CN116693669A

CN116693669A - Antibodies against coronaviruses and uses thereof

Info

Publication number: CN116693669A
Application number: CN202310183373.6A
Authority: CN
Inventors: 张天英; 熊华龙; 朱雨荷; 张金蕾; 江尧; 颜思平; 袁伦志; 罗文新; 袁权; 张军; 夏宁邵
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2022-03-01
Filing date: 2023-03-01
Publication date: 2023-09-05

Abstract

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

Description

Antibodies against coronaviruses and uses thereof

Technical Field

Background

Coronavirus (coronavirus) infection can cause respiratory diseases in humans, mild coronavirus infection can cause influenza-like symptoms, and severe infection can progress to severe viral pneumonia, threatening human life health. Coronaviruses can infect humans and animals simultaneously, and some animal-derived coronaviruses can spread rapidly among humans and cause serious disease if they break through the host barrier to infect humans. For example, severe Acute Respiratory Syndrome (SARS) caused by severe acute respiratory syndrome coronavirus (SARS-CoV, also known as SARS-CoV-1) infection and COVID-19 caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection both bring great impact and burden to the economic and social development of human beings.

At present, no specific medicine for preventing or treating SARS-CoV-2 infection is approved. Patients with pneumonia caused by SARS-CoV-2 infection should be given only general supportive treatment, oxygen therapy and antiviral treatment such as interferon beta-1 b, lopinavir/ritonavir, dexamethasone, etc., and these measures have limited clinical effects. Studies have found that higher levels of SARS-CoV-2 neutralizing antibody production are often accompanied in the recovery of new coronaries. In a new coronavirus pneumonia diagnosis and treatment regimen (trial seventh edition) issued by the national Wei Jian Committee, convalescent plasma treatment was recommended for patients with faster disease progression, severe and critical. There are study data showing that the viral load in patients is rapidly reduced and the clinical symptoms of patients are effectively improved after convalescence plasma treatment with neutralizing antibodies in critically ill patients identified as having covd-19 with concomitant severe respiratory distress syndrome (ARDS). These studies have shown the importance of humoral immunity in SARS-CoV-2 and that in addition to vaccine development, one or more monoclonal antibodies capable of neutralizing SARS-CoV-2 with high efficiency and specificity should be developed, which can be applied to short-term prophylaxis and effective treatment of COVID-19, either alone or in combination, which is of great importance to our country and even global control of COVID-19, while it is expected that, as the continuous mutation of viruses and the rapid and widespread communication of human development speeds are not difficult to predict, newer coronaviruses may be continually emerging, as such, making the development of neutralizing antibodies (nAbs) against broad-spectrum beta-CoV-Bs an item of great strategic importance.

SARS-CoV-1 and SARS-CoV-2 are both beta coronaviruses, which are in the same branch of the tree, and are both enveloped single-stranded sense RNA viruses. Both contain at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2, like SARS-CoV-1, mediates membrane fusion and cell entry of the virus by specific binding of the receptor binding domain (Receptor binding domain, RBD) on the S protein to angiotensin transferase 2 (ACE 2) on the host cell, and plays a vital role in the process of virus infection of cells. Thus, viral infections can be neutralized by interfering with the binding of the S protein to ACE 2. Therefore, S protein and particularly RBD region are the main source and recognition region of neutralizing antibodies against coronaviruses, and development of neutralizing antibodies against RBD region is preferred.

At present, SARS-CoV-2 and related mutant virus strains are being rolled worldwide, greatly influencing the development of human society and economy, and causing great threat to the safety of people's life. Therefore, the development of drugs such as antibodies capable of treating or preventing coronavirus (e.g., SARS-CoV-2) infection is of great importance in controlling the relevant epidemic.

Disclosure of Invention

The present invention provides monoclonal antibodies capable of neutralizing coronaviruses (e.g., SARS-CoV-2 and/or SARS-CoV-1). These monoclonal antibodies bind to epitopes on the S protein RBD region of coronaviruses and neutralize coronaviruses, inhibit the binding of coronavirus RBD proteins to the receptor ACE2, and protect animals to some extent from coronavirus attack in animal models. In particular, the monoclonal antibodies of the invention maintain a stable neutralizing effect against the currently world-wide epidemic SARS-CoV-2 mainstream mutant strain. Thus, the antibodies of the invention have the potential to be used in the prevention and/or treatment of coronavirus (e.g., SARS-CoV-1 and SARS-CoV-2) infections or related diseases, and are of great clinical value.

Antibodies of the invention

In a first aspect, the invention provides an antibody or antigen-binding fragment thereof comprising:

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

(i) VH CDR1 consisting of the sequence: SEQ ID NO. 7, SEQ ID NO. 13 or SEQ ID NO. 19, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared therewith,

(ii) VH CDR2 consisting of the sequence: SEQ ID NO. 8, SEQ ID NO. 14 or SEQ ID NO. 20, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to it, and

(iii) VH CDR3 consisting of the sequence: SEQ ID NO. 5, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

and/or the number of the groups of groups,

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

(iv) VL CDR1, consisting of the sequence: SEQ ID NO. 10, SEQ ID NO. 16 or SEQ ID NO. 22, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) in comparison therewith,

(v) VL CDR2, consisting of the sequence: SEQ ID NO. 11, SEQ ID NO. 17 or SEQ ID NO. 23, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared to that of SEQ ID NO. 23, and

(vi) VL CDR3 consisting of the sequence: SEQ ID NO. 12, SEQ ID NO. 18 or SEQ ID NO. 24, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto.

In certain embodiments, the substitutions described in any one of (i) - (vi) are conservative substitutions.

In certain embodiments, the CDRs are defined according to the Kabat numbering system.

In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is derived from monoclonal antibody 10E9, comprising:

(a) The following 3 heavy chain CDRs: VH CDR1 of SEQ ID No. 7 or a variant thereof, VH CDR2 of SEQ ID No. 8 or a variant thereof, VH CDR3 of SEQ ID No. 9 or a variant thereof; and/or, the following 3 light chain CDRs: VL CDR1 of SEQ ID NO. 10 or a variant thereof, VL CDR2 of SEQ ID NO. 11 or a variant thereof, VL CDR3 of SEQ ID NO. 12 or a variant thereof; wherein the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids, e.g., a conservative substitution) as compared to the sequence from which it is derived;

or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 1; and/or 3 CDRs contained in the light chain variable region (VL) as shown 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.

In certain embodiments, the antibody or antigen binding fragment thereof comprises a Framework Region (FR) derived from a murine immunoglobulin.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as set forth in SEQ ID NO. 1 or a variant thereof and a VL comprising a sequence as set forth in SEQ ID NO. 2 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution. The variants substantially retain the biological function of the sequence from which they were derived.

In certain embodiments, the antibody or antigen binding fragment thereof possesses at least one of the following characteristics:

(1) RBD that specifically binds coronavirus S protein; preferably, the coronavirus is a beta coronavirus, such as SARS-CoV-2, SARS-CoV-1 and/or RaTG13; preferably, the SARS-CoV-2 comprises a mutant strain;

(2) RBD of S protein binding SARS-CoV-2 (e.g., wuhan-Hu-1 strain (GenBank: MN 908947)) with an EC50 of less than about 50ng/mL, such as less than about 40ng/mL, 30ng/mL, 20ng/mL or less, and/or RBD of S protein binding SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) with an EC50 of less than about 100ng/mL, such as less than about 90ng/mL, 80ng/mL, 70ng/mL, 65ng/mL or less; preferably, the EC50 may be determined by ELISA;

(3) K at less than about 10nM, e.g., less than about 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM or less _D RBD binding to S protein of SARS-CoV-2 (e.g., wuhan-Hu-1 virus strain (GenBank: MN 908947)); preferably, the K _D By surface plasmon resonance techniques (e.g., biacore);

(4) Neutralization of SARS-CoV-2 pseudovirus (e.g., wuhan-Hu-1 strain (GenBank: MN 908947)) in vitro at a half-inhibitory concentration (IC 50) of less than about 0.1nM, e.g., less than about 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM or less, e.g., as determined by the neutralization assay described in example 6;

(5) Neutralization of SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) in vitro at a half-inhibitory concentration (IC 50) of less than about 0.1nM, e.g., less than about 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM or less, e.g., as determined by the neutralization assay described in example 6;

(6) Neutralization of SARS-CoV-2-related mutant pseudovirus (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at a half-inhibitory concentration (IC 50) of less than about 2nM, e.g., less than about 1.5nM, 1.4nM, 1.3nM, 1.2nM, 1.1nM, 1nM or less, e.g., as determined by neutralization assay described in example 6;

(7) Neutralizing coronavirus, preventing and/or treating coronavirus infection or a disease caused by coronavirus infection in a subject (e.g., human); preferably, the coronavirus is a beta coronavirus, such as SARS-CoV-2, SARS-CoV-1 and/or RaTG13; preferably, the SARS-CoV-2 comprises a mutant strain.

