CN113583116A

CN113583116A - Antibodies against SARS-CoV-1 or SARS-CoV-2 and uses thereof

Info

Publication number: CN113583116A
Application number: CN202110475354.1A
Authority: CN
Inventors: 袁权; 巫洋涛; 熊华龙; 张天英; 王邵娟; 蒋艺超; 侯汪衡; 施洋; 张雅丽; 罗文新; 夏宁邵
Original assignee: Yang Sheng Tang Co Ltd; Xiamen University
Current assignee: Yang Sheng Tang Co Ltd; Xiamen University
Priority date: 2020-04-30
Filing date: 2021-04-29
Publication date: 2021-11-02

Abstract

The present invention relates to the fields of immunology and molecular virology, in particular the fields of diagnosis, prevention and treatment of coronaviruses (e.g. SARS-CoV-2 and/or SARS-CoV-1). In particular, the invention relates to monoclonal antibodies against coronaviruses, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the invention relates to the use of said antibodies. The antibodies of the invention are useful for the diagnosis, prevention and/or treatment of coronavirus infection and/or diseases caused by said infection.

Description

Antibodies against SARS-CoV-1 or SARS-CoV-2 and uses thereof

Technical Field

The present invention relates to the fields of immunology and molecular virology, in particular the fields of diagnosis, prevention and treatment of coronaviruses. In particular, the invention relates to monoclonal antibodies against coronaviruses, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the invention relates to the use of said antibodies. The antibodies of the invention are useful for the diagnosis, prevention and/or treatment of coronavirus infection and/or diseases caused by said infection.

Background

Coronavirus (coronavirus) infection can cause respiratory diseases of human beings, mild coronavirus infection can cause flu-like symptoms, and severe infection can develop into severe viral pneumonia and threaten the life and health of human beings. Coronaviruses can infect both humans and animals, and some animal-derived coronaviruses, if they breach the host barrier to infect humans, can spread rapidly among people and cause serious disease.

Currently, there is no specific drug approved for the prevention or treatment of SARS-CoV-2 infection. Patients with pneumonia caused by SARS-CoV-2 infection are only given general supportive therapy, oxygen therapy measures and antiviral therapy, such as interferon-alpha, lopinavir/ritonavir, chloroquine phosphate, etc., which have limited clinical effects. Studies have found that higher levels of SARS-CoV-2 neutralizing antibody production are often associated in convalescent patients with new coronary pneumonia. In a novel diagnosis and treatment scheme (trial seventh edition) for coronavirus pneumonia issued by Weijian Wei of China, plasma treatment of convalescent patients is recommended for patients with fast and severe disease progression and critical patients. There are research data showing that after treatment with convalescent plasma containing neutralizing antibodies in critically ill patients with COVID-19 associated severe respiratory distress syndrome (ARDS), the viral load in the patients is rapidly reduced and the clinical symptoms of the patients are effectively improved. These studies indicate the importance of humoral immunity in SARS-CoV-2, and indicate that in addition to vaccine development, a monoclonal antibody capable of neutralizing SARS-CoV-2 with high efficiency and specificity should be developed for short-term prevention and effective treatment of COVID-19, which is of great significance to national and even global prevention and treatment of COVID-19.

Disclosure of Invention

The inventor of the present application has conducted intensive research and creative work to obtain a murine antibody capable of specifically and efficiently neutralizing SARS-CoV-2 and a murine antibody having a broad spectrum for SARS-CoV-2 and SARS-CoV-1, respectively. On the basis of the above, the inventors have made extensive creative efforts and made intensive studies and alterations on these murine antibodies, thereby developing humanized antibodies of these murine antibodies. The humanized antibody of the invention not only retains the similar (even better) functions and properties with the murine parent antibody, but also has higher humanized degree and is not easy to cause immunogenic reaction. Therefore, the antibody of the present invention has a potential for the prevention and/or treatment of coronavirus infection or a disease caused by coronavirus infection, and has a great clinical value.

Antibodies of the invention

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

(a) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:

(i) a VH CDR1, consisting of the sequence: 5, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) a VH CDR2, consisting of the sequence: 6, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) a VH CDR3, consisting of the sequence: 7, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

and/or the presence of a gas in the gas,

(b) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:

(iv) a VL CDR1, consisting of the sequence: 8, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(v) a VL CDR2, consisting of the sequence: 9, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) compared thereto, and

(vi) a VL CDR3, consisting of the sequence: 10, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g. substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain embodiments, the substitution recited in any one of (i) - (vi) is a conservative substitution.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: the following 3 heavy chain CDRs defined according to the Kabat numbering system: the sequence is SEQ ID NO:5, the sequence of VH CDR1 of SEQ ID NO:6, the sequence of VH CDR2 of SEQ ID NO: VH CDR3 of 7; and/or, the following 3 light chain CDRs as defined by the Kabat numbering system: the sequence is SEQ ID NO:8, VL CDR1 of SEQ ID NO:9, VL CDR2 of SEQ ID NO:10 VL CDR 3.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or 3 CDRs contained in the light chain variable region (VL) shown in SEQ ID NO: 2. In certain embodiments, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by Kabat, IMGT or Chothia numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region (VH) comprising an amino acid sequence selected from:

(i) SEQ ID NO: 1;

(ii) and SEQ ID NO:1 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO:1, has 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;

and

(b) a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

(iv) SEQ ID NO: 2;

(v) and SEQ ID NO:2 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO:2, 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.

In certain embodiments, the substitutions recited in (ii) or (v) are conservative substitutions.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 1 and a VL comprising the sequence shown as SEQ ID NO. 2.

In certain embodiments, an antibody or antigen-binding fragment thereof of the invention may be humanized to reduce immunogenicity to a human. Methods for humanizing non-human antibodies are known in the art, and for example, the CDR regions of an antibody or antigen-binding fragment thereof of the invention can be grafted into human framework sequences using methods known in the art.

In certain embodiments, a humanized antibody or antigen-binding fragment thereof of the invention may comprise framework region sequences derived from a human immunoglobulin, which optionally comprise one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) back mutations from human residues to corresponding murine residues.

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

In certain embodiments, the VH of the antibody or antigen-binding fragment thereof comprises: heavy chain framework regions FR1, FR2 and FR3 derived from heavy chain germline sequence IGHV1-3 x 01, and heavy chain framework region FR4 derived from heavy chain germline sequence IGHJ5 x 02; and, the VL of the antibody or antigen-binding fragment thereof comprises: light chain framework regions FR1, FR2 and FR3 derived from the light chain germline sequence IGKV4-1 × 01, and light chain framework region FR4 derived from the light chain germline sequence IGKJ2 × 01. The heavy chain framework 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) back mutations from a human residue to a corresponding murine residue.

(i) SEQ ID NO: 17;

(ii) and SEQ ID NO:17 with one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in (b); or

(iii) And SEQ ID NO:17, has 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;

and

(iv) SEQ ID NOs: 18. 19, or a sequence set forth in any one of seq id nos;

(v) and SEQ ID NOs: 18. 19 with one or more amino acid substitutions, deletions or additions (e.g. 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence set forth in any one of claims; or

(vi) And SEQ ID NOs: 18. 19, 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.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises:

(1) a VH comprising the sequence shown as SEQ ID NO. 17 and a VL comprising the sequence shown as SEQ ID NO. 18; or

(2) A VH comprising the sequence shown as SEQ ID NO. 17 and a VL comprising the sequence shown as SEQ ID NO. 19.

In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect is 36H6 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the monoclonal antibody or antigen-binding fragment thereof from which it is derived.

In certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect has one or more of the following biological functions:

in certain embodiments, the antibody or antigen-binding fragment thereof of the first aspect has one or more of the following characteristics:

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

(2) RBD that does not bind or does not substantially bind to the S protein of SARS-CoV-1;

(3) blocking or inhibiting binding of SARS-CoV-2 to Ace2 receptor, and/or blocking or inhibiting infection of cells by SARS-CoV-2;

(4) does not affect or does not substantially affect the binding of SARS-CoV-1 to the Ace2 receptor;

(5) neutralizing SARS-CoV-2 in vitro or in a subject (e.g., human);

(6) preventing and/or treating SARS-CoV-2 infection or a disease caused by SARS-CoV-2 infection (e.g., COVID-19).

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

(i) a VH CDR1, consisting of the sequence: 11, or a sequence having substitution, deletion or addition of one or several amino acids (e.g.substitution, deletion or addition of 1, 2 or 3 amino acids) thereto,

(ii) a VH CDR2, consisting of the sequence: 12, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(iii) a VH CDR3, consisting of the sequence: 13, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto;

and/or the presence of a gas in the gas,

(iv) a VL CDR1, consisting of the sequence: 14, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids),

(v) a VL CDR2, consisting of the sequence: 15, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto, and

(vi) a VL CDR3, consisting of the sequence: 16, or a sequence having substitution, deletion or addition of one or several amino acids compared thereto (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids).

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: the following 3 heavy chain CDRs defined according to the Kabat numbering system: the sequence is SEQ ID NO:11, VH CDR1 of SEQ ID NO:12, VH CDR2 of SEQ ID NO:13 VH CDR 3; and/or, the following 3 light chain CDRs as defined by the Kabat numbering system: the sequence is SEQ ID NO:14, VL CDR1 of SEQ ID NO:15, the sequence of VL CDR2 of SEQ ID NO:16 VL CDR 3.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises: 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 3; and/or 3 CDRs contained in the light chain variable region (VL) shown in SEQ ID NO: 4. In certain embodiments, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by Kabat, IMGT or Chothia numbering system.

(i) SEQ ID NO: 3;

(ii) and SEQ ID NO:3 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(iii) And SEQ ID NO:3, 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;

and

(iv) SEQ ID NO: 4;

(v) and SEQ ID NO:4 compared to a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions); or

(vi) And SEQ ID NO:4, 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.

In certain exemplary embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 3 and a VL comprising the sequence shown as SEQ ID NO. 4.

In certain embodiments, the antibody or antigen-binding fragment thereof of the second aspect is 2B4 or an antigen-binding fragment thereof, a chimeric antibody thereof, or a humanized antibody thereof, or a variant thereof that substantially retains the biological function of the monoclonal antibody or antigen-binding fragment thereof from which it is derived.

