CN118184774A

CN118184774A - Fully human antibody for neutralizing SARS-CoV-2 and variant strain and application thereof

Info

Publication number: CN118184774A
Application number: CN202410388957.1A
Authority: CN
Inventors: 段良伟; 王辉; 江志华
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2024-06-14
Also published as: CN115043938A

Abstract

The present application relates to the fields of immunology and molecular virology, in particular to the fields of diagnosis, prevention and treatment of SARS-CoV-2 and its mutants. In particular, the application relates to antibodies or antigen-binding fragments thereof against SARS-CoV-2 and mutants thereof, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the application relates to the use of said antibodies or antigen binding fragments. The antibodies or antigen binding fragments of the application are useful in the diagnosis, prevention and/or treatment of SARS-CoV-2 and mutant infections thereof and/or diseases caused by such infections.

Description

Fully human antibody for neutralizing SARS-CoV-2 and variant strain and application thereof

The application is a divisional application of Chinese application patent application with the application date of 2022, 06, 15 and the application number of 202210675885X.

Technical Field

Background

SARS-CoV-2 Spike glycoprotein (Spike, S) plays a key role in the novel coronavirus infection and pathogenesis. The mature S protein of SARS-CoV-2 virus is a highly glycosylated trimer, each of which consists of 1260 amino acids (residues 14-1273), wherein the S1 subunit consists of 672 amino acids (residues 140-685), divided into four domains: one N-terminal domain (NTD), one C-terminal domain (CTD, also called receptor binding domain, RBD) and two subdomains (SD 1 and SD 2).

SARS-CoV-2S glycoprotein is a conformational machine that mediates viral entry by passing from a metastable, non-triggered state, through a pre-hairpin intermediate state, to a stable post-fusion state. Angiotensin converting enzyme 2 (ACE 2) has been shown to be the primary receptor for SARS-CoV-2. The detailed interactions between SARS-CoV-2RBD and its receptor ACE2 have been revealed by the complex structure of both. Structurally, RBD consists of two subdomains: core and outer subdomains. The extension loop (residues 438-506) on one side of the core subdomain presents a slightly concave surface to support the ACE 2N-terminal helix (α1). Analysis of the interface between SARS-CoV-2RBD and ACE2 shows that a total of 17 residues in RBD contact 20 amino acids in ACE2, forming a hydrophilic interaction network, leading to virus binding to the receptor.

The RBD of SARS-CoV-2S glycoprotein binds to the ACE2 receptor on the target cell surface, initiating the membrane fusion and subsequent viral invasion process of SARS-CoV-2. The second cleavage (S2' cleavage site) occurs after S glycoprotein binds ACE2 on plasma membrane (plasma membrane invasion pathway) or subsequent endocytosis of the viral particle by the host cell (endosomal invasion pathway), mediated by the cell surface serine protease TMPRSS2 or endosomal cysteine protease cathepsins B and L, respectively (CATHEPSINS B AND L). Protease cleavage of the S2' site releases the fusion peptide from the N-terminal region of the newly formed S2 subunit, further disrupting the stability of the SARS-CoV-2S glycoprotein trimer and may initiate the S2-mediated membrane fusion cascade. After the second cleavage is completed, the fusion peptide at the N-terminus of the S2 trimer is inserted into the host membrane, forming a pre-hairpin intermediate state. Because the pre-hairpin intermediate state is extremely unstable, the S2 fusion protein refoldes rapidly and irreversibly to a stable post-fusion state. These massive conformational rearrangements bring the viral and host cell membranes closer together, ultimately leading to membrane fusion. Thus, the S protein determines the host range and tissue tropism of the virus. In view of the crucial role of S protein in the virus invasion process and the characteristic that most of S protein is exposed outside the virus envelope and can be directly recognized by the host immune system, S protein is a main target point of host humoral immunity and cellular immunity and is a main acting site of antibodies in the host. Thus, current new coronal vaccine strategies are mostly based on full-length S glycoprotein or RBD domain thereof as immunogens.

However, SARS-CoV-2 has the characteristics of high mutation rate and high evolution speed as RNA virus. Currently, variants (VOCs) of interest have been designated by the World Health Organization (WHO); alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and Omicon (B.1.1.529). The initial variant BA.1 of Omicron was first discovered in Boltwanna and south Africa at 2021, month 11, and rapidly replaced the Delta variant, becoming the most popular strain of SARS-CoV-2 worldwide. Another variant of Omicron, BA.2, spreads faster than the BA.1 variant, and the resulting confirmed cases have exceeded BA.1 cases at the end of 2 months 2022, becoming the major worldwide epidemic variant. Even more terrible, omicron is still mutating rapidly, creating a large number of new intra-subtype variants. Currently well-known Omicron mutants include at least XE, BA.2.12.1, BA.4 and ba.5.