In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is derived from monoclonal antibody 30A4-2, comprising:

(a) The following 3 heavy chain CDRs: VH CDR1 of SEQ ID No. 13 or variant thereof, VH CDR2 of SEQ ID No. 14 or variant thereof, VH CDR3 of SEQ ID No. 15 or variant thereof; and/or, the following 3 light chain CDRs: VL CDR1 of SEQ ID NO. 16 or a variant thereof, VL CDR2 of SEQ ID NO. 17 or a variant thereof, VL CDR3 of SEQ ID NO. 18 or a variant thereof; wherein the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids, e.g., a conservative substitution) as compared to the sequence from which it is derived;

or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO 3; and/or 3 CDRs contained in the light chain variable region (VL) as shown 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.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO 3 or a variant thereof and a VL comprising a sequence as shown in SEQ ID NO 4 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution. The variants substantially retain the biological function of the sequence from which they were derived.

(2) RBD of S protein binding SARS-CoV-2 (e.g., wuhan-Hu-1 strain (GenBank: MN 908947)) with an EC50 of less than about 50ng/mL, such as less than about 40ng/mL, 30ng/mL, 20ng/mL, 15ng/mL or less, and/or RBD of S protein binding SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) with an EC50 of less than about 100ng/mL, such as less than about 90ng/mL or less; preferably, the EC50 may be determined by ELISA;

(3) K at less than about 10nM, e.g., less than about 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM or less _D RBD binding to S protein of SARS-CoV-2 (e.g., wuhan-Hu-1 virus strain (GenBank: MN 908947)); preferably, the K _D By surface plasmon resonance techniques (e.g., biacore);

(4) Neutralization of SARS-CoV-2 pseudovirus (e.g., wuhan-Hu-1 strain (GenBank: MN 908947)) in vitro at a half-inhibitory concentration (IC 50) of less than about 1nM, e.g., less than about 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM or less, e.g., as determined by the neutralization assay described in example 6;

(5) Neutralization of SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) in vitro at a half-inhibitory concentration (IC 50) of less than about 1nM, e.g., less than about 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM or less, e.g., as determined by the neutralization assay described in example 6;

(6) Neutralization of SARS-CoV-2-related mutant pseudovirus (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at a half-inhibitory concentration (IC 50) of less than about 1nM, e.g., less than about 0.9nM, 0.8nM, 0.7nM, 0.6nM or less, e.g., as determined by the neutralization assay described in example 6;

In certain embodiments, the antibodies or antigen-binding fragments thereof of the invention are derived from monoclonal antibody 1C5-2, comprising:

(a) The following 3 heavy chain CDRs: VH CDR1 of SEQ ID No. 19 or a variant thereof, VH CDR2 of SEQ ID No. 20 or a variant thereof, VH CDR3 of SEQ ID No. 21 or a variant thereof; and/or, the following 3 light chain CDRs: VL CDR1 of SEQ ID NO. 22 or a variant thereof, VL CDR2 of SEQ ID NO. 23 or a variant thereof, VL CDR3 of SEQ ID NO. 24 or a variant thereof; wherein the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids, e.g., a conservative substitution) as compared to the sequence from which it is derived;

or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 5; and/or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO. 6; 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.

In certain embodiments, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO. 5 or a variant thereof and a VL comprising a sequence as shown in SEQ ID NO. 6 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution. The variants substantially retain the biological function of the sequence from which they were derived.

(2) RBD of S protein binding SARS-CoV-2 (e.g., wuhan-Hu-1 strain (GenBank: MN 908947)) with an EC50 of less than about 50ng/mL, such as less than about 40ng/mL, 30ng/mL, 20ng/mL, 15ng/mL, 14ng/mL, 13ng/mL, 12ng/mL or less, and/or RBD of S protein binding SARS-CoV-1 (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) with an EC50 of less than about 500ng/mL, such as less than about 400ng/mL, 300ng/mL, 250ng/mL or less; preferably, the EC50 may be determined by ELISA;

(3) K at less than about 10nM, e.g., less than about 9nM, 8nM, 7nM, 6nM, 5nM or less _D RBD binding to S protein of SARS-CoV-2 (e.g., wuhan-Hu-1 virus strain (GenBank: MN 908947)); preferably, the K _D By surface plasmon resonance techniques (e.g., biacore);

(5) Neutralization of SARS-CoV-1 pseudovirus (e.g., CUHK-W1 strain (GenBank: AY 278554.2)) in vitro at a half-inhibitory concentration (IC 50) of less than about 1nM, e.g., less than about 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM or less, e.g., as determined by the neutralization assay described in example 6;

(6) Neutralization of SARS-CoV-2-related mutant pseudovirus (e.g., D614G, B.1.1.7, B.1.351, P.1, B.1.617.2) in vitro at a half-inhibitory concentration (IC 50) of less than about 20nM, e.g., less than about 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM or less, e.g., as determined by neutralization assay described in example 6;

In certain embodiments, the antibody or antigen binding fragment thereof of any of the above embodiments may be humanized to reduce immunogenicity to humans. Methods for humanizing non-human antibodies are known in the art, for example, the CDR regions of an antibody or antigen binding fragment thereof of the invention may be grafted into a human framework sequence using methods known in the art. In certain embodiments, the antibody or antigen binding fragment thereof of any of the above embodiments may comprise a Framework Region (FR) derived from a human immunoglobulin.

In certain embodiments, the antibody or antigen binding fragment thereof of any of the embodiments above may further comprise a constant region sequence derived from a mammalian (e.g., murine or human) immunoglobulin.

In certain embodiments, the heavy chain of the antibody or antigen binding fragment thereof of any of the above embodiments comprises a heavy chain constant region derived from a murine or human immunoglobulin (e.g., igG1, igG2, igG3, or IgG 4). In certain embodiments, the light chain of the antibody or antigen binding fragment thereof of any of the above embodiments comprises a light chain constant region derived from a murine or human immunoglobulin (e.g., kappa or lambda).

In certain embodiments, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, igG2, igG3, or IgG4 heavy chain constant region.

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

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

Herein, an antibody or antigen-binding fragment thereof of the invention may comprise a variant that differs 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), or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and substantially retains the above-described biological function of the antibody or antigen-binding fragment thereof from which it is derived.

Preparation of antibodies

The antibodies of the invention may 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 the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention.

Antigen binding fragments of the invention may be obtained by hydrolysis of intact antibody molecules (see Morimoto et al, J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al, science 229:81 (1985)). Alternatively, these antigen binding fragments can 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') ₂ Fragments (Carter et al, bio/Technology,10:163-167 (1992)). In addition, fv, fab or F (ab') ₂ Fragments may also be isolated directly from recombinant host cell culture broth. 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 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 an antibody or antigen-binding fragment thereof 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 a heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention, and/or a second nucleotide sequence encoding a light chain variable region of an antibody or antigen-binding fragment thereof 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 the heavy chain of an antibody or antigen-binding fragment thereof of the invention, and/or a second nucleotide sequence encoding the light chain of an antibody or antigen-binding fragment thereof 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 embodiments, the host cell is a microorganism, such as a prokaryotic cell or a yeast cell.

In another aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof of the invention comprising culturing a host cell as described above under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

Therapeutic application

The antibody or antigen binding fragment thereof can be used for neutralizing coronavirus in vitro or in a subject, blocking or inhibiting the infection of cells by coronavirus, thereby achieving the purpose of preventing and/or treating coronavirus infection or related diseases of the subject. In particular, the antibodies derived from 10E9, 30A4-2 and 1C5-2 provided in the first aspect of the invention, or antigen binding fragments thereof, may be used in combination for the prevention and/or treatment of coronavirus infections.

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

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

In certain embodiments, the pharmaceutical composition may further comprise additional pharmaceutically active agents, such as additional antiviral agents (e.g., interferon, lopinavir, ritonavir, adefovir, dexamethasone, and the like).

In certain embodiments, the antibody or antigen-binding fragment or composition of the invention and the additional pharmaceutically active agent in the pharmaceutical composition may be provided as separate components or as a mixed component. Thus, the antibody or antigen-binding fragment or composition 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 the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), 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 a coronavirus in a sample comprising contacting a sample comprising the coronavirus with an antibody or antigen-binding fragment thereof, a composition, or a pharmaceutical composition of the invention. In certain embodiments, the methods are performed in vitro. In certain embodiments, the methods are used for non-diagnostic therapeutic purposes.