In certain embodiments, the antibody or antigen-binding fragment thereof of the second aspect has one or more of the following biological functions:

in certain embodiments, the antibody or antigen-binding fragment thereof of the second aspect has one or more of the following characteristics:

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

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

(4) blocking or inhibiting binding of SARS-CoV-1 to Ace2 receptor, and/or blocking or inhibiting infection of cells by SARS-CoV-1;

(5) neutralizing SARS-CoV-2 and/or SARS-CoV-1 in vitro or in a subject (e.g., a human);

(6) preventing and/or treating SARS-CoV-2 and/or SARS-CoV-1 infection or a disease caused by SARS-CoV-2 and/or SARS-CoV-1 infection (e.g. COVID-19, SARS).

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

In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it is derived; and/or the presence of a gas in the gas,

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

In some embodiments, the variants of the heavy chain constant region (CH) may have conservative substitutions of one or more amino acids compared to the sequence from which they are derived. In such embodiments, the variants of the heavy chain constant region (CH) may have the same or substantially the same effector function as compared to the wild-type sequence from which they are derived.

In other embodiments, the variant of the heavy chain constant region (CH) may comprise one or more amino acid mutations to alter one or more of the following properties of the antibody of the invention: fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc. A functional change, e.g., an alteration in the affinity of an antibody for an effector ligand (e.g., FcR or complement C1q), can be produced by replacing at least one amino acid residue in the constant region of the antibody with a different residue, thereby altering (e.g., decreasing) effector function. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, and the like.

In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a murine IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the heavy chain constant region is a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

In certain embodiments, the light chain constant region is a kappa light chain constant region. In certain embodiments, the light chain constant region is a murine kappa light chain constant region. In certain embodiments, the light chain constant region is a human kappa light chain constant region.

In certain exemplary embodiments, the antibodies or antigen-binding fragments thereof of the invention comprise 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.

In certain embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab ', (Fab')₂Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb).

In certain embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

Herein, an antibody or antigen-binding fragment thereof according to the first or second aspect of the invention may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-described biological functions of the antibody or antigen-binding fragment thereof from which it is derived.

Preparation of antibodies

The antibody of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.

The antigen-binding fragments of the invention can be obtained by hydrolysis of the whole antibody molecule (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 be produced directly 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, Fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab')₂Fragments (Carter et al, Bio/Technology,10: 163-. In addition, Fv, Fab or F (ab')₂The fragments may also be isolated directly from the culture medium of the recombinant host cell. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.

Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the first or second aspects of the invention, or a heavy chain variable region and/or a light chain variable region thereof. In certain embodiments, the isolated nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention, or a heavy chain variable region and/or a light chain variable region thereof.

In another aspect, the invention provides a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above. In certain embodiments, the vectors of the invention are, for example, plasmids, cosmids, phages and the like.

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

In certain embodiments, the vector comprises a first nucleotide sequence encoding a heavy chain of the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention, and/or a second nucleotide sequence encoding a light chain of the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention; wherein the first nucleotide sequence and the second nucleotide sequence are provided on the same or different vectors.

In another aspect, the invention provides a host cell comprising an isolated nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E.coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). In certain preferred embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DG 44).

In another aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof according to the first or second aspect of the invention, comprising culturing a host cell as described above under conditions which 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.

Pharmaceutical compositions and therapeutic uses

In another aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first or second aspect of the invention, and a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical compositions may further comprise additional pharmaceutically active agents, such as additional antiviral agents (e.g., interferon, lopinavir, ritonavir, chloroquine phosphate, fabiravir, ridciclovir, and the like).

In certain embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention and the additional pharmaceutically active agent may be provided as separate components or as a mixed component. Thus, the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

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

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

In certain embodiments, the methods are used to neutralize the virulence of SARS-CoV-2 in a sample. In certain embodiments, the method comprises: contacting a sample comprising SARS-CoV-2 with an antibody or antigen-binding fragment thereof according to the first aspect of the invention or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

In certain embodiments, the antibody or antigen-binding fragment thereof is used alone, or in combination with another pharmaceutically active agent (e.g., another antiviral agent).

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

In certain embodiments, the antibody or antigen-binding fragment thereof is used alone, or in combination with another pharmaceutically active agent (e.g., another antiviral agent). The antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to the first aspect of the invention, in the manufacture of a medicament for:

(1) neutralizing SARS-CoV-2 in vitro or in a subject (e.g., human); and/or

(2) For preventing and/or treating SARS-CoV-2 infection or a disease associated with SARS-CoV-2 infection (e.g., COVID-19) in a subject.

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

In certain embodiments, the methods are used to neutralize the virulence of SARS-CoV-2 and/or SARS-CoV-1 in a sample. In certain embodiments, the method comprises: contacting a sample comprising SARS-CoV-2 and/or SARS-CoV-1 with an antibody or antigen-binding fragment thereof according to the second aspect of the invention or a pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

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

In another aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to the second aspect of the invention, in the manufacture of a medicament for:

(1) neutralizing SARS-CoV-2 and/or SARS-CoV-1 in vitro or in a subject (e.g., a human); and/or

(2) For preventing and/or treating SARS-CoV-2 and/or SARS-CoV-1 infection or a disease associated with SARS-CoV-2 and/or SARS-CoV-1 infection (e.g., COVID-19, SARS) in a subject.

The antibody or antigen-binding fragment thereof according to the first or second aspect of the present invention, or the pharmaceutical composition of the present invention may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The antibodies or antigen binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the antibody or antigen-binding fragment thereof of the present invention is incorporated in a suitable solvent in the necessary dosage and, optionally, with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, e.g., 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), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof, prior to use.

The antibody or antigen-binding fragment thereof of the invention, or the pharmaceutical composition of the invention, may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, groin, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, the antibody or antigen-binding fragment thereof or pharmaceutical composition of the invention is administered by intravenous injection or bolus injection.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof of the invention. A "prophylactically effective amount" is an amount sufficient to prevent, or delay the onset of disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. A therapeutically effective amount of an antibody or antigen-binding fragment thereof of the invention may vary according to the following factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g. age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, etc.

In this context, the dosage regimen may be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, the dosage may be given in a single dose, may be given multiple times over a period of time, or may be reduced or increased proportionally with the exigencies of the therapeutic situation.

Herein, the subject may be a mammal, e.g. a human.

Conjugates

The antibody or antigen-binding fragment thereof according to the first or second aspect of the invention may be derivatised, e.g. linked to another molecule (e.g. another polypeptide or protein). In general, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to SARS-CoV-2. Thus, the antibodies or antigen-binding fragments thereof of the present invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present invention may 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, for example to increase serum half-life.

Thus, in certain embodiments, the antibody or antigen-binding fragment thereof according to the first or second aspect of the invention is detectably labeled.

In this context, the detectable label according to the invention may be any substance detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g.,³H、¹²⁵I、³⁵S、¹⁴c or³²P), 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., chemo-luminescent materialsOptical substances such as acridine esters, luminol and its derivatives, ruthenium derivatives such as ruthenium terpyridyl), magnetic beads (e.g.,

) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.

In certain embodiments, the detectable label can be suitable for use in immunological detection (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, and the like). In certain embodiments, the detectable label may be selected from an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, or β -galactosidase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein such as FITC, TRITC, or PE), a radionuclide, or biotin.

In certain embodiments, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via a linker of varying length to reduce potential steric hindrance.

Kit and detection application

In another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof according to the first or second aspect of the invention, or a conjugate according to the invention.

In some embodiments, the kit comprises a conjugate of the invention.

In other embodiments, the kit comprises an antibody or antigen-binding fragment thereof according to the first or second aspects of the invention. In certain embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In certain embodiments, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the first or second aspect of the 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., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.

In certain embodiments, the second antibody is specific for an antibody of the species (e.g. murine or human) from which the constant region comprised by the antibody or antigen binding fragment thereof of the first or second aspect of the invention is derived.

In certain embodiments, the second antibody is an anti-immunoglobulin (e.g., human or murine immunoglobulin) antibody, such as an anti-IgG antibody. In certain embodiments, the second antibody is an anti-mouse IgG antibody or an anti-human IgG antibody.

In certain embodiments, the kits of the invention may further comprise reagents for allowing the detection of the corresponding detectable label. For example, when the detectable label is an enzyme, the kit may further comprise a chromogenic substrate for the corresponding enzyme, such as o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), ABTS or luminol-type 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., an acridinium ester compound), the kit may further comprise a pre-excitation liquid and/or an excitation liquid for chemiluminescence.

In another aspect, the invention provides a method of detecting the presence or level of SARS-CoV-2 or S protein thereof or RBD of S protein, or a cell infected with SARS-CoV-2 in a sample comprising using an antibody or antigen-binding fragment thereof according to the first aspect of the invention.

In certain embodiments, the method is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

In some embodiments, the method comprises the use of a conjugate of the invention comprising an antibody or antigen-binding fragment thereof according to the first aspect of the invention.

In other embodiments, the method comprises the use of an antibody or antigen-binding fragment thereof according to the first aspect of the invention. In certain embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In certain embodiments, the methods further comprise detecting the antibody or antigen-binding fragment thereof using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin).

In certain embodiments, the second antibody is specific for an antibody of the species (e.g., murine or human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the first aspect of the invention is derived.

In certain embodiments, the method comprises: (1) contacting the sample with an antibody or antigen-binding fragment thereof according to the first aspect of the invention; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of said immune complex. The formation of the immune complex indicates the presence of SARS-CoV-2 or a cell infected with SARS-CoV-2.

In certain embodiments, the methods can be used for diagnostic purposes, e.g., a subject can be diagnosed as infected with SARS-CoV-2 based on the presence or level of SARS-CoV-2 in the sample. In such embodiments, the sample may be a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

In certain embodiments, the methods may be used for non-diagnostic purposes, e.g., the sample is not a sample from a subject, e.g., a vaccine sample.