Omicron has a strong immune escape capacity for vaccines and neutralizing antibodies. Studies have shown that the neutralizing activity of most monoclonal antibodies (mAbs) on ba.1 and ba.2 is completely or substantially lost. Of the eleven monoclonal antibodies approved or authorized, REGN10987(imdevimab)、REGN10933(casirivimab)、LY-CoV555(bamlanivimab)、CB6/LY-CoV016(etesevimab)、S309(sotrovimab)、COV2-2130(cilgavimab)、COV2-2196(tixagevimab)、CT-P59(regdanvimab)、BRII-196(amubarvimab)、BRII-198(romlusevimab) and LY-CoV1404 (bebtelovimab), S309 retained most of the neutralizing activity to ba.1 (only about 2-fold reduction), but their activity was further escaped by ba.2. S309 (sotrovimab) is a representative member of a class of neutralizing antibodies that target the highly conserved region of sarbecovirus, the core domain of RBD (with more than 85% identical amino acid residues), and thus typically exhibit broad sarbecovirus neutralizing activity, although neutralizing activity is generally relatively low. The S371F, D N and R408S mutations possessed by ba.2 may contribute to the escape ability of such sarbecovirus neutralizing antibodies. In contrast, some neutralizing antibodies such as COV2-2130 (cilgavimab) completely lost activity on BA.1, but restored activity on BA.2.

Disclosure of Invention

In the present application, the inventors developed a human antibody having excellent properties capable of neutralizing SARS-CoV-2 and its mutants (Omicron BA.1 mutant, omicron BA.2 mutant, beta mutant and Delta mutant), blocking or inhibiting the binding of SARS-CoV-2 and its mutant to receptor ACE2, and not easily eliciting an immunogenic response in a subject. Therefore, the antibody or antigen binding fragment provided by the application has potential for detecting, diagnosing, preventing and/or treating SARS-CoV-2 infection or diseases caused by SARS-CoV-2 infection, and has great clinical value.

Antibodies of the application

In a first aspect, embodiments of the present application disclose an antibody or antigen-binding fragment thereof that specifically binds to the Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2 and mutants thereof, comprising:

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

(a) VH CDR1 consisting of the sequence: SEQ ID NO.8, or a sequence having a conservative substitution, deletion or addition of one or several amino acids (e.g., a conservative substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto,

(B) VH CDR2 consisting of the sequence: SEQ ID NO.9, or a sequence having a conservative substitution, deletion or addition of one or several amino acids (e.g.a conservative substitution, deletion or addition of 1,2 or 3 amino acids) as compared to it, and

(C) VH CDR3 consisting of the sequence: SEQ ID NO.10, 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,

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

(d) VL CDR1, consisting of the sequence: SEQ ID NO.14, or a sequence having conservative substitutions, deletions or additions of one or several amino acids (e.g., substitutions, deletions or additions of 1,2 or 3 amino acids) as compared thereto,

(E) VL CDR2, consisting of the sequence: SEQ ID NO.15, or a sequence having conservative substitutions, deletions or additions of one or several amino acids (e.g.substitutions, deletions or additions of 1, 2 or 3 amino acids) as compared to it, and

(F) VL CDR3 consisting of the sequence: SEQ ID No.16, or a sequence having conservative substitutions, deletions or additions of one or several amino acids (e.g., conservative substitutions, deletions or additions of 1,2 or 3 amino acids) as compared thereto.

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

Three Complementarity Determining Regions (CDRs) defining a heavy chain variable region (VH) according to the Kabat numbering system: a VH CDR1 as shown in SEQ ID No.8, a VH CDR2 as shown in SEQ ID No.9, and a VH CDR3 as shown in SEQ ID No. 10; and

Three Complementarity Determining Regions (CDRs) defining a light chain variable region (VL) according to the Kabat numbering system: VL CDR1 as shown in SEQ ID No.14, VL CDR2 as shown in SEQ ID No.15, and VL CDR3 as shown in SEQ ID No. 16.

In a second aspect, embodiments of the application provide an antibody or antigen-binding fragment thereof that specifically binds to the Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2 and mutants thereof. The antibody or antigen binding fragment thereof comprises:

(I) A heavy chain variable region comprising an amino acid sequence selected from any one of (a) to (c) as set forth in seq id no:

(a) An amino acid sequence as shown in SEQ ID NO. 2;

(b) Sequences having conservative substitutions, deletions or additions of one or several amino acids (e.g., conservative substitutions, deletions or additions of 1, 2, three, 4 or 5 amino acids) compared to the sequence shown in SEQ ID No. 2; or (b)

(C) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 2; and

(II) a light chain variable region comprising an amino acid sequence selected from any one of (d) to (f) below:

(d) A sequence shown in SEQ ID NO. 4;

(e) Sequences having conservative substitutions, deletions or additions of one or several amino acids (e.g., conservative substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the sequence shown in SEQ ID No. 4; or (b)

(F) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 4;

Alternatively, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the sequence shown as SEQ ID No.2 and a light chain variable region comprising the sequence shown as SEQ ID No. 4.