In certain embodiments, the coronavirus is a β genus coronavirus. In certain embodiments, the coronaviruses SARS-CoV-2, SARS-CoV-1 and/or RaTG13. In certain embodiments, the coronaviruses SARS-CoV-2 and/or SARS-CoV-1.

In certain embodiments, the SARS-CoV-2 comprises a mutant strain. In certain embodiments, the mutant S protein contains a mutation, e.g., one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In certain embodiments, the mutant S protein comprises one or more amino acid substitutions selected from the group consisting of D614G, K417N/T, E484K, N501Y, L452R, T K. In certain embodiments, the mutant strain is selected from the group consisting of a D614G strain, an Alpha strain (e.g., b.1.1.7), a Beta strain (e.g., b.1.351), a Gamma strain (e.g., p.1), a Delta strain (e.g., b.1.617.2), or any combination thereof.

In another aspect, the invention provides a method for neutralizing coronavirus or preventing and/or treating coronavirus infection or a disease associated with coronavirus infection in a subject, comprising: administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment, composition or pharmaceutical composition of the invention.

In certain embodiments, the subject is administered a composition of the invention, wherein the various antibodies comprised by the composition can be administered simultaneously, separately or sequentially.

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

In certain embodiments, the antibody or antigen-binding fragment, composition, or pharmaceutical composition thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent, such as interferon, lopinavir, ritonavir, adefovir, dexamethasone, and the like).

In another aspect, the invention relates to the use of an antibody or antigen binding fragment, composition or pharmaceutical composition of the invention in the manufacture of a medicament for: (1) Neutralizing the coronavirus in vitro or in vivo in a subject (e.g., human); and/or, (2) for preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject.

The antibodies of the invention, or antigen-binding fragments thereof, or the pharmaceutical compositions of the invention, may be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and injectable concentrated solutions), 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 the necessary amount in a suitable solvent, and optionally, simultaneously with other desired ingredients (including, but not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonizing agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, the sterile injectable solutions may be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for convenient storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier prior to use, such as water for injection (WFI), water for bacteriostatic injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The antibodies of the invention, or antigen-binding fragments thereof, or the pharmaceutical compositions 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, inguinal, 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 injection 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 antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention are 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 or composition of the invention. "prophylactically effective amount" means an amount sufficient to prevent, arrest, or delay the onset of a 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. The therapeutically effective amount of an antibody or antigen binding fragment thereof of the invention may vary depending 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 such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

In this context, the dosing regimen may be adjusted to obtain the optimal target response (e.g., therapeutic or prophylactic response). For example, the dosage may be administered in a single dose, may be administered multiple times over a period of time, or may be proportionally reduced or increased as the degree of urgency of the treatment situation.

Herein, the subject may be a mammal, such as a human.

Conjugate(s)

The antibodies or antigen binding fragments thereof of the invention may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). Typically, derivatization (e.g., labeling) of the antibody or antigen-binding fragment thereof does not adversely affect its binding to the coronavirus S protein. Thus, the antibodies or antigen binding fragments thereof of the invention are also intended to include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the invention may be functionally linked (by chemical coupling, gene fusion, non-covalent linkage, or otherwise) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and/or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule. Furthermore, the antibodies of the invention or antigen binding fragments thereof 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 antibodies, such as increasing serum half-life.

Accordingly, the present invention also provides a conjugate comprising an antibody or antigen-binding fragment thereof of the present invention and a detectable label attached thereto.

In this context, a detectable label according to the invention may be any substance that is 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., ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ c or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dot or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridine esters, luminol and derivatives thereof, ruthenium derivatives such as ruthenium terpyridyl), magnetic beads (e.g.,) A calorimetric marker such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads,And biotin for binding to the above-mentioned label-modified avidin (e.g., streptavidin).

In certain embodiments, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). 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., an acridine ester compound, luminol and derivatives thereof, 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 may be attached to an antibody or antigen binding fragment thereof of the invention by linkers of different lengths to reduce potential steric hindrance.

Detection application

The antibodies or antigen binding fragments thereof of the invention are capable of specifically binding to the RBD of the coronavirus S protein, and thus can be used to detect coronaviruses or the RBD of the S protein or S protein thereof, and optionally to diagnose whether a subject is infected with coronavirus based on the detection results described above.

Thus, in another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention, or a conjugate of 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 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 an antibody or antigen-binding fragment thereof of the invention; optionally, the secondary antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound, luminol and derivatives thereof, 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 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 causing 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), tetramethyl benzidine (TMB), ABTS, or luminol for horseradish peroxidase, or p-nitrophenyl phosphate (p-NPP) or AMPPD for alkaline phosphatase. The kit may further comprise a pre-excitation and/or excitation liquid for chemiluminescence, for example when the detectable label is a chemiluminescent reagent, such as an acridine ester compound.

In another aspect, the invention provides a method of detecting the presence or level of coronavirus or its S protein or RBD of S protein, or coronavirus infected cells in a sample, comprising using an antibody or antigen binding fragment thereof of the invention.

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

In some embodiments, the methods comprise using the conjugates of the invention.

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

In certain embodiments, the method comprises: (1) Contacting the sample with an antibody or antigen binding fragment thereof 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 coronavirus or cells infected with coronavirus.

In certain embodiments, the methods may be used for diagnostic purposes, e.g., whether a subject is infected with coronavirus may be diagnosed based on the presence or level of coronavirus 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 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 of the invention in the preparation of a detection reagent for detecting the presence or level of coronavirus or its S protein or RBD of S protein, or cells infected with coronavirus in a sample, and/or for diagnosing whether a subject is infected with coronavirus.

In certain embodiments, the detection reagent detects the presence or level of coronavirus or its S protein or RBD of S protein, or cells infected with coronavirus in the sample by a detection method as described above, and optionally diagnoses whether the subject is infected with coronavirus 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 from a subject (e.g., a mammal, preferably a human).

In certain embodiments, the coronavirus described in any one of the above aspects is a β genus coronavirus. In certain embodiments, the coronavirus described in any one of the above aspects is SARS-CoV-2, SARS-CoV-1 and/or RaTG13. In certain embodiments, the coronavirus described in any one of the above aspects is SARS-CoV-2 and/or SARS-CoV-1. In certain embodiments, the SARS-CoV-2 comprises a mutant strain. In certain embodiments, the mutant S protein contains a mutation, e.g., one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In certain embodiments, the mutant S protein comprises one or more amino acid substitutions selected from the group consisting of D614G, K417N/T, E484K, N501Y, L452R, T K. In certain embodiments, the mutant strain is selected from the group consisting of a D614G strain, an Alpha strain (e.g., b.1.1.7), a Beta strain (e.g., b.1.351), a Gamma strain (e.g., p.1), a Delta strain (e.g., b.1.617.2), or any combination thereof.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Further, laboratory procedures such as virology, biochemistry, nucleic acid chemistry, immunology and the like, as used herein, are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus, sars-CoV-2)", is known as "novel coronavirus" or "2019-nCov". The disease caused by SARS-CoV-2 is known as novel coronavirus pneumonia (COVID-19). As used herein, the term "SARS-CoV-2" encompasses known isolates, including, for example, both the original strain (e.g., the first sequenced isolate GenBank: MN 908947.3) and subsequently discovered mutants, e.g., the concern mutant (Variants of Concern, VOC). In certain embodiments, the mutants include D614G (e.g., genBank: MN 908947), alpha (B.1.1.7 and Q lines, e.g., genBank: MW 624725.1), beta (B.1.351 and descendent lineages, e.g., genBank: MZ 314998.1), gamma (P.1 and descendent lineages, e.g., genBank: MZ 427312.1), and Delta (B.1.617.2 and AY lines, e.g., genBank: OM 444216.1). In certain embodiments, the term "SARS-CoV-2" encompasses both isolates whose spike protein does not comprise a mutation (e.g., as compared to reference strain MN 908947.3) and isolates that comprise a mutation in their spike protein (e.g., an amino acid substitution as compared to reference strain MN908947.3, such as D614G, K417N/T, E484K, N501Y, L452R, T478K, or any combination thereof). In certain embodiments, the mutant strain is preferably selected from isolates comprising a mutation (e.g., an amino acid substitution, such as D614G, K417N/T, E484K, N501Y, L452R, T478K, or any combination thereof) in their spike protein. In certain exemplary embodiments, the mutant strain is selected from the group consisting of a D614G strain, an Alpha strain (e.g., B.1.1.7), a Beta strain (e.g., B.1.351), a Gamma strain (e.g., P.1), a Delta strain (e.g., B.1.617.2).

