molecule of claim 9.

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

12. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-8, comprising culturing the host cell of claim 11 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.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8, and a pharmaceutically acceptable carrier and / or excipient.

14. The pharmaceutical composition of claim 13, further comprising an additional pharmaceutically active agent.

15. The pharmaceutical composition of claim 14, wherein the additional pharmaceutically active agent is interferon, lopinavir, ritonavir, chloroquine phosphate, favipiravir, or remdesivir.

16. A method for neutralizing the virulence of SARS-CoV-2 in a sample, comprising contacting a sample containing SARS-CoV-2 with an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8 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.

18. The use as described in claim 17, wherein, The disease in question is COVID-19.

19. The use as described in claim 17, wherein, The subjects were mammals.

20. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, and a detectable label linked to said antibody or antigen-binding fragment thereof.

21. The conjugate of claim 20, wherein the detectable marker is selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides, or biotin.

22. The conjugate of claim 20, wherein the detectable marker is selected from horseradish peroxidase or alkaline phosphatase, acridine esters, luminol and its derivatives, ruthenium derivatives, fluorescein or fluorescent protein.

23. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-8 or the conjugate as described in any one of claims 20-22.

24. The kit of claim 23, wherein the kit comprises the conjugate of any one of claims 20-22.

25. The kit of claim 23, wherein the kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1-8, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.

26. The kit of claim 25, wherein the second antibody further comprises a detectable label selected from enzymes, chemiluminescent reagents, fluorescent dyes, radionuclides or biotin.

27. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-8 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.

28. The use according to claim 27, wherein the sample is a blood sample, excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from the subject.

29. The use according to claim 27, wherein the sample is whole blood, plasma or serum from a human.

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