In certain embodiments of the application, the antibody or antigen binding fragment thereof further comprises a framework region sequence derived from a human immunoglobulin. In certain embodiments of the application, the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence. In certain embodiments of the application, the antibody or antigen binding fragment thereof comprises: heavy chain framework region sequences derived from human rearranged antibody sequences and light chain framework region sequences derived from human rearranged antibody sequences. In certain embodiments of the application, the antibody or antigen binding fragment thereof comprises: heavy chain framework region sequences derived from human heavy chain germline sequences and light chain framework region sequences derived from human light chain germline sequences.

In certain embodiments of the application, the antibody or antigen binding fragment thereof comprises: a heavy chain variable region (VH) as shown in SEQ ID No. 2; and a light chain variable region (VL) as shown in SEQ ID NO. 4.

In certain embodiments of the application, the antigen binding fragment of the first or second aspect 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 rabbit antibody, chimeric antibody, humanized antibody, bispecific antibody or multispecific antibody.

In certain embodiments of the application, the antibody or antigen binding fragment thereof of the first or second aspect is provided with one or more of the following features:

(1) An RBD that specifically binds SARS-CoV-2, or a mutant strain thereof, or an S protein or an RBD of an S protein thereof, or an S1 subunit or an RBD of an S1 subunit thereof, said mutant strain comprising a SARS-CoV-2 Omicron BA.1 mutant strain, an Omicron BA.2 mutant strain, a Beta mutant strain and a Delta mutant strain;

(2) Blocking or inhibiting the binding of SARS-CoV-2, or a mutant thereof, or the RBD of S protein or S protein thereof, or the RBD of S1 subunit or S1 subunit thereof, to the Ace2 receptor, and/or blocking or inhibiting the infection of cells by SARS-CoV-2, or a mutant thereof, or the RBD of S protein or S protein thereof, or the RBD of S1 subunit or S1 subunit thereof;

(3) Neutralizing SARS-CoV-2, or a mutant strain thereof, or an RBD of S protein or S protein thereof, or an RBD of S1 subunit or S1 subunit thereof, in vitro or in vivo in a subject (e.g., human);

(4) Preventing and/or treating SARS-CoV-2, or a mutant thereof, or an S protein or RBD of an S protein, or an S1 subunit or RBD of an S1 subunit thereof, or a disease associated with SARS-CoV-2, or a mutant thereof, or an RBD of an S protein or S protein, or an RBD of an S1 subunit or S1 subunit thereof (e.g., COVID-19).

In this context, an antibody or antigen-binding fragment thereof according to any aspect of the application 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity 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.

Derived antibodies

The antibodies or antigen binding fragments thereof of any aspect of the application may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of a monoclonal antibody or antigen-binding fragment thereof has no effect on its binding to SARS-CoV-2. Thus, the antibodies or antigen binding fragments thereof just disclosed of the application may also include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the application 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 application 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.

In certain embodiments of the application, the monoclonal antibodies or antigen binding fragments thereof of the application bear a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye (e.g., isothiocyanate or fluorescent protein), a radionuclide, or biotin.

In this context, a detectable label according to the application 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 enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridine esters), magnetic beads (e.g., dynabeads ^@), thermolabels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, biotin for binding to the label-modified avidin (e.g., streptavidin).

In certain embodiments of the application, the detectable label can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluoroimmunoassay, chemiluminescent immunoassay, etc.).

In certain embodiments of the application, a detectable label as described above may be attached to an antibody or antigen binding fragment thereof of the application by linkers of different lengths to reduce potential steric hindrance.

Preparation of antibodies

Antibodies according to any aspect of the application may be prepared by various methods known in the art, for example by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy chain variable region and the light chain variable region genes of the antibodies of the application 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 application.

The antigen binding fragments of any of the aspects of the application may be obtained by hydrolysis of the intact antibody molecule (see Morimoto et al, J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al, science 229:81 (1985)). In addition, these antigen binding fragments can also be produced directly from recombinant host cells (preparation of different fragments of SARS-CoV-2N protein and use in fluorescence chromatography, journal of bioengineering, published time 2021.06.28). For example, fab' fragments may be obtained directly from host cells; fab 'fragments can be chemically coupled to form F (ab') 2 fragments (Carter et al, bio/Technology,10:163-167 (1992)). Alternatively, fv, fab or F (ab') 2 fragments may 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.