As used herein, "severe acute respiratory syndrome coronavirus (SARS-CoV)" may also be referred to as "SARS-CoV-1", a causative agent of Severe Acute Respiratory Syndrome (SARS). As used herein, the term "SARS-CoV-1" encompasses known various isolates, such as the CUHK-W1 strain (GenBank: AY 278554.2) or GenBank: AAP13567.1. The disease caused by SARS-CoV-1 is known as Severe Acute Respiratory Syndrome (SARS).

SARS-CoV-1 and SARS-CoV-2 both belong to the genus beta coronavirus and are enveloped single-stranded plus-sense RNA viruses. They contain at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Both are membrane fusion and cell entry of the virus through specific binding of the receptor binding domain (Receptor binding domain, RBD) on the S protein to angiotensin-transferase 2 (ACE 2) on the host cell, which plays a vital role in the process of virus infection of cells.

As used herein, "RaTG13" is a bat coronavirus, available in, for example, genBank: MN996532.1.

The term "antibody" as used herein refers to an immunoglobulin derived molecule capable of specifically binding to a target antigen, which immunoglobulin derived molecule binds to the target antigen through at least one antigen binding site located in its variable region. When referring to the term "antibody", it includes not only whole antibodies, but also antigen-binding fragments capable of specifically binding to a target antigen, unless the context clearly indicates. An "intact antibody" typically consists 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 isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises 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 (CH 1, 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 antibodies to antigens, but exhibit multiple effector functions, such as may mediate immunoglobulins to host tissues or factors, including various immune systems Binding of cells (e.g., effector cells) to the first component (C1 q) of the classical complement system. VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist 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 antigen binding sites, respectively. The amino acid assignment in each region or domain can be followed by Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as 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. Comparat. 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, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

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

As used herein, the term "framework region" or "FR" residues refer 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 antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

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 an antigen, also referred to as an "antigen-binding portion. See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabodies (diabodies), single domain antibodies (single domain antibody), chimeric antibodies, linear antibodies (linear antibodies), nanobodies (technology from Dommantis), probody and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23:1126-1136.

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 a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises 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-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-to C-terminal direction. The 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 or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., 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 complete 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 of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -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) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments of the invention, an scFv may form a di-scFv, which refers to two or more individual scFv in tandem to form an antibody. In certain embodiments of the invention, scFv may be formed (scFv) ₂ It refers to the formation of antibodies from two or more individual scfvs in parallel.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. Et al, structures 2:1121-1123 (1994)).

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 monomer variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which a 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., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen-binding fragments in the same manner as used for intact antibodies.

As used herein, the term "chimeric antibody (Chimeric 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 belong to a particular class or subclass of antibody) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different class or subclass of antibody), but which nevertheless retains binding activity for the antigen of interest (u.s.p 4,816,567to Cabilly et al.; morrison et al, proc.Natl. Acad.Sci.USA,81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies (e.g., human murine chimeric antibodies) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., human antibody).

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part 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 typically all three, acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody are replaced by donor CDRs. Immunoglobulins that provide CDRs are referred to as "donors" and immunoglobulins that provide frameworks are referred to as "acceptors". 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 the desired properties (e.g., antigen specificity, affinity, reactivity, etc.).

In the present application, the expected properties of the antibodies of the present application include: (1) RBD that specifically binds to S protein of coronavirus (e.g., SARS-CoV-2 and/or SARS-CoV-1); (2) Neutralizing coronaviruses (e.g., SARS-CoV-2 and/or SARS-CoV-1) in vitro or in vivo in a subject (e.g., human); (3) Preventing and/or treating coronavirus (such as SARS-CoV-2 and/or SARS-CoV-1) infection or related diseases. The antibodies of the application have one or more of the above-described desirable properties.

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

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 can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present application, the term "K _D "refers to the dissociation equilibrium constant of 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 tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen. Specific binding properties between two molecules can be used The determination is performed by methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of 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 such that 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 Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (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 also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, 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 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 matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out 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 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a 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 substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. 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 USA94:412-417 (1997), which are incorporated herein by reference).

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, 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. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations 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 a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates 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, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier 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 the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), 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 in order to prevent or delay the occurrence of a disease or disorder or symptom (e.g., coronavirus infection) in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the 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., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the 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, a coronavirus infection or a related disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount of a disease (e.g., a coronavirus infection) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., a coronavirus infection); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the 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 such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment that binds to an antigenic protein on a virus, thereby preventing the maturation of virus-infected cells and/or virus progeny and/or the release of virus progeny, and an antibody or antibody fragment having neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

Advantageous effects of the invention

The present invention provides monoclonal antibodies capable of neutralizing coronaviruses, which have at least the following advantages: (1) Has strong neutralizing ability to SARS-CoV-2 and SARS-CoV-1; (2) Maintaining stable neutralization effect against SARS-CoV-2 related mutant in the current world; (3) RBD capable of cross-combining SARS-CoV-1, SARS-CoV-2 and RaTG13, has broad spectrum; (4) Protection against SARS-CoV-2 related mutant b.1.351 challenge was demonstrated in animal models. The antibody of the present invention has important clinical value for the prevention/treatment and diagnosis of coronavirus infection.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting 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 following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows ELISA reactivity of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 to SARS-CoV-2, SARS-CoV and RaTG13-CoV viral RBD recombinant proteins.

Fig. 2A shows the ligand coupling level calculation formula in the Biacore affinity assay.

FIG. 2B shows the results of affinity assays of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 for the SARS-CoV-2 virus RBD recombinant protein.

FIG. 3 shows the results of fluorescence detection of SARS-CoV-2VSVpp and SARS-CoV VSVpp infected BHK21-hACE2 cells.

FIG. 4 shows the measurement of the neutralization activity of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 in SARS-CoV-2 and SARS-CoV VSV pp pseudovirus infection models.

FIG. 5 shows the measurement of the neutralizing activity of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 against SARS-CoV-2-related mutant strain (Variants of Concern, VOC) in SARS-CoV-2.

FIG. 6 shows the measurement of neutralizing activity of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 against the true virus of the relevant mutant strain Beta (B.1.351).

FIG. 7 shows the evaluation of therapeutic effect of a combination of murine mab 10E9, 30A4-2 and 1C5-2 three antibodies on hamster vaccinated with Beta (B.1.351).

FIG. 8 shows hamster lung histopathology following combination therapy with murine mab 10E9, 30A4-2 and 1C 5-2.

Sequence information

Information relating to the sequences of the present invention is provided in table 1 below.

Table 1: description of the sequence

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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, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

EXAMPLE 1 construction of SARS-CoV and SARS-CoV-2 pseudovirus on the basis of VSV vector

1.1 construction of plasmid expressing SARS-CoV and SARS-CoV-2Spike Gene

The spike protein gene C end 18 amino acids of a novel coronavirus Wuhan-Hu-1 virus strain (GenBank: MN 908947) is truncated and mutated, and then subjected to codon optimization cloning to eukaryotic expression plasmid vector pCAG to obtain pCAG-nCoVS-del18 plasmid (the Wuhan-Hu-1-spike gene sequence is shown as SEQ ID NO:25, and the nCoVS-del18 gene sequence is shown as SEQ ID NO: 26); the spike protein gene C end 18 amino acids of the CUHK-W1 strain (GenBank: AY 278554.2) of coronavirus SARS-CoV is truncated and mutated, then codon optimized cloned into eukaryotic expression plasmid vector pCAG to obtain pCAG-SARS-Sdel18 plasmid (SARS CoV CUHK-W1 spike gene sequence is shown as SEQ ID NO:27, SARS-Sdel18 gene sequence is shown as SEQ ID NO: 28).