In certain embodiments, the subject is a mammal, e.g., a human.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof according to the first aspect of the invention in the preparation of a kit for detecting the presence or level of SARS-CoV-2 or the S protein thereof or the RBD of the S protein, or a cell infected with SARS-CoV-2, in a sample, and/or for diagnosing whether a subject is infected with SARS-CoV-2.

In certain embodiments, the kit detects the presence or level of SARS-CoV-2 or its S protein or the RBD of S protein, or cells infected with SARS-CoV-2 in a sample by a detection method as described above, and optionally diagnoses whether the subject is infected with SARS-CoV-2 based on the detection result.

In certain embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

In another aspect, the invention provides a method of detecting the presence or level of a coronavirus, or its S protein or RBD of S protein, or a cell infected with a coronavirus, in a sample, comprising using an antibody or antigen-binding fragment thereof according to the second aspect of the invention, said coronavirus being selected from SARS-CoV-2 and/or SARS-CoV-1.

In some embodiments, the method comprises the use of a conjugate of the invention comprising an antibody or antigen-binding fragment thereof according to the second aspect of the invention.

In other embodiments, the method comprises the use of an antibody or antigen-binding fragment thereof according to the second aspect of the invention. In certain embodiments, the antibody or antigen-binding fragment thereof does not comprise a detectable label. In certain embodiments, the methods further comprise detecting the antibody or antigen-binding fragment thereof using a second antibody with a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin).

In certain embodiments, the second antibody is specific for an antibody of the species (e.g., murine or human) from which the constant region comprised by the antibody or antigen-binding fragment thereof of the second aspect of the invention is derived.

In certain embodiments, the method comprises: (1) contacting the sample with an antibody or antigen-binding fragment thereof according to the second aspect of the invention; (2) detecting the formation of an antigen-antibody immune complex or detecting the amount of said immune complex. The formation of the immune complex indicates the presence of the coronavirus or a cell infected with the coronavirus.

In certain embodiments, the methods can be used for diagnostic purposes, e.g., whether a subject is infected with the coronavirus can be diagnosed based on its presence or its level in a sample. In such embodiments, the sample may be a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

In certain embodiments, the subject is a mammal, e.g., a human.

In another aspect, there is provided the use of an antibody or antigen-binding fragment thereof according to the first aspect of the invention in the preparation of a kit for detecting the presence or level of an RBD of a coronavirus or S protein thereof, or a cell infected with a coronavirus in a sample, and/or for diagnosing whether a subject is infected with a coronavirus selected from SARS-CoV-2 and/or SARS-CoV-1.

In certain embodiments, the kit detects the presence or level of the coronavirus, or its S protein or the RBD of the S protein, or a cell infected with the coronavirus, in a sample by a detection method as described above, and optionally diagnoses whether the subject is infected with the coronavirus based on the detection result.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the laboratory procedures of virology, biochemistry, nucleic acid chemistry, immunology, etc. used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "novel coronavirus" or "2019-nCov", belongs to its genus β -coronavirus, and is an enveloped, single-stranded, positive-sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Like SARS-CoV, the SARS-CoV-2 Receptor is specifically bound to angiotensin transferase 2(ACE2) on host cells via Receptor Binding Domain (RBD) on S protein, and then is connected to viral membrane fusion and cell entry, and plays a crucial role in the process of viral infection of cells.

As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia resulting from SARS-CoV-2 infection, which have the same meaning and are used interchangeably.

As used herein, "Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), belonging to the genus beta coronavirus thereof, is an envelope-containing single-stranded positive-sense RNA virus. The genomic sequence of SARS-CoV-1 is known to those skilled in the art and can be found, for example, in GenBank: AAP 13567.1. The pneumonia caused by SARS-CoV-1 is called SARS.

As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complementCombination of the first component of the system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each V_HAnd V_LBy the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antigen-binding sites, respectively. The distribution of amino acids in each region or domain may follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia&Lesk (1987) J.mol.biol.196: 901-917; chothia et al (1989) Nature 342: 878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs, named CDR1, CDR2, and CDR3, in the variable regions of the heavy and light chains, respectively. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, for example, as defined in 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.Complex.Immunol.27: 55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al, Dev. company. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies of the present invention or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat, Chothia, or IMGT numbering system. In certain embodiments, the CDRs contained by the antibodies or antigen binding fragments thereof of the present invention are preferably determined by the Kabat numbering system.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch.7(Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes₂Fd, Fv, Complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (single domain antibodies), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technology from Domantis), probodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding capability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol,23: 1126-.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein "full-length heavy chain" refers to a polypeptide chain consisting of, in the N-terminal to C-terminal direction, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "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. Two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric antibodies or humanized antibodies are also possible. The full-length antibody of the present invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, that specifically recognize/bind to the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al, Nature 341: 544546 (1989)); the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab')₂Fragment "means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reductively linked F (ab')₂The fragment obtained after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete Fd fragment of the light and heavy chains, consisting of the VH and CH1 domains.

As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that form an entire antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity on the antibody. However, even one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the entire binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain and the second and third constant regions of a second heavy chain of an antibody. The Fc fragment of an antibody has a number of different functions, but is not involved in antigen binding.

As used hereinAs used herein, The term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein The VL and VH are linked by a linker (linker) (see, e.g., Bird et al, Science 242: 423-. Such scFv molecules can have the general structure: NH (NH)₂-VL-linker-VH-COOH or NH₂-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS)₄But variants thereof can also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in the present invention are described by Alfthan et al (1995), Protein Eng.8: 725-. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the invention, the scFv may form a di-scFv, which refers to two or more individual scFv connected in tandem to form an antibody. In certain embodiments of the invention, the scFv may form a (scFv)₂It refers to two or more individual scfvs connected in parallel to form an antibody.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but that a linker is used that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

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

Each of the above antibody fragments retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or competes with the full length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) 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 antibodies are specifically screened for specificity in the same manner as for intact antibodies.

Herein, when the term "antibody" is referred to, it includes not only intact antibodies, but also antigen-binding fragments of antibodies, unless the context clearly indicates otherwise.

As used herein, the term "Chimeric antibody" (scieric antibody) "refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belonging to a particular antibody class or subclass) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest (u.s.p. 4,816,567to harvesting cam et al.; Morrison et al., proc.natl.acad.sci.usa,81: 68516855 (1984)). In certain embodiments, the term "chimeric antibody" may include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Typically, at least one or two but usually all three acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDRs is referred to as the "donor" and the immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, the donor immunoglobulin is a non-human (e.g., murine) antibody and the acceptor framework may be a naturally occurring human framework or a sequence having about 85%, 90%, 95%, 99% or more identity thereto. Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having a desired property (e.g., antigen specificity, affinity, reactivity, etc.).

The chimeric antibody or humanized antibody of the present invention can be prepared based on the sequence of a monoclonal antibody produced by immunizing an animal (e.g., a mouse). The DNA encoding the heavy and light chains can be obtained from a hybridoma of interest or a specific B cell from an immunized animal and engineered to contain human immunoglobulin sequences using standard molecular biology techniques.

To prepare chimeric antibodies, immunoglobulin variable regions of an immunized animal (e.g., a mouse) can be linked to human immunoglobulin constant regions using methods known in the art (see, e.g., U.S. Pat. No.4,816,567 to Cabilly et al). For example, DNA encoding a VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the Human heavy chain constant region gene is known in the art (see, e.g., 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 comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is typically preferably an IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full-length light chain gene (as well as the Fab light chain gene). The sequence of the Human light chain constant region gene is known in the art (see, e.g., 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 comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is typically preferably a kappa constant region.

To prepare humanized antibodies, CDR regions of an immunized animal (e.g., a mouse) can be grafted into human framework sequences using Methods known in the art (see, U.S. Pat. No.5,225,539 to Winter; U.S. Pat. No.5,530,101 to Queen et al; 5,585,089; 5,693,762 and 6,180,370; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004).

As used herein, the term "germline antibody gene (germline antibody gene)" or "germline antibody gene segment (germline antibody gene segment)" refers to immunoglobulin-encoding sequences present in the genome of an organism that have not undergone a maturation process that can lead to genetic rearrangements and mutations that express specific immunoglobulins. In the present invention, the expression "heavy chain germline gene" means the germline antibody gene or gene segment encoding the immunoglobulin heavy chain, which includes the V gene (variable), the D gene (diversity), the J gene (conjugation), and the C gene (constant); similarly, the expression "light chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin light chains, which include the V gene (variable), the J gene (junction), and the C gene (constant). In the present invention, the amino acid sequence encoded by the germline antibody gene or germline antibody gene segment is also referred to as "germline sequence", the amino acid sequence encoded by the heavy chain germline gene is referred to as heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene is referred to as 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 specialized databases (e.g., IMGT, unsmig, NCBI, or VBASE 2).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction_D) And (4) showing. In the present invention, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Specific binding properties between two molecules can be determined using methods well known in the art, for example in a BIACORE instrument using Surface Plasmon Resonance (SPR).

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, and includes, but is 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 fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percent 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 x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48: 444-.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., 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).

The twenty conventional amino acids referred to herein are written following conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R.Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)) which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations as is well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19th ed. pennsylvania: mach Publishing Company,1995), and include, but are not limited to: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate salts and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, including, but not limited to, sodium 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 degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.

As used herein, the term "preventing" refers to a method performed to prevent or delay the onset of a disease or disorder or symptom (e.g., SARS-CoV-2 infection) in a subject. As used herein, the term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to expected survival (if not treated).

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

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, an amount effective to prevent a disease (e.g., SARS-CoV-2 infection) is an amount sufficient to prevent, or delay the onset of a disease (e.g., SARS-CoV-2 infection); a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

As used herein, the term "neutralizing activity" means that the antibody or antibody fragment has a functional activity of binding to an antigenic protein on the virus, thereby preventing the virus from infecting cells and/or maturation of viral progeny and/or release of viral progeny, and the antibody or antibody fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.