To this end, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen binding fragment thereof of the application, or a heavy chain variable region and/or a light chain variable region thereof. In certain embodiments of the application, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the application, or a heavy chain variable region and/or a light chain variable region thereof.

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

In another aspect, the application provides a host cell comprising an isolated nucleic acid molecule of the application or a vector of the application. Such host cells include prokaryotic cells, such as E.coli cells (TG 1), and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells (e.g., CHO-K1, CHO-S, CHO DG 44), e.g., mouse cells, human cells, etc.).

In another aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof of the application comprising culturing a host cell of the application 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.

Antibody use

In another aspect, embodiments of the application disclose a kit comprising an antibody or antigen-binding fragment thereof provided herein.

In certain embodiments of the application, the antibodies or antigen binding fragments thereof provided herein further comprise a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridine ester compound), a fluorescent dye (e.g., isothiocyanate or fluorescent protein), a radionuclide, or biotin.

To this end, the disclosed kits further comprise a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. 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), a fluorescent dye (e.g., isothiocyanate or fluorescent protein), a radionuclide, or biotin.

In another aspect, embodiments of the application disclose a method for detecting the presence or level of SARS-CoV-2, or a mutant strain thereof, or an S protein or RBD of an S protein thereof, or an S1 subunit or RBD of an S1 subunit thereof, in a sample, the mutant strain being selected from the group consisting of a SARS-CoV-2 Omicron BA.1 mutant strain, an Omicron BA.2 mutant strain, a Beta mutant strain and a Delta mutant strain, the method comprising using an antibody or antigen binding fragment thereof disclosed herein.

In certain embodiments of the application, the assay is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay; for example, the antibody or antigen binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin; for example, the method further comprises detecting the antibody or antigen binding fragment thereof using a secondary antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin.

In another aspect, the present embodiments disclose the use of the antibody or antigen binding fragment thereof in the preparation of a kit for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or an S protein or RBD of an S protein, or an S1 subunit or RBD of an S1 subunit in a sample, or for diagnosing whether a subject is infected with SARS-CoV-2, or a mutant thereof, or an RBD of an S protein or S protein, or an RBD of an S1 subunit or S1 subunit thereof, selected from the group consisting of an Omicron BA.1 mutant, an Omicron BA.2 mutant, a Beta mutant and a Delta mutant;

In certain embodiments of the application, the kit detects the presence or level of SARS-CoV-2 in a sample by the method. In some 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, optionally a human).

In another aspect, embodiments of the application disclose a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier and/or excipient; optionally, the pharmaceutical composition further comprises additional pharmaceutically active agents, such as famprivir, adefovir, interferon, and the like.

In certain embodiments of the application, the antibody or antigen-binding fragment thereof in the pharmaceutical composition and the additional pharmaceutically active agent may be provided as separate components or as mixed components for simultaneous, separate or sequential administration. In some embodiments, the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In some 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, embodiments of the application disclose a method for neutralizing toxicity of SARS-CoV-2, or a mutant strain thereof, or an RBD of S protein or S protein thereof, or an RBD of S1 subunit or S1 subunit thereof, in a sample. The method comprises contacting a sample comprising SARS-CoV-2, or a mutant strain thereof, or an RBD of S protein or S protein thereof, or an S1 subunit thereof, or an RBD of S1 subunit thereof, with an antibody or antigen-binding fragment thereof disclosed in the examples of the present application.

In another aspect, the present examples disclose the use of the antibody or antigen binding fragment thereof for the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample, or for preventing or treating SARS-CoV-2, or a mutant thereof, or an RBD of S protein or S protein, or an RBD infection of S1 subunit or S1 subunit thereof, or a disease associated with an RBD of SARS-CoV-2, or a mutant thereof, or an RBD of S protein or S1 subunit thereof, in a subject (e.g., COVID-19).

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

In some embodiments, the antibody or antigen binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent such as interferon, lopinavir, ritonavir, chloroquine phosphate, fampride Weirui darcy, etc.).

The antibodies or antigen-binding fragments thereof disclosed herein, or the pharmaceutical compositions of the application, 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, injectable sterile powders and injectable concentrated solutions), inhalants, sprays, and the like. The alternative dosage form depends on the intended mode of administration and the therapeutic use. The pharmaceutical compositions of the present application should be sterile and stable under the conditions of manufacture and storage. An alternative 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 recombinant proteins of the present application are incorporated in the necessary amount in a suitable solvent, and optionally, other desired ingredients (including pH modifiers, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof) are incorporated simultaneously, 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, or antigen-binding fragments thereof, or pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, including, orally, buccally, sublingually, ocularly, topically, parenterally, rectally, intrathecally, intracytoplasmic omentum, inguinal, intravesical, topically (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In an alternative embodiment, the antibody or antigen-binding fragment thereof, or pharmaceutical composition of the application is administered by intravenous injection or bolus injection.