1.2 SARS-CoV based on VSV vector and pseudo-viral packaging of SARS-CoV-2

The present study successfully constructed recombinant VSV using the VSV reverse genetics operating system: VSVdG-EGFP-G. VSVdG-EGFP-G is a recombinant virus form, the G gene of the genome of which is replaced by a fluorescent reporter gene EGFP, and the recombinant virus can be used for generating rVSV pseudoviruses containing heterologous envelope proteins, and the heterologous proteins can be viral proteins with high biological protection requirements, because the replication capacity of the VSVdG-EGFP-G pseudoviruses is limited to Shan Dai replications, so that viruses required by BSL-3/BSL-4 laboratories, such as SARS-CoV, SARS-CoV-2 and the like, can be studied in BSL-2 laboratories. The Vero-E6 cells are transfected with pCAG-nCoVS-del18 plasmid, the virus VSVdG-EGFP-G is infected after 24 hours, the cell supernatant is replaced by 5% FBS DMEM containing anti-VSV-G rat serum after one hour of infection, the infection of residual VSVdG-EGFP-G is blocked, the cell supernatant is harvested after 24 hours, the pseudo virus carrying SARS-CoV-2-Spike-del18 based on the VSV vector is obtained, and the split package is frozen at the temperature of-80 ℃ for standby; in the same packaging mode, the pCAG-SARS-Sdel18 plasmid is utilized to obtain the pseudo virus carrying SARS-CoV-Spike-del18 based on VSV vector, and the pseudo virus is split-packaged and frozen at-80 ℃ for standby.

EXAMPLE 2 screening of monoclonal antibodies that neutralize both SARS-CoV and SARS-CoV-2

2.1 immunization of mice

2.1.1 immunogen preparation: the immunogen was a pseudo virus obtained in example 1 and based on the VSV vector carrying SARS-CoV-2-Spike-del18 and carrying SARS-CoV-Spike-del18, and the immunological dose was 5X 10 ⁶ PFU/dose, and Freund's adjuvant by mixing, and mixing thoroughly in an injection emulsifier to form a water-in-oil emulsion. The primary immunization used Freund's complete adjuvant, and the subsequent booster immunization used Freund's incomplete adjuvant.

2.1.2 immunization of mice: double-sided inguinal subcutaneous multipoint injection immunization is carried out on the BALB/c female mice with the age of 6-8 weeks by using the prepared immunogen, the injection volume is 300 mu L/time, a sequential immunization mode is adopted in the immunization program, the immunization is carried out in 1, 3 and 5 weeks on the basis of the pseudo virus of the VSV carrier carrying SARS-CoV-Spike-del18, the pseudo virus of the SARS-CoV-2-Spike-del18 is carried by the VSV carrier in 2, 4 and 6 weeks, about 200 mu L of orbital venous blood is collected before each immunization for titer measurement, the neutralization titer of serum on the SARS-CoV and the SARS-CoV-2 pseudo virus is detected by using a pseudo virus neutralization method, and a fusion experiment is carried out in 7 weeks.

2.2 antibody screening

2.2.1 preparation before fusion: mouse myeloma cells (SP 2/0) were recovered and cultured in RPMI1640 medium containing 10% fetal bovine serum to logarithmic growth phase for fusion.

2.2.2 preparation and screening of fusion hybridomas

The spleen of the mouse is taken to prepare a cell suspension, and the cell suspension is fused with the myeloma cell SP2/0 of the mouse to obtain hybridoma cells. And preparing feeder cells and hybridoma cells for co-culture, wherein a large number of unfused myeloma cells and mouse spleen cells die in an RPMI1640-HAT screening culture medium in the culture process of the hybridoma cells, and a small number of hybridoma cells are not easy to survive and can survive by adding other cells, and the added cells are feeder cells. The laboratory used mouse peritoneal macrophages and small week old mouse thymocytes as feeder cells.

2.2.2.1 preparation of mouse peritoneal macrophages: (i) Killing a BALB/C mouse with 6 weeks old by introducing a neck, soaking in 75% alcohol for 3-5min, placing into a super clean workbench, lifting the abdominal skin of the mouse with forceps, cutting a small opening, and using 2 hemostatic forceps to kill the outer skin of the mouse in the vertical direction at the cut-off position to expose the peritoneum of the mouse; (ii) Lifting the peritoneum by using a sterile forceps, injecting 5mL of RPM 1640 culture medium into the abdominal cavity of a mouse by using a syringe, shaking the mouse to mix the culture medium uniformly in the abdominal cavity, and carefully sucking out the culture medium in the abdominal cavity by using the syringe; (iii) Macrophage-containing medium was added to RPMI1640-HAT screening medium containing 20% fetal bovine serum and mixed with the fused cells.

2.2.2.2 preparation of mouse thymocytes: (i) Killing a BALB/C mouse with 3 weeks old by introducing a neck, soaking in 75% alcohol, sterilizing for 3-5min, placing into a super clean workbench, lifting the skin of the chest of the mouse with forceps, cutting a small opening, and killing the outer skin of the mouse with 2 hemostatic forceps in the vertical direction of the cut-out position to expose the inner skin of the chest of the mouse; (ii) Clamping the chest with another sterile forceps, and cutting the chest with scissors; (iii) The opalescent thymus in the chest was removed with clean sterile forceps, placed in a 70 μm cell screen and ground to give thymus feeder cells, which were added to RPMI1640-HAT screening medium containing 20% fetal bovine serum and mixed with the fused cells.

Preparation of 2.2.2.3 mouse myeloma cells: SP2/0 cells grown to log phase were selected for fusion. Myeloma cells were removed from the flask and washed 1 time (1500 rpm. Times.5 min) with RPMI-1640 medium in centrifuge tubes prior to fusion. Cells were resuspended in RPMI-1640 medium and counted.

2.2.2.4 preparation of spleen cells from immunized mice: (i) The BALB/C mice boosted in 3.1.3 were collected, whole blood was collected, and serum was collected. (ii) Then introducing a neck to kill the mice, soaking the mice in 75% alcohol to sterilize for 3-5min, and then placing the mice in an ultra-clean workbench to enable the mice to lie on the right side; (iii) Opening the abdominal cavity of the mouse by using sterile forceps and surgical scissors, cutting the spleen of the mouse, cutting the spleen into small blocks, and grinding the small blocks in a 70 mu m cell screen to obtain spleen cells; (iv) Placing spleen cells into a 50mL centrifuge tube, sucking off adipose tissue by using a glass pipette (bent tube), then supplementing RPMI-1640 medium to 30mL, centrifuging at 1500rpm for 5min, and repeating for 3 times; (v) RPMI-1640 medium resuspended spleen cells and counted.

2.2.2.5PEG fusion preparation of hybridomas: (i) 1mL PEG1450 (from SIGMA) and 40mL RPM 1640 medium were incubated to 37℃for use prior to fusion; (ii) Mixing the prepared myeloma cells and spleen cells in a 50mL centrifuge tube, centrifuging at 1500rpm for 5min, discarding the supernatant, and lightly flicking the bottom of the tube to loosen the cells into paste; (iii) Slowly dripping the incubated PEG into the cells, shaking the cells while adding, uniformly mixing the cells, and terminating the fusion with the incubated RPMI1640 medium after 1 min; (iv) Centrifugation at 1500rpm for 5min, adding cells to RPMI1640-HAT screening medium containing feeder cells and 20% fetal bovine serum, then adding to 96-well plates with 200 μl per well, and culturing in a 5% CO2 incubator; (v) After 5 days of culture, 20% fetal bovine serum RPMI1640-HAT medium was aspirated, and 10% fetal bovine serum RPMI1640-HT medium was used instead, and after 5 days of culture, the cell supernatant was aspirated for detection.

Screening of 2.2.2.6 hybridomas: pseudo virus neutralization screening, namely, pseudo viruses of SARS-CoV and SARS-CoV-2 based on the VSV vector in example 1 are utilized, 50 mu L of fusion cell culture supernatant is taken for pseudo virus neutralization experiment, positive cloning holes capable of simultaneously inhibiting pseudo virus infection of SARS-CoV and SARS-CoV-2 are selected, and monoclonal hybridoma cells are cloned and screened.

2.3 production of mouse monoclonal antibody ascites

5 BALB/C mice are taken, and the mice are sensitized by intraperitoneal injection of 0.5mL of liquid paraffin oil and can be used after 3 days of sensitization. The hybridoma cells in logarithmic growth phase were centrifuged at 1500rpm for 5min, the supernatant was discarded, and resuspended to 1-2X 10-6 cells/mL with PBS, and 0.5mL of cells were intraperitoneally injected per mouse. After 7 days, the abdomen of the mice was obviously distended, the mice were sacrificed by cervical diversion, the abdominal cavity of the mice was dissected, and all ascites in the abdominal cavity of the mice were carefully aspirated.

2.4 purification of monoclonal antibody ascites

The monoclonal antibody ascites was centrifuged at high speed, the supernatant was taken, an equal volume of saturated ammonium sulfate solution was added, and after 30min of precipitation on ice, centrifugation was carried out at 25000rpm for 10min, the precipitate was dissolved with 0.2M disodium hydrogen phosphate buffer, and then purified by Protein A affinity chromatography column (purchased from GE company, USA) to obtain a purified mouse monoclonal antibody. Three antibodies 10E9 (type IgG2 a), 30A4-2 (type IgG 1) and 1C5-2 (type IgG 1) were finally obtained.