Advantageous effects of the invention

The present invention provides monoclonal antibodies that neutralize SARS-CoV-2. Specifically, these monoclonal antibodies bind to an epitope on the RBD region of the S protein of SARS-CoV-2 and neutralize SARS-CoV-2. The monoclonal antibody of the invention can inhibit the combination of the RBD protein of SARS-CoV-2 and the receptor ACE 2. The monoclonal antibody provided by the invention has the following advantages: (1) monoclonal antibody 36H6 has a strong neutralizing capacity, with a half inhibitory concentration (IC50) of 0.041nM in the neutralization assay of SARS-CoV-2 pseudovirus SARS-CoV 2-LvPP. (2) The monoclonal antibody 2B4 can cross-bind to RBD of SARS-CoV-1 and SARS-CoV-2, and can cross-neutralize SARS-CoV-2 and SARS-CoV-1, and has half-inhibitory concentration of 0.739nM and 0.503nM respectively in neutralization test of pseudoviruses SARS-CoV1-LvPP and SARS-CoV2-LvPP, and broad spectrum property.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.

Drawings

FIG. 1 shows SDS-PAGE electrophoresis of the RBD protein of SARS-CoV-2.

FIG. 2 shows the results of ELISA assays for RBD protein binding activity of mAbs 36H6 and 2B4 against SARS-CoV-2 (FIG. 2A), SARS-CoV-1 (FIG. 2B) and RaTG13-CoV (FIG. 2C).

FIG. 3 shows the results of affinity assay (Biacore) of mAbs 36H6 and 2B4 for the RBD proteins of SARS-CoV-2, SARS-CoV-1 and RaTG 13-CoV.

FIG. 4 shows the test of the ability of SARS-CoV1-LvPP pseudovirus at different doses to infect H1299ACE2hR cells.

FIG. 5 shows the test of the ability of SARS-CoV2-LvPP pseudovirus at different doses to infect H1299ACE2hR cells.

FIG. 6 shows the dose-response relationship between mAbs 2B4 and 36H6 for infection Neutralization (NAT) of SARS-CoV1-LvPP (FIG. 6A) and SARS-CoV2-LvPP (FIG. 6B).

FIG. 7 shows the results of the determination of neutralizing activity of mAbs 2B4 and 36H6 in a model of SARS-CoV2VSVPp pseudovirus infection.

FIG. 8 shows the analysis of the ability of Fab fragments of mAbs 2B4 and 36H6 to block SARS-CoV-2RBD binding to ACE 2. Wherein, FIG. 8A is mAb 2B 4; FIG. 8B shows mAb 36H 6; figure 8C is an irrelevant control antibody.

FIG. 9 shows the results of ELISA assays for RBD-His binding activity of humanized antibodies 36H6-10 and 36H 6-12.

FIG. 10 shows the results of the determination of the neutralizing activity of humanized antibodies 36H6-10 and 36H6-12 in a SARS-CoV2VSVPp pseudovirus infection model.

Sequence information

Information on the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequences

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1: synthesis of anti-SARS-CoV-2 receptor binding region RBD gene and construction of expression vector

The nucleotide sequence encoding the RBD protein of SARS-CoV2-2 receptor binding domain (corresponding to amino acid 316-550 of the S protein) was optimized according to human codon preference with reference to the SARS-CoV2-2 complete gene sequence (MN 908947.3). And a signal peptide coding sequence (a leader sequence of human B2M) is connected to the N end of the optimized nucleic acid sequence, and a polyhistidine polypeptide (6 XHis) which is convenient for affinity chromatography purification is connected to the C end, the protein is named as RBD-His, the nucleotide sequence of the protein is shown as SEQ ID NO:22, and the amino acid sequence of the protein is shown as SEQ ID NO: 23. The nucleotide sequence is connected to an expression vector EIRBsMie-C18hA2dtSCT (enzyme cutting site AgeI/BglII) by adopting NEBuilder HiFi DNA Assembly Master Mix (NEB company), and the sequence is proved to be completely consistent with the design through DNA sequencing, so that the expression vector EIRBsMie-RBDhis of the RBDhis of SARS-CoV-2 is finally obtained.

Example 2: expression and purification of RBD antigen of SARS-CoV-2

Expression of the RBD antigen of SARS-CoV-2

At 3x10⁶The density of ExpicHO cells in appropriate amount of culture Medium ExpicHO^TMExpression Medium (Thermo Scientific Co.) was cultured in a triangular flask at 37 ℃ with 8% CO₂Culturing in constant temperature shaking table at proper rotation speed for 24 hr until cell density reaches 6x10⁶The density of (c).

Expifeacmine was used according to the kit instructions^TMThe vector EIRBsMie-RBDhis obtained in example 1 was transfected into ExpicHO cells by CHO Transfection Kit (Thermo Scientific Co.). After further culturing for 17-24h under the same conditions, the supplement and enhancer provided in the kit were added and the cells were replaced to 32 ℃ with 5% CO₂And continuously culturing for 6 days in a constant-temperature shaking table with proper rotating speed.

Purification of the RBD antigen of SARS-CoV-2

After 6 days of culture, the expichho-expressing cell suspension was collected, centrifuged at 12000rpm for 30min at room temperature, the supernatant was left and dialyzed against PBS, and then filtered through a 0.22 μm filter.

The supernatant sample dialyzed into PBS was purified by medium pressure Ni-excel chromatography (GE medium), 30mM imidazole removed impure protein, 250mM imidazole eluted target protein. The target protein is the RBD antigen of SARS-CoV-2 and is named as SARS-CoV-2 RBD. SDS-PAGE shows that the purity of SARS-CoV-2RBD is more than 90% (FIG. 1). The obtained target protein was dialyzed into PBS buffer and stored at-20 ℃.

Example 3: obtaining murine monoclonal antibodies

1. Immunization of mice

Using standard in vivo immunization protocols, detailed procedures are described in Ed Harlow et al, "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988, brief procedures are as follows:

the SARS-CoV-2RBD protein obtained in example 2 was used and mixed in equal volumes with Freund's adjuvant and emulsified. BALB/c female mice 6-8 weeks old were immunized by bilateral inguinal subcutaneous multi-point injections, and boosted once at 2-week intervals after primary immunization. Serum antibody titers were determined by indirect ELISA and fusion experiments were performed after 4 weeks.

The final spleen booster immunization was performed 72 hours before the fusion of mouse spleen cells and mouse myeloma cells (SP2/0), and the antigen of this immunization was an adjuvant-free antigen and was diluted to 1 mg/mL. 50 μ L of protein was injected longitudinally along the spleen. Meanwhile, mouse myeloma cells (SP2/0) were recovered and cultured to logarithmic growth phase in RPMI1640 medium containing 10% fetal bovine serum, and used for fusion.

2. Preparation and screening of hybridoma cells

Taking the spleen to boost the mice 72 hours later, taking the spleen to prepare a cell suspension, and fusing the cell suspension with a mouse myeloma cell SP2/0 to obtain a hybridoma cell. And preparing feeder cells for co-culture with the hybridoma cells, wherein mouse abdominal cavity macrophages and small-week-old mouse thymocytes are used as the feeder cells in the laboratory.

Screening hybridoma cells by indirect ELISA, coating 50 ng/well of SARS-CoV-2RBD protein in example 2, sealing, adding 60 μ L fusion cell culture supernatant, reacting at 37 deg.C for 1 hr, adding horseradish peroxidase-labeled secondary goat-anti-mouse antibody (GAM-HRP), reacting at 37 deg.C for 30min, developing, and selecting positive clone wells.

5 BALB/C mice were subjected to intraperitoneal injection to obtain ascites. Two purified mouse monoclonal antibodies (Table 2) were obtained, designated 36H6 (also designated M36H6) and 2B4 (also designated M2B4), respectively, by subjecting the supernatant obtained by high-speed centrifugation to high-speed centrifugation, adding an equal volume of saturated ammonium sulfate solution, precipitating the supernatant on ice for 30min, then centrifuging the supernatant at 25000rpm for 10min, dissolving the precipitate in 0.2M disodium phosphate dodecahydrate buffer solution, and then purifying the precipitate with a Protein A affinity chromatography column (purchased from GE, USA).

TABLE 2 monoclonal antibody information

Example 4: obtaining murine monoclonal antibody sequences

36H6 and 2B4 hybridoma cells cultured to the logarithmic growth phase were RNA-extracted by Trizol method (Invitrogen), and the final extracted RNA pellet was dissolved in 50. mu.l DEPC water. Then, reverse transcription PCR is carried out on the heavy chain variable region and the light chain variable region respectively, and sequencing is carried out after PCR products are recovered. The sequences are blast compared to determine the heavy chain variable region sequence and the light chain variable region sequence of the antibody. The amino acid sequences of the variable regions of 36H6 and 2B4 that were finally determined are shown in tables 1 and 2.

Further, the CDR Sequences of mouse monoclonal antibodies 36H6 and 2B4 were also determined using the method described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, national institutes of Health, Besserda, Maryland (1991), p.647-669). The amino acid sequences of the CDRs of the variable regions of 36H6 and 2B4 that were finally determined are shown in tables 1 and 3.

TABLE 3 sequences of antibodies 36H6 and 2B4

Table 3: variable region sequence of monoclonal antibody

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

With reference to the viral genome sequences SARS-CoV-1(AAP13567.1) and RaTG13(MN996532.1) which have been published on Genebank, respectively, the RBD sequences of the above 2 coronaviruses were constructed in the manner described in reference to example 1, and recombinant proteins were prepared by the method described in reference to example 2. Wherein, the RBD recombinant protein of SARS-CoV-1 is named as SARS-CoV1-RBD, and the RBD recombinant protein of RaTG13-CoV is named as RaTG 13-RBD.

The SARS-CoV2-RBD, SARS-CoV1-RBD and RaTG13-RBD proteins obtained in example 2 and above were treated with 50mM CB buffer (NaHCO) pH9.6₃/Na₂CO₃Buffer, final concentration 50mM, pH 9.6) diluted to a final concentration of 2. mu.g/mL. Adding 100 mu L of coating solution into each hole of a 96-hole enzyme label plate, coating for 16-24 hours at 2-8 ℃, and then coating for 2 hours at 37 ℃. Washing with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20) 1 time, then adding 200. mu.L of blocking solution (20mM Na pH 7.4 containing 20% calf serum and 1% casein) per well₂HPO₄/NaH₂PO₄Buffer solution), sealing at 37 deg.C for 2 hr; the blocking solution was discarded. Drying, and packaging in aluminum foil bag at 2-8 deg.C.