The pharmaceutical compositions disclosed herein may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof of the present application. "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 disclosed herein may vary depending on the factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

In embodiments of the application, the dosing regimen may be adjusted to achieve an 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.

Definition of terms

In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

As described herein, "Severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)", which is known as "novel coronavirus" or "2019-nCoV", belongs to the genus Beta coronavirus, which is a single-stranded positive sense RNA virus containing an envelope. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found in, for example, genBank: MN908947.SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 is, like SARS-CoV, specifically combined with angiotensin transferase 2 (ACE 2) on host cell by receptor binding domain (Receptor binding domain, RBD) on S protein, and then grafted with membrane fusion and cell entry of virus, which plays a vital role in the process of virus infection of cells.

As used herein, the term "COVID-19" refers to pneumonia caused by SARS-CoV-2 infection.

As used herein, the terms "S protein" and "spike protein" both refer to the surface spike protein of SARS-CoV-2, having a Receptor Binding Domain (RBD) thereon, both having the same meaning and being used interchangeably.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair comprising a light chain (L) and a heavy chain (H). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as mu, delta, gamma, alpha or epsilon, and the isotypes of antibodies can be defined as IgM, igD, igG, igA and IgE, respectively, depending on the heavy chain. 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 three or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (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 region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). 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 VH and VL is prepared from the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of three CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to regions or domains follows Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883. 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 types, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As described herein, the term: "complementarity determining regions" or "CDRs" refer to the amino acid residues in the variable regions of an antibody that are responsible for antigen binding. The exact boundaries of these amino acid residues 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)、Chothia (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 the person skilled in the art (see, for example, LEFRANC ET al. Dev. Comparat. Immunol.27:55-77,2003).

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. Non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') 2, fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabody, single domain antibody (single domain antibody), chimeric antibody, linear antibody (technology from Domanis), probody and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide.

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" means 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, a heavy chain constant region CH3 domain in the direction from the N-terminus to the C-terminus; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Alternatively, 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 application may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the application 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 antigen-binding fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antigen-binding fragment consisting of a VH domain (Ward et al Nature 341:544546 (1989)); the term "Fab fragment" means an antigen-binding fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') 2 fragment" means an antigen binding fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means the fragment obtained after reduction of the disulfide bond joining the two heavy chain fragments of the F (ab') 2 fragment, consisting of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antigen-binding fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antigen-binding 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 antigen binding fragment formed by disulfide bonding of a second and third constant region of a first heavy chain of an antibody to a second and third constant region 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. USA 85:5879-5883 (1988); and Pluckaphun, the Pharmacology of Monoclonal Antibodies, vol. 113, roseburg and Moore, springer-Verlag, new York, pp 269-315 (1994)). Such scFv molecules may have the general structure: 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 linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al (1993), proc.Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present application 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 application, 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 application, an scFv may form (scFv) 2, which refers to two or more individual scFv that are connected in parallel to form an antibody.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but 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, structure2:1121-1123 (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 antigen-binding 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 antigen-binding fragments described above 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 antigen binding fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the application) 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 of antibodies in the same manner as used for intact antibodies.

As used herein, the term "chimeric antibody (Chimeric antibody)" means that a portion of its light or/and heavy chains 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 its light or/and heavy chains 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 in any event it retains binding activity to the antigen of interest (u.s.p4, 816,567to cabill et al; morrison et al, proc.Natl. Acad.Sci.USA, 81:68553 (1984)). In certain embodiments of the application, the term "chimeric antibody" may include, for example, a human murine chimeric antibody in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody). In certain embodiments of the application, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., an individual human antibody sequence) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human consensus germline antibody sequence).

To prepare chimeric antibodies, the variable region of one antibody can be linked to the constant region of another antibody (e.g., a human immunoglobulin) using methods known in the art. For example, the DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full length heavy chain gene. The sequence of a human heavy chain constant region gene is known in the art (see, e.g., kabat, e.a. et al (1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH PublicationNo.91-3242),, DNA fragments comprising these regions may be obtained by standard PCR amplification, the heavy chain constant region may be an IgG1, igG2, igG3, igG4, igA, igE, igM or IgD constant region, but is typically selected as an IgG1 or IgG4 constant region, e.g., DNA encoding VL is operably linked to another DNA molecule encoding light chain constant region CL to obtain a full length light chain gene (and Fab light chain gene).

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 usually only 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 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.).

To prepare humanized antibodies, CDR regions of immunized animals (e.g., mice) can be grafted into a human framework sequence using methods known in the art (see, humanized antibody library design for anti-liver cancer single chain antibodies based on homology modeling techniques [ J ]. J. Senior.in China, 2015, 16 th edition, ISSN: 1005-9202).