EXAMPLE 3 amplification sequencing of the light and heavy chain variable region genes of monoclonal antibodies 10E9, 30A4-2 and 1C5-2

Hybridoma RNA preparation: the 10E9, 30A4-2 and 1C5-2 hybridoma cells cultured to the logarithmic growth phase were blown up and transferred into a 15mL centrifuge tube, centrifuged at 1500rpm for 3min to collect the cells, resuspended in 200. Mu.l of sterile PBS (pH 7.45), and transferred into a new 1.5mL centrifuge tube free of RNase, added with 800. Mu.l of Trizol solution (Invitrogen), vigorously shaken for 30S, and allowed to stand at 4℃for 10min. Frozen in-80℃refrigerator and sent to sequencing company for sequencing.

The heavy and light chain variable region amino acid sequences of 10E9, 30A4-2 and 1C5-2 were determined by sequencing. The VH and VL sequences of 10E9 are shown as SEQ ID NO. 1 and 2, the VH and VL sequences of 30A4-2 are shown as SEQ ID NO. 3 and 4, and the VH and VL sequences of 1C5-2 are shown as SEQ ID NO. 5 and 6.

Further, the CDR sequences of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 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, bezida, maryland (1991), pages 647-669). The amino acid sequences of the CDRs of the 10E9 heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 7-12, the amino acid sequences of the CDRs of the 30A4-2 heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 13-18, and the amino acid sequences of the CDRs of the 1C5-2 heavy chain variable region and the light chain variable region are shown in SEQ ID NO. 19-24.

EXAMPLE 4 ELISA binding Activity of monoclonal antibodies 10E9, 30A4-2 and 1C5-2 against SARS-CoV-2, SARS-CoV-1 and RaTG13

4.1 expression and purification of RBD proteins of SARS-CoV, SARS-CoV-2 and RaTG13

The RBD sequences of the 3 coronaviruses are respectively referred to as SARS-CoV-2Wuhan-Hu-1 (GenBank: MN 908947), SARS-CoV (AAP 13567.1) and RaTG13 (MN 996532.1) to construct respective RBD protein expression clones to prepare recombinant proteins, wherein the RBD recombinant proteins of the SARS-CoV-2 are named as SARS-CoV-2-RBD, and the RBD recombinant proteins of the SARS-CoV are named as SARS-CoV-RBD and the RBD recombinant proteins of the RaTG13-CoV are named as RaTG13-RBD.

4.2 preparation of reaction plate

The SARS-CoV-2-RBD, SARS-CoV-RBD and RaTG13-RBD proteins obtained above are treated with 50mM CB buffer (NaHCO) at pH9.6 ₃ /Na ₂ CO ₃ Buffer solution with final concentration of 50mM and pH value of 9.6) and final concentration of 2 mug/mL; adding 100 mu L of coating liquid into each hole of a 96-hole ELISA plate, coating for 16-24 hours at 2-8 ℃ and then coating for 2 hours at 37 ℃; washing 1 time with PBST washing solution (20mM PB7.4, 150mM NaCl,0.1% Tween 20); then 200. Mu.L of blocking solution (20 mM Na with pH 7.4 containing 20% calf serum and 1% casein) was added to each well ₂ HPO ₄ /NaH ₂ PO ₄ Buffer solution), and sealing at 37 ℃ for 2 hours; the blocking solution was discarded. Drying, and packaging in aluminum foil bag at 2-8deg.C.

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

The mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 obtained in example 3 were diluted to 10. Mu.g/mL in PBS containing 20% of neonatal bovine serum, and then diluted to 8 gradients in 3-fold or 5-fold gradient, and ELISA was performed as follows:

(1) Sample reaction: the ELISA plates coated with SARS-CoV-2-RBD, SARS-CoV-RBD and RaTG13-RBD proteins, respectively, were prepared by adding 100. Mu.L of the diluted sample to each well, and allowing the mixture to react in a 37℃incubator for 30 minutes.

(2) Enzyme-labeled reaction: after completion of the sample reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), 100. Mu.L of HRP-labeled goat anti-mouse IgG (GAM) reaction solution was added to each well, and the mixture was allowed to react in a 37℃incubator for 30 minutes.

(3) Color reaction: after completion of the enzyme-labeled reagent reaction step, the ELISA plate was washed 5 times with PBST wash (20mM PB7.4, 150mM NaCl,0.1%Tween20), 50. Mu.L of TMB developer (available from Beijing Wantai Biodrug Co., ltd.) was added to each well, and the mixture was allowed to react in an incubator at 37℃for 15 minutes.

(4) Termination reaction and read measurements: after completion of the color reaction step, 50. Mu.L of a stop solution (available from Wantai Biopharmaceutical Co., ltd. In Beijing) was added to each well of the reacted microplate, and the OD450/630 value of each well was measured on a microplate reader.

Reactivity determination of mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 with SARS-CoV-2-RBD, SARS-CoV-RBD and RaTG 13-RBD: and judging according to the read value after the reaction. If the detection value/background value is larger than 5, the test result is positive.

(5) Analysis of results: ELISA reactivity results are shown in FIG. 1, and mouse monoclonal antibodies 10E9, 30A4-2 and 1C5-2 have strong binding activity to SARS-CoV-2-RBD and SARS-CoV-RBD (FIGS. 1-A and 1-B); wherein 10E9 and 1C5-2 also have certain reactivity (figure 1-C) to RaTG13-RBD, have certain spectral properties, and the corresponding reactivity EC50 is summarized as shown in figure 1-D.

EXAMPLE 5 Biacore affinity assay of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 for RBD recombinant proteins of SARS-CoV-2

The present study uses surface plasmon resonance (Surface Plasmon Resonance, SPR) techniques to detect the affinity of 3 strains of monoclonal antibodies 10E9, 30A4-2 and 1C5-2 obtained in example 2 against SARS-CoV and SARS-CoV-2 with the RBD antigen of SARS-CoV-2. The detection method used in this example was a capture method, and a Protein G chip (GE company) was used to capture the murine monoclonal antibody.

5.1 determination of ligand response value (RU)

The ligand coupling level calculation formula is shown in FIG. 2A, and the ligand response value is about 1000RU according to the molecular weight of the murine monoclonal antibody and the molecular weight of the three RBD analytes.

The 3-strain antibodies 10E9, 30A4-2 and 1C5-2 were diluted with PBS to appropriate concentrations, respectively, wherein 30A4-2 was 50. Mu.g/mL, 30A4-2 was 40. Mu.g/mL, and 1C5-2 was 40. Mu.g/mL, so that the response value of the binding with Protein G chip was stabilized at about 1000 RU.

5.2 affinity assay

SARS-CoV-2RBD antigen was diluted 2-fold down from an initial concentration of 1000nM (10 dilution gradients total, 1000nM maximum and 1.6nM minimum) and tested for antigen antibody affinity using a surface plasmon resonance detector Biacore 8000 (GE). The results are shown in FIG. 2B.

5.3 analysis of results

The 3-strain antibodies 10E9, 30A4-2 and 1C5-2 have better affinity to the RBD antigen of SARS-CoV-2 virus, wherein the affinity of the 10E9 monoclonal antibody to the RBD antigen of SARS-CoV-2 is highest, the dissociation constant Kd=0.99 nM, and the dissociation constants of 30A4-2 and 1C5-2 to the RBD antigen of SARS-CoV-2 virus are Kd=2.79 nM and Kd=4.69 nM. The murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 demonstrated better cross-binding activity against both coronavirus RBDs.