The mouse monoclonal antibodies 36H6 and 2B4 obtained in example 3 were diluted to 10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL, 0.01. mu.g/mL in a diluted gradient of 20% newborn bovine serum in PBS and subjected to ELISA detection as follows:

(1) sample reaction: a100. mu.L of diluted sample was added to each well of an ELISA plate coated with SARS-CoV2-RBD, SARS-CoV1-RBG and RaTG13-RBG proteins, respectively, and the mixture was reacted at 37 ℃ for 30 minutes.

(2) Enzyme label reaction: after completion of the sample reaction step, the microplate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), and 100. mu.L of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (GAM) reaction solution was added to each well, and the mixture was placed in an incubator at 37 ℃ for 30 minutes.

(3) And (3) color development reaction: after completion of the enzyme-labeled substance reaction step, the plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 50. mu.L each of TMB color developing agents (purchased from Beijing Wantai Bio-pharmaceuticals Co., Ltd.) was added to each well, and the plate was left to react in an incubator at 37 ℃ for 15 minutes.

(4) Termination of the reaction and measurement of the reading: after the color reaction step was completed, 50. mu.L of stop solution (purchased from Beijing Wantai biological pharmaceuticals Co., Ltd.) was added to each well of the reacted microplate, and the OD450/630 value of each well was measured on a microplate reader. Determination of reactivity of mouse monoclonal antibodies 36H6 and 2B4 with SARS-CoV2-RBD, SARS-CoV1-RBG and RaTG 13-RBG: the determination was made based on the reading after the reaction. If the detection value/background value is greater than 5, the test result is determined to be positive.

And (4) analyzing results: as shown in FIG. 2, 36H6 showed strong binding activity to SARS-CoV2-RBD and no binding activity to SARS-CoV1-RBD and RaTG13-RBD at concentrations of 10. mu.g/mL, 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL. 2B4 has strong binding activity to SARS-CoV2-RBD and SARS-CoV1-RBD and no binding activity to RaTG13-RBD when the concentration is 10 mug/mL, 1 mug/mL, 0.1 mug/mL and 0.01 mug/mL. The results show that 36H6 can specifically recognize the RBD of SARS-CoV-2, while 2B4 can cross-recognize the RBD of SARS-CoV-2 and SARS-CoV-1, and has a certain broad spectrum.

Example 6: detection of affinity constants of monoclonal antibodies 2B4 and 36H6 for receptor binding region protein RBD of SARS-CoV-2, SARS-CoV-1 and RaTG13

This study used Surface Plasmon Resonance (SPR) technology to detect the affinity of mAbs 2B4 and 36H6 to the RBD proteins of SARS-CoV-2, SARS-CoV-1 and RaTG13, respectively. The detection method used in this example was a capture method in which a mouse monoclonal antibody was captured using a Protein G chip (GE). And calculating by using a ligand coupling level calculation formula according to the molecular weight of the mouse monoclonal antibody and the molecular weights of the three RBD analytes to obtain a ligand response value of about 1000 RU. Two antibodies, 2B4 and 36H6, were diluted with PBS to appropriate concentrations, wherein 2B4 was 50. mu.g/mL and 36H6 was 40. mu.g/mL, so that the response value of the binding to the Protein G chip was stabilized at about 1000 RU. The RBD proteins of SARS-CoV-2, SARS-CoV-1 and RaTG13 were diluted 2-fold in order from an initial concentration of 200nM (11 dilution gradients were tested, the highest concentration tested was 200nM and the lowest concentration tested was 0.19nM), respectively, and the affinity of antigen and antibody was determined using a surface plasmon resonance detector Biacore 8000(GE corporation).

As shown in FIG. 3, 2B4 has the highest affinity for the RBD protein of SARS-CoV-2 and the equilibrium dissociation constant (K)_D) 3.05nM (FIG. 3A), 2B4 also has some degree of affinity for the RBD protein of SARS-CoV-1 and RaTG13, equilibrium dissociation constant (K)_D) 34.1nM (FIG. 3B) and 67nM (FIG. 3C). 36H6 has better affinity only for RBD protein of SARS-CoV-2, equilibrium dissociation constant (K)_D) It was 5.84nM (FIG. 3D). Therefore, the monoclonal antibody 36H6 is an antibody which is specific to the RBD of SARS-CoV-2, and 2B4 has certain cross-binding activity to other two coronavirus RBDs.

Example 7: pseudovirus neutralization assay for SARS-CoV-1 and SARS-CoV-2 with monoclonal antibodies 2B4 and 36H6

1. Pseudovirus packaging cell preparation

The 293T cells were recovered and cultured to logarithmic growth phase in DMEM medium containing 10% fetal bovine serum at 7X 10⁶(iii) 293T cells were seeded on 10cm cell culture plates in 5% CO₂The cells were cultured in a cell incubator at 37 ℃ for 12h and transfected when the cells reached 95-99% confluence.

Lipofectamine was used according to the kit instructions^TM3000(ThermoFisher Scientific Co.) plasmid psPAX2 (lentivirus packaging plasmid), pLvEF1 α mNGNNL (lentivirus shuttle plasmid carrying green fluorescent reporter gene) and pCMV-SARS1-S (full length Spike protein expressing SARS-CoV-1) were co-transfected into 293T cells to package SARS-CoV-1 pseudovirus (abbreviated SARS-CoV 1-LvPP); the plasmids psPAX2, pLvEF1 alpha mNGNL and EIRBsMie-SARS2-SFL were co-transfected into 293T cells to package SARS-CoV-2 pseudovirus (SARS-CoV 2-LvPP for short). After 6 hours of transfection, the packaging medium was removed and replaced. After 24 hours of transfection, pseudoviruses were collected for the first time, and the whole cell supernatant was collected and stored at 4 ℃. And replaced with preheated fresh medium at 37 deg.C with 5% CO₂The cell culture box was cultured for 24 hours. I.e., 48 hours after transfection, pseudovirus was collected for a second time and the whole cell supernatant was collected and mixed with the supernatant collected for the first time. 20Centrifuge at 00rpm for 30 minutes and filter the supernatant through a 0.45 μm pore size filter. Viral titers were determined using a H1299 cell line overexpressing human ACE2 (H1299ACE2hR), split and stored at-80 ℃. The two pseudovirus infectious titer determination methods were as follows:

(1) h1299ACE2hR cells in logarithmic growth phase at 1.2X 10⁴The cells were seeded at a density of 100. mu.L/well in 96-well plates at 37 ℃ with 5% CO₂The cells were cultured in a cell incubator overnight.

(2) Two pseudoviral supernatants, SARS-CoV1-LvPP and SARS-CoV2-LvPP, were added to each well at 30. mu.L infected cells. Viral infections of different titers were performed according to the conditions of serial 3-fold dilutions, with 5 dilution gradients, one-fold (30. mu.L), 3-fold (10. mu.L), 9-fold (3.3. mu.L), 27-fold (1.1. mu.L) and 81-fold (0.37. mu.L) dilutions.

(3) After 36-48 hours of infection of H1299ACE2hR cells, fluorescence imaging (20X water immersion lens, 25 field of view) was performed on infected cells using a high content imaging system based on disc confocal (Opera phenix or Operetta CLS, available from Perkinelmer). After the detection is finished, quantitative analysis is carried out on the obtained fluorescence image by adopting Columbus image management analysis software to detect the number of green fluorescence (mNeonGreen) positive cells. Results of gradient dilution infection of H1299ACE2hR cells with two pseudoviruses are shown in fig. 4 and 5. The result proves that the obtained pseudoviruses SARS-CoV1-LvPP and SARS-CoV2-LvPP can efficiently infect H1299-hAce2 cells, and the number of green fluorescence positive cells and the percentage of green fluorescence positive cells are linearly related to the amount of the infected virus.

(4) Through linear regression analysis, the initial pore infection titer of SARS-CoV1-LvPP can be measured to be 1.4 multiplied by 10 by combining the number of cells which can be lightened after infection with different doses of pseudoviruses⁵TU/mL (pseudovirus original concentration 4.67X 10)⁵TU/mL), while SARS-CoV2-LvPP pseudovirus primary pore infection was 3.03X 10⁵TU/mL (pseudovirus original concentration 1.01X 10)⁶TU/mL)。

2. Pseudovirus neutralizing ability of monoclonal antibodies 2B4 and 36H6 against SARS-CoV-1 and SARS-CoV-2

To test whether the two monoclonal antibodies developed in accordance with the present invention could neutralize SARS-CoV-1 and SARS-CoV-2 in vitro, we used two pseudoviruses SARS-CoV1-LvPP and SARS-CoV2-LvPP to perform the test on H1299ACE2hR cells. The specific test method is as follows:

(2) The two antibodies were diluted in DMEM medium containing 10% fetal bovine serum at different concentrations, up to 1000nM, in 2-fold serial dilutions.

(3) 60 μ L of antibody dilutions at different concentrations were added to 1.0X 10⁵Equal amounts of SARS-CoV1-LvPP and SARS-CoV1-LvPP were mixed and incubated at 37 ℃ for 1 hour to allow sufficient binding of antibody to pseudovirus.

(4) Adding 100 μ L of the virus antibody incubation solution obtained in the previous step into the well-paved H1299ACE2hR cells, and culturing at 37 deg.C with 5% CO₂Infection was cultured in a cell incubator.

(5) After 36-48 hours of infection, the infected cells were fluorescence imaged (20 x water immersion lens, 25 field of view) using a high content imaging system based on rotary disc confocal (Opera phenix or Operetta CLS, available from Perkinelmer). After the fluorescent image is obtained, quantitative analysis is carried out on the obtained fluorescent image by adopting Columbus image management analysis software to calculate the number of green fluorescent protein (mNeonGreen) expression positive cells in each cell hole.

(6) The average number of positive cells in the infected control wells to which no antibody was added was compared, and the infection inhibition rate per well was calculated. The calculation formula is as follows: (number of green fluorescence positive cells in positive control well-number of green fluorescence positive cells in test well)/number of green fluorescence positive cells in positive control well x 100%. After the inhibition rates of the two antibodies under different dosage conditions are calculated, an inhibition curve is drawn by adopting Graphpad Prism 8 software, and the half maximum inhibition concentration (IC50) is calculated by adopting a 4-parameter curve fitting model.