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 "germline antibody gene (germline antibody gene)" or "germline antibody gene fragment (germline antibody GENE SEGMENT)" refers to an immunoglobulin-encoding sequence present in the genome of an organism that has not undergone a maturation process that can result in the genetic rearrangement and mutation of the expressed specific immunoglobulin. Accordingly, the term "rearranged antibody sequence" refers to a sequence of a specific antibody that has undergone a maturation process that results in the expression of genetic rearrangements and mutations of a specific immunoglobulin. In the present application, the expression "heavy chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin heavy chains, which include V genes (variable), D genes (diversity), J genes (joining), and C genes (constant); similarly, the expression "light chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin light chains, including the V gene (va ria ble), the J gene (joining), and the C gene (constant). In the present application, the amino acid sequence encoded by the germline antibody gene or germline antibody gene fragment is also referred to as "germline sequence (germline sequence)", the amino acid sequence encoded by the heavy chain germline gene is referred to as a heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene is referred to as a light chain germline sequence. Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from professional databases (e.g., IMGT, UNSWIg, NCBI or VBASE 2).

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: a plasmid; phagemid; 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, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including 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, and includes, for example, a prokaryotic cell such as E.coli or B.subtilis, a fungal cell such as a yeast cell or a Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast, CHO cell, COS cell, NSO cell, heLa cell, BHK cell, HEK293 cell or human cell.

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. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with an affinity (KD) of less than about 10 ^-5 M, such as less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less.

As used herein, the term "KD" 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. Typically, antibodies bind antigen with a dissociation equilibrium constant (KD) of less than about 10 ^-5 M.

As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antigen binding fragment that binds to an antigen 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 antigen binding fragment with neutralizing activity may prevent the amplification of a virus, thereby inhibiting or eliminating the infection by a virus.

As used herein, the term "identity" is used to refer to the match of sequences between two proteins, two polypeptides, or 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 (three of the total 6 positions match). Typically, the comparison is made when two sequences are aligned for 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 algorithm of E.Meyers and W.Miller (Comput. Appl biosci.,4:11-17 (1988)) which has 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)) algorithms that have 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 the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the 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, another amino acid residue from the same side chain family may be selected to replace the corresponding amino acid residue. Methods for identifying amino acid conservative substitutions 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,Sunderland,Mass.(1991)),, incorporated by reference herein. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present application, 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.Edited by Gennaro AR,19th.ed.Pennsylvania:Mack Publishing Company,1995), and includes: for example, pH adjusting agents include phosphate buffers, surfactants include cationic, anionic or nonionic surfactants such as Tween-80, ionic strength enhancers include sodium chloride, preservatives include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid and the like, agents to maintain osmotic pressure include sugars, naCl and the like, agents to delay absorption include monostearates and gelatin, diluents include water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol) and the like, preservatives include various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid and the like, stabilizers have the meanings commonly understood by those skilled in the art that are capable of stabilizing the desired activity of active ingredients in pharmaceuticals including sodium, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, dextran, glycine, glutamic acid, such as lactose, glycine, lactose, glutamic acid, whey, or glucose, lactose, glutamic acid, whey (e.g., lactose), and the like, 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 that is performed in order to prevent or delay the occurrence of a disease or disorder or symptom 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 application, beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of disease progression, amelioration or palliation of the state of the disease, 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 "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of a disease; 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.

The beneficial effects of the application are that

The present application provides antibodies or antigen binding fragments having specific binding ability to SARS-CoV-2, or a mutant thereof, or an RBD of S protein or S1 subunit thereof, or an RBD of S1 subunit or S1 subunit thereof, or to SARS-CoV-2, or a mutant thereof, or an RBD of S protein or S protein thereof, or an RBD of S1 subunit or S1 subunit thereof. Wherein the mutant strain of SARS-CoV-2 comprises SARS-CoV-2 Omicron BA.1 mutant strain, SARS-CoV-2 Omicron BA.2 mutant strain, SARS-CoV-2 Beta mutant strain and SARS-CoV-2 Delta mutant strain. In particular, these monoclonal antibodies bind to an epitope on the RBD region of the S protein of SARS-CoV-2 and neutralize SARS-CoV-2. The monoclonal antibody of the application can inhibit the combination of RBD protein of SARS-CoV-2 and receptor ACE 2.

Drawings

FIG. 1 shows the results of protein electrophoresis of antibodies Amb1 (left panel) and Amb2 (right panel) provided in the examples of the present application.

FIG. 2 shows ELISA detection results of binding activities of antibodies Amb1, amb2 and recombinant protein ACE2-hFc provided by the embodiment of the application with wild-type novel coronavirus RBD protein respectively.