EXAMPLE 6 pseudo-virus neutralization experiments of monoclonal antibodies 10E9, 30A4-2 and 1C5-2 on SARS-CoV and SARS-CoV-2

6.1 construction of pseudo-viral neutralization System based on SARS-CoV VSVpp and SARS-CoV 2VSVpp

To construct a VSV pseudovirus carrying SARS-CoV-2spike protein, the spike gene of the Wuhan-Hu-1strain (sequence source GenBank: MN 908947) was codon optimized for expression in human cells, and the spike gene of SARS-CoV-2, which was C-terminally truncated by 18 amino acids, was cloned into the eukaryotic expression vector pCAG to yield pCAG-nCoVSde18. Plasmid pCAG-nCoVSde18 was transfected into Vero-E6. 48 hours after transfection, VSVdG-EGFP-G (Addgene, 31842) virus was inoculated into cells expressing SARS-CoV-2Sde18 truncated protein 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 residual VSVdG-EGFP-G infection. The progeny virus will carry SARS-CoV-2Sde18 truncated protein to obtain pseudovirus VSV-SARS-CoV-2VSVpp. And SARS-CoV-1 pseudovirus SARS-CoV VSVpp was constructed in the same manner. After 24 hours post-VSVdG-EGFP-G infection, cell supernatants were collected, then centrifuged and filtered (pore size 0.45- μm, millipore, SLHP033 RB) to remove cell debris, and stored at-80℃for later use. Viral titers were determined by the number of GFP positive cells after infection of BHK21-hACE2 with the gradient diluted supernatant. The gene for hACE2 was integrated in BHK21 cells by the PiggyBac transposon system. The transposon vector (SBI system biosciences, PB 514B-2) containing the hACE2 gene and the transposase plasmid were co-transfected into BHK21 cells, and the cells BHK21-hACE2 stably expressing hACE2 were obtained by screening with puromycin resistance and red fluorescence. The fluorescence patterns of pseudoviruses SARS-CoV-2VSVpp and SARS-CoV VSVpp infected BHK21-hACE2 cells are shown in FIG. 3A.

Analysis of results: as shown in FIG. 3B, when SARS-CoV-2VSVpp and SARS-CoV VSVpp infect BHK21-hACE2 cells, the GFP positive cell number decreases correspondingly with the increase of dilution of infection, and within the GFP positive cell number range of 1000-20000, the positive cell number shows better correlation with dilution of infection after pseudo virus infection (SARS-CoV-2 VSVpp: R ² ＝0.98,SARS-CoV VSVpp:R ² =0.98). From viral titer = positive cell number x dilution/virus fluid volume, the corresponding viral titer was calculated: SARS-CoV-2 vsvpp=2.97x10 ⁷ PFU/mL；SARS-CoV VSVpp＝2.73×10 ⁷ PFU/mL。

6.2 pseudo-virus neutralization experiments on SARS-CoV VSVpp and SARS-CoV 2VSVpp

Antibodies 10E9, 30A4-2 and 1C5-2 were diluted to 666.7nM as gradient 1, 3-fold down gradient dilutions for 11 gradients, respectively, and the gradient diluted antibodies were mixed with diluted VSVpp virus (moi=0.05) and incubated for 1h at 37 ℃. All samples and viruses were diluted with 10% FBS-DMEM. mu.L of the mixture was added to the pre-plated BHK21-hACE2 cells. After 12 hours incubation, the infected cells were fluorescent imaged using a high content imaging system based on turntable confocal (Opera phenoix or operaetta CLS, available from Perkinelmer corporation). And after the completion, quantitatively analyzing the obtained fluorescence image by using Columbus image management analysis software to detect the number of green fluorescence positive cells. The decrease (%) in the number of GFP positive cells in the antibody-treated group compared to the untreated control well was calculated, and the inhibition ratio was calculated. The IC50 of the antibodies was calculated using nonlinear regression analysis.

As shown in FIG. 4, 10E9, 30A4-2 and 1C5-2 have strong neutralization activities on SARS-CoV-2VSVpp and SARS-CoV VSVpp, wherein 10E9 has better neutralization activity, the neutralization IC50 of SARS-CoV VSVpp is up to 0.03nM, and the neutralization IC50 of SARS-CoV VSVpp is up to 0.04nM; the neutralizing IC50 of the monoclonal antibody 30A4-2 to the SARS-CoV and SARS-CoV-2 pseudoviruses is 0.11nM and 0.29nM, respectively, and the neutralizing IC50 of the monoclonal antibody 1C5-2 to the SARS-CoV and SARS-CoV-2 pseudoviruses is 0.34nM and 0.37nM, respectively.

6.3 New coronavirus SARS-CoV-2 related mutant (Variants of Concern, VOC) pseudovirus neutralization assay currently prevalent in the world

To examine the neutralizing effect of the monoclonal antibodies produced in this patent on the currently world-wide pandemic novel coronavirus SARS-CoV-2-related mutant strain (Variants of Concern, VOC), the present study was based on the production of pseudoviruses of example 6.1.1.1, by cloning the spike gene of the SARS-CoV-2-related mutant strain into the eukaryotic expression vector pCAG to obtain the corresponding plasmid, replacing plasmid pCAG-nCoVSde18, producing the corresponding pseudoviruses comprising D614G (sequence source GenBank: MN 908947), alpha (B.1.1.7 and Q-lineages, genBank: MW 624725.1), beta (B.1.351 and descendent lineages, genBank: MZ 314998.1), gamma (P.1 and descendent lineages, genBank: MZ 427312.1) and Delta (B.1.617.2 and AY-lineages, genBank: 444216.1), and incubating the 10E9, 30A 4-C2 and C5-C2 to a gradient of 1.67.5 nM to a dilution gradient of 3nM, and diluting the antibodies at a gradient of 3nM to 11.37 times, respectively. All samples and viruses were diluted with 10% FBS-DMEM. mu.L of the mixture was added to the pre-plated BHK21-hACE2 cells. After 12 hours incubation, the infected cells were fluorescent imaged using a high content imaging system based on turntable confocal (Opera phenoix or operaetta CLS, available from Perkinelmer corporation). And after the completion, quantitatively analyzing the obtained fluorescence image by using Columbus image management analysis software to detect the number of green fluorescence positive cells. The decrease (%) in the number of GFP positive cells in the antibody-treated group compared to the untreated control well was calculated, and the inhibition ratio was calculated. The IC50 of the antibodies was calculated using nonlinear regression analysis.

As shown in FIG. 5 and Table 2, the combination of 10E9, 30A4-2, 1C5-2 and the three antibodies has a stable and strong neutralization effect on the novel coronavirus SARS-CoV-2-related mutant strain (Variants of Concern, VOC) which is currently prevalent in the world, the neutralization effect is not affected by the mutation of the virus, and the IC50 corresponding to the neutralization effect is summarized in the following Table.

Table 2: neutralization IC50 summary of SARS-CoV-2-related mutant (Variants of Concern, VOC) by 10E9, 30A4-2 and 1C5-2

EXAMPLE 7 neutralization Effect of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 on New Guanyuan mutant Beta (B.1.351) strain true Virus

To further evaluate the neutralizing effect of murine monoclonal antibodies 10E9, 30A4-2 and 1C5-2 on SARS-CoV-2, the neutralizing effect of antibodies on the novel crown-block mutant Beta (B.1.351) strain true virus was examined in this example as follows:

10E9, 30A4-2 and 1C5-2 were diluted to 6666.67nM as gradient 1, 5-fold down-gradient dilution, 5 gradients total, and the gradient diluted antibodies were mixed with the New crown-block mutant Beta (B.1.351) strain true virus and incubated for 1h at 37 ℃. All samples and viruses were diluted with 10% fbs-DMEM and the mixtures were added to pre-plated Vero cells and subsequently virus titers were characterized by the formation of viral plaques. The reduction (%) in the number of plaques in the antibody-treated group compared to the untreated control well was calculated, and the inhibition ratio was calculated. The IC50 of the antibodies was calculated using nonlinear regression analysis.

Analysis of results: as shown in FIG. 6, 10E9, 30A4-2 and 1C5-2 have strong neutralization activity on the novel coronavirus-related mutant strain Beta (B.1.351) strain, wherein the 10E9 has the best neutralization activity, the neutralization IC50 of the novel coronavirus-related mutant strain Beta (B.1.351) strain is up to 30.2nM, and the neutralization IC50 of the monoclonal antibodies 30A4-2 and 1C5-2 are 103.4nM and 132.9nM, respectively.

EXAMPLE 8 evaluation of therapeutic protective Effect of murine mab 10E9, 30A4-2 and 1C5-2 Triantibody in combination on hamster model after challenge with novel coronaviruses

To evaluate the therapeutic effects of murine mab 10E9, 30A4-2 and 1C5-2, the present example used a nasal drip to combat toxicity to vaccinate hamsters at 1X 10 ⁴ After 24 hours of the PFU novel coronavirus-related mutant Beta (b.1.351), the murine mab 10E9, 30A4-2 and 1C5-2 were used in combination, and 1ml of antibody was intraperitoneally injected at a dose of 35mg/kg, continuously observing the occurrence of hamster signs of lung inflammation associated therewith and viral load and histopathological characteristics in the respiratory tract after antibody treatment.