As shown in FIG. 6, both mAbs 2B4 and 36H6 have strong neutralizing activity against SARS-CoV-2, wherein 36H6 is stronger than 2B4, and IC50 reaches 0.041nM, but the antibody has no neutralizing effect against SARS-CoV-1; the monoclonal antibody 2B4 has strong neutralizing activity to SARS-CoV-1 and SARS-CoV-2, and the antibody has IC50 of 0.739nM and 0.503nM to two viruses. The above results demonstrate that 36H6 is a highly neutralizing antibody specific for SARS-CoV-2, while 2B4 is a neutralizing antibody universal for SARS-CoV-1/2.

Example 8: analysis of neutralizing ability of monoclonal antibodies 2B4 and 36H6 and SARS-CoV2VSVPp pseudovirus

SARS-CoV2-VSVPp pseudovirus neutralization reference methods were performed (doi: https:// doi.org/10.1101/2020.04.08.026948). The main experimental process is briefly described as follows, in order to construct VSV pseudovirus carrying SARS-CoV-2spike protein, the spike gene of SARS-CoV-2 (sequence source GenBank: MN908947) with 18 amino acids truncated at C-terminal of the spike gene of SARS-CoV-2 is cloned into eukaryotic expression vector pCAG to obtain pCAG-nCoVSde 18. Plasmid pCAG-nCoVSde18 was transfected into Vero-E6. 48 hours after transfection, VSVDG-EGFP-G (Addgene, 31842) virus was inoculated into cells expressing the truncated protein of SARS-CoV-2Sde18 and incubated for 1 hour. The supernatant was then removed of VSVdG-EGFP-G virus and anti-VSV-G rat serum was added to block infection by residual VSVdG-EGFP-G. The progeny virus will carry the truncated protein of SARS-CoV-2Sde18 to obtain the pseudovirus VSV-SARS-CoV2 VSVPp. After 24 hours post-infection with VSVDG-EGFP-G, cell supernatants were collected, centrifuged and filtered (0.45- μm pore size, Millipore, SLHP033RB) to remove cell debris, and stored at-80 ℃ until use. The supernatant, diluted by a gradient, was infected with BHK21 cell line BHK21-hACE2 overexpressing human ACE 2. Viral titers were determined by the number of GFP positive cells after infection.

Antibodies 36H6 and 2B4 were diluted to 106.4nM as 1, 2-fold down gradient dilutions for a total of 16 gradients, mixed with diluted SARS-CoV2VSVpp virus (MOI ═ 0.05) and incubated at 37 ℃ for 1H. All samples and viruses were diluted with 10% FBS-DMEM. 80 μ L of the mixture was added to pre-plated BHK21-hACE2 cells. After 12 hours of incubation, the infected cells were fluorescence imaged using a high content imaging system based on confocal rotating disk (Opera phenix or Operetta CLS, available from Perkinelmer). And after the detection is finished, quantitative analysis is carried out on the obtained fluorescence image by adopting Columbus image management analysis software to detect the number of green fluorescence positive cells. The reduction (%) in the number of GFP-positive cells in the antibody-treated group compared to the untreated control well was calculated, and the inhibition rate was calculated.

IC50 of the antibody was calculated using non-linear regression analysis. The results of inhibition of SARS-CoV2VSVPp virus by antibodies m36H6 and m2B4 are shown in FIG. 7, with IC50 at 0.021nM and 0.893nM, respectively.

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

To assess whether monoclonal antibodies 2B4 and 36H6 blocked the binding of the SARS-CoV-2spike protein to the ACE2 receptor in vitro, we performed an analysis using Biacore 8000(GE corporation). The method comprises the following steps:

(1) 2B4, 36H6 and an irrelevant control antibody (control mAb) were digested with papain (from Sigma), and the samples were chromatographed using MabSelect Sure, with successfully digested Fab fragments not binding to the MabSelect Sure media, while the remaining antibody and Fc fragments that were not digested were captured and removed by the MabSelect Sure media. The sample was chromatographed through MabSelect SuRe, dialyzed to 20mM PB7.4, and concentrated using a Millipore 10kd concentration tube, and the sample was ready for use after concentration.

(2) The ACE2 recombinant Protein (ACE2-Ig) fused to the Fc fragment of IgG antibody at the C-terminal was diluted to an appropriate concentration and ACE2-Ig was captured using Protein A chip (GE Co.).

(3) The SARS-CoV-2RBDhis antigen prepared in example 2 was diluted to 20mM PB7.4 at a concentration of 200nM and incubated for 60 min after mixing in equal volumes with samples of different concentrations of 2B4, 36H6, control antibody Fab (800nM, 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM), respectively.

(4) The series of protein samples prepared in step (3) were subjected to Biacore testing to obtain respective binding and dissociation kinetic data, and the graph is shown in fig. 8.

As shown in FIG. 8, the Fab of 2B4 and 36H6 can directly block the combination of SARS-CoV-2RBD and ACE2, wherein the 36H6 Fab has strong blocking effect, can show obvious blocking effect under the condition that the molar concentration ratio to RBD is 1:2, and can almost completely block under the condition that the molar concentration ratio to RBD is 1:1 or higher; meanwhile, Fab of the 2B4 antibody shows a remarkable blocking effect under the condition that the molar concentration ratio of the Fab to RBD is 1:1, and complete blocking can be realized under the condition of higher molar ratio. In contrast, the Fab fragment of the control antibody exhibited no significant blocking effect even at high concentrations. The above results demonstrate that antibodies 2B4 and 36H6 directly block the binding of SARS-CoV-2RBD to ACE2, which may be responsible for their high neutralizing activity.

Example 10: preparation of humanized antibody of 36H6

The gene database was searched by IMGT to find the sequence of the variable region of the human germline gene having the highest homology with the FR region of the murine antibody 36H 6. Through homology analysis, the sequences of IGHV1-3 x 01 and IGKV4-1 x 01 embryonic genes are determined to be used as heavy chain and light chain modification templates of the humanized antibody respectively, meanwhile, because the embryonic genes do not contain FR4 required by a modification part, the FR4 part needs to be aligned separately, and finally, the sequences of IGHJ5 x 02 and IGKJ2 x 01 embryonic genes are determined to be used as the modification templates of the heavy chain and light chain FR4 of the humanized antibody respectively. The heavy chain and light chain CDR regions of the mouse antibody 36H6 are respectively transplanted to FR frameworks of humanized templates vH and vK, simultaneously, the sequence alignment of the mouse antibody and the FR regions of embryonic genes is carried out, the selective back mutation is carried out on the different amino acids, and 1 humanized heavy chain and 2 humanized light chains are finally designed, and 2 humanized antibodies are combined, wherein the variable region sequences are shown in the following table:

the 36H6 humanized antibody light and heavy chain variable region genes were obtained by overlapping extension PCR (SOE-PCR). The amplified products were analyzed by agarose gel electrophoresis, and the purified PCR products were recovered with a DNA purification recovery kit (Tianggen, DP 118-02). The heavy chain variable region fragment of the murine antibody was constructed into a PTT5-H vector (restriction enzyme sites AgeI/SalI) containing the coding sequence of the human heavy chain constant region (SEQ ID NO:20) and the light chain variable region fragment was constructed into a PTT5-K vector (restriction enzyme sites AgeI/BsiWI) containing the coding sequence of the human light chain constant region (SEQ ID NO:21) by the Gibson assembly method. The recombinant vector was transformed into DH 5. alpha. competent cells (Shenzhen concatamer), and after 12h of growth on ampicillin resistant LB plates, single clones were picked and sequenced (Shanghai Production). Recombinant plasmids with correct sequencing were extracted in large quantities using an endotoxin-free plasmid macroextraction kit (Tianggen, DP 117). The humanized antibodies of H36H6-10 and H36H6-12 are obtained after expression and purification.

Example 11: expression and purification of H36H6-10 and H36H6-12 humanized antibodies

11.136H 6 humanized antibodies eukaryotic expression

Transient transfection of Expi-293F cells with dual plasmids expressed the H36H6-10 and H36H6-12 humanized antibodies. Preparing Expi-293F cells with a viability rate higher than 95% at 4X 10⁶200ml of the suspension was inoculated into a 1L cell culture flask. Taking 0.5mg of PTT5-36H6-H3 recombinant plasmid and 0.5mg of PTT5-36H6-K1 recombinant plasmid as light-heavy chain plasmids for expressing H36H 6-10; 0.5mg of PTT5-36H6-H3 recombinant plasmid and 0.5mg of PTT5-36H6-K1 recombinant plasmid are used as light and heavy chain plasmids for expressing H36H 6-10. The mixed 1mg of light and heavy chain plasmid and 2mg of PEI are mixed, vigorously shaken for 8s and then kept stand for 8min, and the mixture is added into 200ml of cells. After 4h, 200mL Freestyle medium was supplemented and placed in 5% CO₂The expression was carried out in an incubator at 37 ℃ for 7 days. The cell supernatant was collected, centrifuged at 10000rpm for 30min, and the supernatant was collected and subjected to subsequent purification.

11.236 purification and preparation of H6 humanized antibody:

filtering the cell supernatant with a 0.22 μm filter; opening the AKTA instrument, washing the pipeline A and the pipeline B with solution A (200mM disodium hydrogen phosphate dodecahydrate) and solution B (100mM citric acid monohydrate), respectively, and installing a protein A column; balancing protein A column with solution A at a flow rate of 8mL/min for more than 15min, and performing the next step after the UV value, pH value and conductivity detected by the instrument are stable; the sample is loaded at a flow rate of 6-10mL/min, then the UV value will rise, the peak is the breakthrough peak, and the column is washed with liquid A continuously while collecting the breakthrough peak sample for detection. Feeding liquid B at the flow rate of 6-10mL/min after the pH value is not changed, then lowering the pH value, raising the UV value, taking the peak as an elution peak, mainly containing the antibody in the elution peak, and collecting the elution peak sample to be detected; equilibrating the column with solution A, filling the tube and protein A column with 20% ethanol, removing the column, and storing at 4 deg.C. The purified antibody was dialyzed overnight against 20mM PBS buffer and dispensed into 1.5mL tubes using UV spectroscopy or BCA measurements and stored at-20 ℃ until needed.