FIG. 3 is a graph showing the measurement of the affinity of antibodies Amb1, amb2 and recombinant protein ACE2 provided in the examples of the present application with wild-type novel coronavirus RBD protein and novel coronavirus Omicron BA.1 mutant strain RBD protein, respectively.

FIG. 4 shows the measurement curves of neutralization activities of antibodies Amb1 and Amb2 provided in the examples of the present application, respectively, with Omicron BA.1 mutant, omicron BA.2 mutant and wild-type novel coronavirus.

FIG. 5 shows the neutralization activity measurement curves of the antibodies Amb1 and Amb2 provided in the examples of the present application, respectively, with the Beta mutant strain and the Delta mutant strain pseudovirus.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present application 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 application by way of example and are not intended to limit the scope of the application as claimed.

Construction and screening of fully human anti-novel coronavirus scFv antibody library

Total RNA was extracted by lysis of human Peripheral Blood Mononuclear Cells (PBMCs) isolated from 10mL whole blood extracted from inactivated vaccinators (three needles) using the one-step total RNA extraction reagent TRIzol. Then, reverse transcription PCR was performed using oligo-dT as a primer to prepare cDNAs, respectively, and the cDNAs were mixed in equal volumes. Antibody genes VH, V.kappa.and V.lambda.were amplified using human antibody signature primers (see volume 275, page 46, 36073-8 of Generation and Characterization of a Recombinant Human CCR5-specific Antibody:a phage display approach for rabbit antibody humanization,"The Journal of biological chemistry"2000), V.kappa.and V.lambda.linker-VH were further obtained by overlap extension Polymerase Chain Reaction (PCR), and the cut fragments were ligated into the pre-cut phage display vector pComb3XSS using T4 DNA ligase after single cleavage with restriction enzyme Sfi I. The linked product was desalted by ethanol precipitation and transferred to TG1 bacteria competent by electrotransformation to construct single chain antibody (scFv) libraries with a pool capacity of 1.5×10 ⁹ and 2.2×10 ⁹, respectively, for subsequent screening. The process is basically referred to (Barbas,C.F.,III;Burton,D.R.;Scott,J.K.,Silverman,G.J.Eds.(2001)Phage Display:A Laboratory Manual;Cold Spring Harbor Laboratory Press:Cold Spring Harbor,New York,736pages.)

Enrichment screening of phage antibody library and induced expression of scFV antibody

The screening antigen used was purified novel coronavirus wild-type RBD protein (RBD-WT, cat#Z03479, available from Gensript company) and was screened as follows:

Diluting the antigen with PBS solution at ph=7.4, coating 96-well plate immunowells, 100 μl per well; coating overnight at 4 ℃. PBST was washed three times, 200. Mu.L/well of blocking buffer was added, and the mixture was left overnight at 4 ℃. PBST wash 5 times, 200 u L per well, with blocking buffer dilution library phage to 1X 10 ¹¹, per well 100 u L,37 degrees C1.5 h. PBST was washed 5 times, then washed 3 times with PBS, and 100. Mu.L of glycine-HCl eluent at pH 2.2 was added to each well, followed by the immediate neutralization by the addition of 15. Mu.L of Tris-HCl at pH 9.1. The remaining eluate was mixed with 5ml log phase TG1 at 37℃and 220rpm for 45min. Transferring the bacterial solution to 20mL 2YT/A+/G culture medium, culturing to logarithmic phase, adding auxiliary Phage VCSM13 (Stratagene in U.S.) for infection, incubating at 37 ℃ for 1h, adding kanamycin (final concentration of 50 mug/mL) and shaking overnight at 30 ℃ for culturing to prepare Phage single-chain antibody, coating an ELISA plate with 0.1 mug/hole SARS-CoV-2 RBD protein, and using PBS buffer (containing 5G/mL skimmed milk powder) to dilute HRP-labeled anti-M13 antibody according to 1:2000 for Phage-ELISA identification and OD450 value determination. The screening was repeated 3 times. Specific enrichment screening method and induction expression process basic reference of scFv segment (Barbas,C.F.,III;Burton,D.R.;Scott,J.K.,Silverman,G.J.Eds.(2001)Phage Display:A Laboratory Manual;Cold Spring Harbor Laboratory Press:Cold Spring Harbor,New York,736pages.).

Through the above experiments, scFv segment 1, scFv segment 2, scFv segment 3 and scFv segment 4 were obtained. The scFv segment 1 has a heavy chain variable region with an amino acid sequence shown as SEQ ID NO.1 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 3. The scFv segment 2 has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO. 4. The scFv segment 3 has a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO.1 and a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO. 4.scFv segment 4, a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO.2 and a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 3.