The results are shown in FIG. 7. As shown in FIG. 7A, 12 hamsters were vaccinated with 1X 10 out of 18 hamsters ⁴ 24 hours after the PFU novel coronavirus-related mutant strain Beta (B.1.351), 6 hamsters were treated with the triple antibody combined single injection at a dose of 35mg/kg/dose, and the other 6 hamsters were not treated after virus inoculation as untreated groups, and the remaining 6 hamsters were not treated at all as blank groups. As can be seen from the weight monitoring data (fig. 7B), the placebo group grew normally and the weight increased gradually over time without any treatment, while the untreated group had a sustained weight decrease over time in hamsters of 9.43% on average on the fifth day, while the hamster weight decreased by 13.76% on average on the fifth day after challenge, whereas the three antibody combination treatment group had only a weak weight decrease over time, and the hamster weight decreased by 2.28% on average on the fifth day after challenge, significantly lower than the untreated group (second day: p=0.0022; third day: p=0.0043; fourth day: p=0.0022; fifth day: p=0.0043), while all hamsters in the untreated group showed significant symptoms of weakness, hair standing, humpback, or abdominal respiration, which did not appear in the group receiving antibody treatment and the placebo group; from the survival rate of hamsters (fig. 7C), 1 hamster in the untreated group died on day 4, 2 on day 5, and 50% on day 5, while the antibody-treated group and the placebo group did not die, and 100% on day 5, which demonstrated that the combination treatment of murine mab 10E9, 30A4-2, and 1C5-2 three antibodies had significant therapeutic effects on hamsters vaccinated with Beta (b.1.351).

Further, on the fifth day after challenge, sacrificial hamsters were selected to assess viral load and histopathological features in the respiratory tract, and SARS-CoV-2 viral RNA was quantified by fluorescent quantitative PCR. The detection results of the ORF-ab1 gene and the N gene are shown in FIG. 7D and FIG. 7E, respectively. The results showed that viral RNA levels in hamster lung tissue receiving antibody treatment were significantly reduced by 2.80 log10 (ORF-ab 1 copies/ml) and 1.57 log10 (N copies/ml) compared to untreated group; hamsters treated with the antibodies showed a more pronounced viral RNA reduction in the tracheal and turbinate tissues compared to untreated groups, which were reduced by 1.30 log10 (ORF-ab 1 copies/ml) and 1.31 log10 (N copies/ml) in the turbinate tissue; 1.90 log10 (ORF-ab 1 copies/ml) and 1.83 log10 (N copies/ml) were reduced in the tracheal tissue.

The lung tissue pathology of hamsters after combination therapy with the 10E9, 30A4-2 and 1C5-2 tri-antibodies is shown in FIG. 8. The lung overview shows that untreated lungs exhibit multifocal diffuse hyperemia and hyperemia, whereas the lung tissue lesions in the group receiving antibody treatment are reduced (fig. 8A). Histopathology it was observed that untreated groups showed 100% hamster lung metaplasia, alveolar destruction and diffuse inflammation, whereas groups receiving antibody treatment showed only minor inflammation in limited lung areas and few pneumologic pathology in most lung areas. Hamsters in the group receiving antibody treatment had a higher pathology severity score than the uninfected hamsters (average score of 2.83 versus 0.88), but significantly lower than the untreated group (average score of 10.68, fig. 8b, p=0.0022). These results indicate that combination treatment of murine mab 10E9, 30A4-2 and 1C5-2 tri-antibodies has significant therapeutic effects on hamsters vaccinated with Beta (b.1.351).

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof comprising:

And/or the number of the groups of groups,

(vi) VL CDR3 consisting of the sequence: SEQ ID NO. 12, SEQ ID NO. 18 or SEQ ID NO. 24, or a sequence having a substitution, deletion or addition of one or several amino acids (for example a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

preferably, the substitutions described in any one of (i) - (vi) are conservative substitutions.

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

Or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 1; and/or 3 CDRs contained in the light chain variable region (VL) as shown 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;

preferably, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as set forth in SEQ ID NO. 1 or a variant thereof and a VL comprising a sequence as set forth in SEQ ID NO. 2 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution.

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

Or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO 3; and/or 3 CDRs contained in the light chain variable region (VL) as shown 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;

preferably, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO 3 or a variant thereof and a VL comprising a sequence as shown in SEQ ID NO 4 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution.

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

Or alternatively, the first and second heat exchangers may be,

(b) 3 CDRs contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 5; and/or 3 CDRs contained in the light chain variable region (VL) as shown in SEQ ID NO. 6; 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;

preferably, the antibody or antigen binding fragment thereof comprises: a VH comprising a sequence as shown in SEQ ID NO. 5 or a variant thereof and a VL comprising a sequence as shown in SEQ ID NO. 6 or a variant thereof; the variant has a substitution, deletion, or addition of one or more amino acids (e.g., a substitution, deletion, or addition of 1, 2, or 3 amino acids) as compared to the sequence from which it is derived, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, e.g., a conservative substitution.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, further comprising a constant region derived from a murine or human immunoglobulin;

preferably, the heavy chain of the antibody or antigen binding fragment thereof comprises a heavy chain constant region derived from a murine or human immunoglobulin (e.g., igG1, igG2, igG3, or IgG 4), and the light chain of the antibody or antigen binding fragment thereof comprises a light chain constant region derived from a murine or human immunoglobulin (e.g., kappa or lambda);

Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, igG2, igG3 or IgG4 heavy chain constant region.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antigen-binding fragment is selected from the group consisting of Fab, fab ', (Fab') ₂ Fv, disulfide-linked Fv, scFv, diabody (diabody) and single domain antibody (sdAb); and/or the antibody is a murine antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

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

8. A vector comprising the nucleic acid molecule of claim 7; preferably, the vector is a cloning vector or an expression vector.

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

10. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising culturing the host cell of claim 9 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.

11. A composition comprising at least two selected from the group consisting of the antibody or antigen-binding fragment thereof of claim 2, the antibody or antigen-binding fragment thereof of claim 3, and the antibody or antigen-binding fragment thereof of claim 4;

preferably, the composition comprises the antibody or antigen-binding fragment thereof of claim 2, the antibody or antigen-binding fragment thereof of claim 3, and the antibody or antigen-binding fragment thereof of claim 4;

preferably, the components of the composition are provided separately or as mixed components.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6 or the composition of claim 11, 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, adefovir, dexamethasone, etc.).

13. A method for neutralizing coronavirus in a sample comprising contacting a sample comprising coronavirus with the antibody or antigen-binding fragment thereof of any one of claims 1-6 or the composition of claim 11;

Preferably, the coronavirus is a beta coronavirus, such as SARS-CoV-2, SARS-CoV-1 and/or RaTG13;

preferably, the SARS-CoV-2 comprises a mutant strain; preferably, the S protein of the mutant contains a mutation, such as an amino acid substitution, deletion or addition; preferably, the S protein of the mutant comprises one or more amino acid substitutions selected from the group consisting of D614G, K417N/T, E484K, N501Y, L452R, T478K; preferably, the mutant strain is selected from the group consisting of a D614G strain, an Alpha strain (e.g., B.1.1.7), a Beta strain (e.g., B.1.351), a Gamma strain (e.g., P.1), a Delta strain (e.g., B.1.617.2), or any combination thereof.

14. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6, the composition of claim 11, or the pharmaceutical composition of claim 12 for the manufacture of a medicament for neutralizing a coronavirus, or preventing and/or treating a coronavirus infection or a disease associated with a coronavirus infection in a subject;

preferably, the SARS-CoV-2 comprises a mutant strain; preferably, the mutant S protein contains a mutation, e.g., one or several (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions; preferably, the S protein of the mutant comprises one or more amino acid substitutions selected from the group consisting of D614G, K417N/T, E484K, N501Y, L452R, T478K; preferably, the mutant strain is selected from the group consisting of a D614G strain, an Alpha strain (e.g., B.1.1.7), a Beta strain (e.g., B.1.351), a Gamma strain (e.g., P.1), a Delta strain (e.g., B.1.617.2), or any combination thereof;

Preferably, the subject is a mammal, such as a human;

preferably, the antibody or antigen-binding fragment, composition or pharmaceutical composition thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent such as interferon, lopinavir, ritonavir, dexamethasone, and the like).

15. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-6, and a detectable label attached 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., acridine esters, luminol and derivatives thereof, or ruthenium derivatives), fluorescent dyes (e.g., fluorescein or fluorescent protein), radionuclides, or biotin.

16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-6 or the conjugate of claim 15 in the preparation of a detection reagent for detecting the presence or level of coronavirus in a sample and/or for diagnosing whether a subject is infected with coronavirus;

preferably, the detection reagent detects the presence or level of coronavirus in the sample by immunological detection (e.g., enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay), and optionally diagnoses whether the subject is infected with coronavirus based on the detection result;

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