Example 12: ELISA binding Activity of H36H6-10 and H36H6-12 humanized antibodies against SARS-CoV-2

12.1 preparation of reaction plates

Diluting SARS-CoV2-RBD (His-tagged) protein with 50mM CB buffer (NaHCO3/Na2CO3 buffer, final concentration 50mM, pH 9.6) pH9.6 to a final concentration of 2 μ g/mL; adding 100 mu L of coating solution into each hole of a 96-hole enzyme label plate, coating for 16-24 hours at 2-8 ℃, and then coating for 2 hours at 37 ℃; wash 1 time with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween 20); then 200. mu.L of a blocking solution (20mM Na2HPO4/NaH2PO4 buffer solution containing 20% calf serum and 1% casein and having a pH of 7.4) was added to each well, and the mixture was blocked at 37 ℃ for 2 hours; the blocking solution was discarded. Drying, and packaging in aluminum foil bag at 2-8 deg.C.

12.236 ELISA detection of H6 humanized antibodies

Humanized antibodies H36H6-10 and H36H6-12 obtained in example 11 were taken, and diluted in a gradient of SD-1 starting from 10ug/mL for a total of 7 gradients. A diluted sample (100. mu.L) was added to each well of an ELISA plate coated with SARS-CoV2-RBD protein, and the mixture was reacted at 37 ℃ for 60 minutes in an incubator. The plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 100. mu.L of HRP-labeled goat anti-human IgG reaction solution was added to each well, and the mixture was incubated at 37 ℃ for 30 minutes. After completion of the enzyme-labeled substance reaction step, the plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl, 0.1% Tween20), 50. mu.L each of TMB color developing agents (purchased from Beijing Wantai Bio-pharmaceuticals Co., Ltd.) was added to each well, and the plate was left to react in an incubator at 37 ℃ for 15 minutes. After the color reaction step was completed, 50. mu.L of stop solution (purchased from Beijing Wantai biological pharmaceuticals Co., Ltd.) was added to each well of the reacted microplate, and the OD450/630 value of each well was measured on a microplate reader. Determination of reactivity of the humanized antibody 36H6 with SARS-CoV 2-RBD: the determination was made based on the reading after the reaction. If the detection value/background value is greater than 5, the test result is determined to be positive.

As shown in FIG. 9, the two humanized antibodies of 36H6 showed strong binding activity to SARS-CoV2-RBD at concentrations of 10. mu.g/mL, 3.33. mu.g/mL, 1.11. mu.g/mL, 0.37. mu.g/mL and 0.12. mu.g/mL. The results show that the H36H6-10 and H36H6-12 after the humanized modification can keep stronger binding activity with SARS-CoV-2 RBD.

Example 13: determination of neutralizing Activity of H36H6-10 and H36H6-12 humanized antibodies in SARS-CoV2VSVPp pseudovirus infection model

The neutralizing activities of humanized antibodies H36H6-10 and H36H6-12, chimeric antibody 36H6-Cab (whose heavy and light chains comprise the heavy chain constant region shown in SEQ ID NO:21 and the light chain constant region shown in SEQ ID NO:22, respectively), and parent murine mAb 36H6(mAb-36H6) were determined in a pseudoviral infection model by the detection method described in example 8, respectively.

The test antibodies were diluted to 1.333nM as 1, 3-fold downward gradient dilutions for a total of 6 gradients, mixed with diluted SARS-CoV2VSVpp virus (MOI ═ 0.05) and incubated at 37 ℃ for 1 h. All samples and viruses were diluted with 10% FBS-DMEM. 80 μ L of the mixture was added to pre-plated BHK21-hACE2 cells. After 12 hours of incubation, the infected cells were fluorescence imaged using a high content imaging system based on confocal rotating disk (Opera phenix or Operetta CLS, available from Perkinelmer). And after the detection is finished, quantitative analysis is carried out on the obtained fluorescence image by adopting Columbus image management analysis software to detect the number of green fluorescence positive cells. The reduction (%) in the number of GFP-positive cells in the antibody-treated group compared to the untreated control well was calculated, and the inhibition rate was calculated. IC50 of the antibody was calculated using non-linear regression analysis.

As shown in FIG. 10, both of the humanized antibodies 36H6 showed strong neutralizing activity against SARS-CoV-2. Among them, murine mAb 36H6 had an IC50 of 0.017nM (comparable to the results obtained in example 8), humanized antibody H36H6-10 had an IC50 of 0.026nM, whereas H36H6-12 was even stronger than H36H6-10, with an IC50 of 0.016 nM. The above results indicate that the neutralizing activity of the two humanized antibodies 36H6 is not weaker than that of the parent murine mAb 36H 6.

While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

SEQUENCE LISTING

<110> health preserving house Co., Ltd; xiamen university

<120> antibody against SARS-CoV-1 or SARS-CoV-2 and use thereof

<130> IDC210043

<150> 202010369877.3

<151> 2020-04-30

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35 40 45

Gly Gly Thr Cys Thr Gly Thr Ala Cys Gly Cys Cys Ala Gly Cys Ala

50 55 60

Ala Cys Thr Thr Cys Cys Gly Cys Gly Thr Gly Cys Ala Gly Cys Cys

65 70 75 80

Cys Ala Cys Cys Gly Ala Gly Ala Gly Cys Ala Thr Cys Gly Thr Gly

85 90 95

Cys Gly Cys Thr Thr Cys Cys Cys Cys Ala Ala Cys Ala Thr Cys Ala

100 105 110

Cys Cys Ala Ala Cys Cys Thr Gly Thr Gly Cys Cys Cys Cys Thr Thr

115 120 125

Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Thr Thr Cys Ala Ala Cys

130 135 140

Gly Cys Cys Ala Cys Cys Cys Gly Cys Thr Thr Cys Gly Cys Cys Ala

145 150 155 160

Gly Cys Gly Thr Gly Thr Ala Cys Gly Cys Cys Thr Gly Gly Ala Ala

165 170 175

Cys Cys Gly Cys Ala Ala Gly Cys Gly Cys Ala Thr Cys Ala Gly Cys

180 185 190

Ala Ala Cys Thr Gly Cys Gly Thr Gly Gly Cys Cys Gly Ala Cys Thr

195 200 205

Ala Cys Ala Gly Cys Gly Thr Gly Cys Thr Gly Thr Ala Cys Ala Ala

210 215 220

Cys Ala Gly Cys Gly Cys Cys Ala Gly Cys Thr Thr Cys Ala Gly Cys

225 230 235 240

Ala Cys Cys Thr Thr Cys Ala Ala Gly Thr Gly Cys Thr Ala Cys Gly

245 250 255

Gly Cys Gly Thr Gly Ala Gly Cys Cys Cys Cys Ala Cys Cys Ala Ala

260 265 270

Gly Cys Thr Gly Ala Ala Cys Gly Ala Cys Cys Thr Gly Thr Gly Cys

275 280 285

Thr Thr Cys Ala Cys Cys Ala Ala Cys Gly Thr Gly Thr Ala Cys Gly

290 295 300

Cys Cys Gly Ala Cys Ala Gly Cys Thr Thr Cys Gly Thr Gly Ala Thr

305 310 315 320

Cys Cys Gly Cys Gly Gly Cys Gly Ala Cys Gly Ala Gly Gly Thr Gly

325 330 335

Cys Gly Cys Cys Ala Gly Ala Thr Cys Gly Cys Cys Cys Cys Cys Gly

340 345 350

Gly Cys Cys Ala Gly Ala Cys Cys Gly Gly Cys Ala Ala Gly Ala Thr

355 360 365

Cys Gly Cys Cys Gly Ala Cys Thr Ala Cys Ala Ala Cys Thr Ala Cys

370 375 380

Ala Ala Gly Cys Thr Gly Cys Cys Cys Gly Ala Cys Gly Ala Cys Thr

385 390 395 400

Thr Cys Ala Cys Cys Gly Gly Cys Thr Gly Cys Gly Thr Gly Ala Thr

405 410 415

Cys Gly Cys Cys Thr Gly Gly Ala Ala Cys Ala Gly Cys Ala Ala Cys

420 425 430

Ala Ala Cys Cys Thr Gly Gly Ala Cys Ala Gly Cys Ala Ala Gly Gly

435 440 445

Thr Gly Gly Gly Cys Gly Gly Cys Ala Ala Cys Thr Ala Cys Ala Ala

450 455 460

Cys Thr Ala Cys Cys Thr Gly Thr Ala Cys Cys Gly Cys Cys Thr Gly

465 470 475 480

Thr Thr Cys Cys Gly Cys Ala Ala Gly Ala Gly Cys Ala Ala Cys Cys

485 490 495

Thr Gly Ala Ala Gly Cys Cys Cys Thr Thr Cys Gly Ala Gly Cys Gly

500 505 510

Cys Gly Ala Cys Ala Thr Cys Ala Gly Cys Ala Cys Cys Gly Ala Gly

515 520 525

Ala Thr Ala Thr Ala Cys Cys Ala Gly Gly Cys Cys Gly Gly Cys Ala

530 535 540

Gly Cys Ala Cys Cys Cys Cys Cys Thr Gly Cys Ala Ala Cys Gly Gly

545 550 555 560

Cys Gly Thr Gly Gly Ala Gly Gly Gly Cys Thr Thr Cys Ala Ala Cys

565 570 575

Thr Gly Cys Thr Ala Cys Thr Thr Cys Cys Cys Cys Cys Thr Gly Cys

580 585 590

Ala Gly Ala Gly Cys Thr Ala Cys Gly Gly Cys Thr Thr Cys Cys Ala

595 600 605

Gly Cys Cys Cys Ala Cys Cys Ala Ala Cys Gly Gly Cys Gly Thr Gly

610 615 620

Gly Gly Cys Thr Ala Cys Cys Ala Gly Cys Cys Cys Thr Ala Cys Cys

625 630 635 640

Gly Cys Gly Thr Gly Gly Thr Gly Gly Thr Gly Cys Thr Gly Ala Gly

645 650 655

Cys Thr Thr Cys Gly Ala Gly Cys Thr Gly Cys Thr Gly Cys Ala Cys

660 665 670

Gly Cys Cys Cys Cys Cys Gly Cys Cys Ala Cys Cys Gly Thr Gly Thr

675 680 685

Gly Cys Gly Gly Cys Cys Cys Cys Ala Ala Gly Ala Ala Gly Ala Gly

690 695 700

Cys Ala Cys Cys Ala Ala Cys Cys Thr Gly Gly Thr Gly Ala Ala Gly

705 710 715 720

Ala Ala Cys Ala Ala Gly Thr Gly Cys Gly Thr Gly Ala Ala Cys Thr

725 730 735

Thr Cys Ala Ala Cys Thr Thr Cys Ala Ala Cys Gly Gly Cys Cys Thr

740 745 750

Gly Ala Cys Cys Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Gly Ala

755 760 765

Thr Cys Thr Gly Gly Thr Cys Ala Cys Cys Ala Cys Cys Ala Cys Cys

770 775 780

Ala Cys Cys Ala Cys Cys Ala Cys

785 790

<210> 23

<211> 264

<212> PRT

<213> artificial

<220>

<223> amino acid sequence of RBD-His protein

<400> 23

Met Ala Arg Ser Val Thr Leu Val Phe Leu Val Leu Val Ser Leu Thr

1 5 10 15

Gly Leu Tyr Ala Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val

20 25 30

Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn

35 40 45

Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser

50 55 60

Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser

65 70 75 80

Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys

85 90 95

Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val

100 105 110

Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr

115 120 125

Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn

130 135 140

Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu

145 150 155 160

Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu

165 170 175

Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn

180 185 190

Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val

195 200 205

Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His

210 215 220

Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys

225 230 235 240

Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Arg

245 250 255

Ser Gly His His His His His His

260

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the receptor-binding Region (RBD) of the S protein of SARS-CoV-2, the antibody or antigen-binding fragment thereof comprising:

(a) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs):

and/or the presence of a gas in the gas,

(b) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs):

(vi) a VL CDR3, consisting of the sequence: 10, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) thereto;

preferably, the CDRs in any of (i) - (vi) are defined according to the Kabat numbering system;

preferably, the substitution recited in any one of (i) - (vi) is a conservative substitution.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising:

(a) the following 3 heavy chain CDRs: the sequence is SEQ ID NO:5, the sequence of VH CDR1 of SEQ ID NO:6, the sequence of VH CDR2 of SEQ ID NO: VH CDR3 of 7; and/or, the following 3 light chain CDRs: the sequence is SEQ ID NO:8, VL CDR1 of SEQ ID NO:9, VL CDR2 of SEQ ID NO:10 VL CDR 3;

or the like, or, alternatively,

(b) 3 CDRs contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1; and/or, 3 CDRs contained in the light chain variable region (VL) as set forth in SEQ ID NO: 2; preferably, the 3 CDRs contained in the VH and/or the 3 CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:

(i) SEQ ID NO: 1;

and

(iv) SEQ ID NO: 2;

(vi) And SEQ ID NO:2, 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;

preferably the substitutions described in (ii) or (v) are conservative substitutions;

preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 1 and a VL comprising the sequence shown as SEQ ID NO. 2.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, which is humanized;

preferably, the antibody or antigen-binding fragment thereof comprises a framework region sequence derived from a human immunoglobulin;

preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein,

the VH of the antibody or antigen-binding fragment thereof comprises: heavy chain framework regions FR1, FR2 and FR3 derived from heavy chain germline sequence IGHV1-3 x 01, and heavy chain framework region FR4 derived from heavy chain germline sequence IGHJ5 x 02; and the combination of (a) and (b),

the VL of the antibody or antigen-binding fragment thereof comprises: light chain framework regions FR1, FR2 and FR3 derived from the light chain germline sequence IGKV4-1 × 01, and light chain framework region FR4 derived from the light chain germline sequence IGKJ2 × 01.

6. The antibody or antigen-binding fragment thereof of claim 4 or 5, comprising:

(i) SEQ ID NO: 17;

and

(iv) SEQ ID NOs: 18. 19, or a sequence set forth in any one of seq id nos;

(vi) And SEQ ID NOs: 18. 19, 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;

preferably, the antibody or antigen-binding fragment thereof comprises:

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, further comprising a constant region derived from a human immunoglobulin;

preferably, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., 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 a human immunoglobulin (e.g., κ or λ);

preferably, the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids or any combination thereof) as compared to the wild type sequence from which it is derived; and/or

(b) A light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10, or up to 5 amino acids or any combination thereof; e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids or any combination thereof) compared to the wild type sequence from which it is derived;

preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region;

preferably, the light chain constant region is a kappa light chain constant region;

preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 20;

preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as set forth in SEQ ID NO: 21.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', (Fab')₂Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb); and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

9. An antibody or antigen-binding fragment thereof that specifically binds to the receptor-binding Region (RBD) of the S protein of SARS-CoV-1 and/or SARS-CoV-2, the antibody or antigen-binding fragment thereof comprising:

and/or the presence of a gas in the gas,

(vi) a VL CDR3, consisting of the sequence: 16, or a sequence having one or more amino acid substitutions, deletions or additions thereto (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;

10. The antibody or antigen-binding fragment thereof of claim 9, comprising:

(a) the following 3 heavy chain CDRs: the sequence is SEQ ID NO:11, VH CDR1 of SEQ ID NO:12, VH CDR2 of SEQ ID NO:13 VH CDR 3; and/or, the following 3 light chain CDRs: the sequence is SEQ ID NO:14, VL CDR1 of SEQ ID NO:15, the sequence of VL CDR2 of SEQ ID NO:16 VL CDR 3;

or the like, or, alternatively,

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

11. The antibody or antigen-binding fragment thereof of claim 9 or 10, comprising:

(i) SEQ ID NO: 3;

and

(iv) SEQ ID NO: 4;

(vi) And SEQ ID NO:4, 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;

preferably, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO. 3 and a VL comprising the sequence shown as SEQ ID NO. 4.

12. The antibody or antigen-binding fragment thereof of any one of claims 9-11, which is humanized;

13. The antibody or antigen-binding fragment thereof of any one of claims 9-12, further comprising a constant region derived from a human immunoglobulin;

preferably, the antibody or antigen-binding fragment thereof comprises:

14. The antibody or antigen-binding fragment thereof of any one of claims 9-13, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', (Fab')₂Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb); and/or, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

15. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-14, or a heavy chain variable region and/or a light chain variable region thereof.

16. A vector comprising the nucleic acid molecule of claim 15; preferably, the vector is a cloning vector or an expression vector.

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

18. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising culturing the host cell of claim 17 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.

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

preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an additional antiviral agent (e.g., interferon, lopinavir, ritonavir, chloroquine phosphate, fabiravir, ridciclovir, and the like).

20. A method for neutralizing the virulence of a coronavirus in a sample comprising contacting a sample comprising a coronavirus with the antibody or antigen-binding fragment thereof of any one of claims 1-14;

for example, the coronavirus is SARS-CoV-2, and the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of any one of claims 1-8;

for example, the coronavirus is SARS-CoV-2 and/or SARS-CoV-1, and the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of any one of claims 9-14.

21. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 for the preparation of a medicament for neutralizing the virulence of a coronavirus in a sample, or for preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject;

for example, the coronavirus is SARS-CoV-2, and the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of any one of claims 1-8; preferably, the disease associated with coronavirus infection is COVID-19;

for example, the coronavirus is SARS-CoV-2 and/or SARS-CoV-1, and the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of any one of claims 9-14; preferably, the disease associated with coronavirus infection is selected from COVID-19 and/or SARS;

preferably, the subject is a mammal, e.g., a human;

preferably, the antibodies or antigen-binding fragments thereof are used alone or in combination with additional pharmaceutically active agents (e.g., additional antiviral agents such as interferon, lopinavir, ritonavir, chloroquine phosphate, fabiravir, ridciclovir, and the like).

22. A method for preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject (e.g., a human), comprising: administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the pharmaceutical composition of claim 19;

for example, the coronavirus is SARS-CoV-2 and/or SARS-CoV-1, and the antibody or antigen-binding fragment thereof is selected from the antibody or antigen-binding fragment thereof of any one of claims 9-14; preferably, the disease associated with coronavirus infection is selected from COVID-19 and/or SARS.

23. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14, and a detectable label linked to the antibody or antigen-binding fragment thereof;

preferably, the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent protein), radionuclides, or biotin.

24. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the conjugate of claim 23;

preferably, the kit comprises the conjugate of claim 23;

preferably, the kit comprises the antibody or antigen-binding fragment thereof of any one of claims 1-14, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; 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., acridinium esters, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or fluorescent protein), a radionuclide or biotin.

25. A method for detecting the presence or level of a coronavirus in a sample, comprising using the antibody or antigen-binding fragment thereof of any one of claims 1-14 or the conjugate of claim 23;

preferably, the method is an immunological detection, such as an enzyme immunoassay (e.g. ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay or a radioimmunoassay;

preferably, the coronavirus is SARS-CoV-2, the method comprising: using the antibody or antigen-binding fragment thereof of any one of claims 1-8, optionally comprising a detectable label;

preferably, the coronavirus is SARS-CoV-2 and/or SARS-CoV-1, the method comprising: use of the antibody or antigen-binding fragment thereof of any one of claims 9-14, optionally comprising a detectable label.

26. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-14 in the preparation of a kit for detecting the presence or level of a coronavirus in a sample, and/or for diagnosing whether a subject is infected with a coronavirus;

preferably, the coronavirus is SARS-CoV-2, the kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-8, optionally comprising a detectable label;

preferably, the coronavirus is SARS-CoV-2 and/or SARS-CoV-1, the kit comprising the antibody or antigen-binding fragment thereof of any one of claims 9-14, optionally comprising a detectable label;

preferably, the kit detects the presence or level of a coronavirus in a sample by the method of claim 25;

preferably, the sample is a blood sample (e.g., whole blood, plasma or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).