Further, the CDR sequences in scFv fragment 1 and scFv fragment 2, scFv fragment 3scFv fragment 4 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, besselda, maryland (1991), pages 647-669). Wherein, the heavy chain CDR 1-3 shown in SEQ ID NO.1 is shown in sequence as SEQ ID NO. 5-7, the heavy chain CDR 1-3 shown in SEQ ID NO.2 is shown in sequence as SEQ ID NO. 8-10, the light chain CDR 1-3 shown in SEQ ID NO.3 is shown in sequence as SEQ ID NO. 11-13, and the light chain CDR 1-3 shown in SEQ ID NO.4 is shown in sequence as SEQ ID NO. 14-16.

Preparation of whole antibodies

Plasmids were extracted from the bacterial solutions of the positive clones using a plasmid extraction kit (QIAGEN MINIPREP KIT, QIAGEN, germany) and sent to commercial company for sequencing. The sequencing result is compared with IgG gene sequences in databases such as V-Base gene library (http://www.vbase2.org/)、IMGT(http://www.imgt.org/)、V-QUEST(http://www.imgt.org/IMGT_vquest/share/textes/)、Ig BLAST(http://www.ncbi.nlm.nih.gov/igblast/). The whole antibody variable region gene sequence obtained by sequencing (the coding sequence of the amino acid sequences shown in SEQ ID NO. 1-4 is shown in SEQ ID NO. 17-20) is converted into the form of an IgG1 whole antibody, the whole antibody is connected into a secretory mammal expression vector pHL-Sec (purchased from Addgene, #99845 and modified from classical mammal expression plasmid pCAGGS) by a seamless cloning method after total gene synthesis, a leader peptide sequence (Murine Ig kappa-CHAIN LEADER sequence, METDTLLLWVLLLWVPGSTGDJ) of a murine kappa chain is used for replacing a signal peptide sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) carried on the vector, and a stop codon is added after the sequence. Thus, heavy chain expression plasmid pHL-HC and light chain expression plasmid pHL-LC were constructed, respectively.

The pHL-HC and pHL-LC which are correctly sequenced are subjected to endotoxin-free mass extraction. HEK293F cells were cultured in suspension in FreeStyle ^TM expression medium, inoculated at a density of 1.0X10 ⁶ cells/mL one day before transfection in disposable conical flasks, and the inoculated cells were placed in a 5% CO ₂ incubator at 37℃and shake cultured at 125 rpm. Cell counts were performed on the day of transfection, transient transfection was performed at a cell density of 2.0X10 ⁶ cells/mL (diluted with medium if the density was exceeded) with viability greater than 95%. The heavy chain plasmid pHL-HC, light chain plasmid pHL-LC and PEI (Polysciences, 1. Mu.g/. Mu.L) were mixed in a ratio of 1:1:8 (wherein "1" means 1. Mu.g/mL cells) to prepare a DNA-PEI complex, which was added to a cell culture flask to be transfected, gently shaken, and placed in a 5% CO ₂ incubator for continuous shake culture at 37 ℃. After 4d of transfection, supernatants were collected daily and cells were dynamically analyzed for recombinant protein expression, and after 7d of transfection, supernatants were collected.

After suction filtration with a 0.22 μm microporous filter, 1/10 volume of 10 XpH adjusting buffer (0.5 MNA ₂HPO₄, pH 7.4) was added, the supernatant of the recombinant antibody-containing medium was directly passed through two 5mL HiTrap Protein G HP pre-packed columns connected in series in a protein purification system (AKTA pure 25) at a flow rate of 5mL/min, and after completion of the binding at a low flow rate, the UV absorbance was no longer changed by rinsing with binding buffer (20 mM Na ₂HPO₄, pH 7.0) to 280 nM. Then eluting with elution buffer (0.1M glycine-HCl, pH 2.7), and adding 1/10 volume of neutralization buffer (1M Tris-HCl, pH 9.0) into the collection tube. Detecting the purification condition by SDS-PAGE coupled bromophenol blue, determining the peak of the target protein, collecting the target protein, concentrating, further purifying by an anion exchange column and a molecular sieve, and then using the obtained product in the next experiment. Optionally, collecting supernatant, centrifuging to remove cells and fragments thereof, and purifying with Protein G HP SPINTRAP centrifugal small amount antibody affinity chromatography column to obtain small amount of monoclonal antibody. The purification method is as follows: the centrifuged supernatant was mixed with an equal volume of binding buffer (20 mM Na ₂HPO₄, pH 7.0). 600. Mu.L of binding buffer was added and 100g centrifuged for 30s to equilibrate the column. 600. Mu.L of pre-equilibrated sample was added repeatedly, centrifuged at 100g for 30s, gently mixed, and allowed to stand for 4min to bind the antibody to the column. 600. Mu.L of binding buffer was added, and the mixture was centrifuged at 100g for 30s to wash out impurities. The procedure was repeated twice. 400. Mu.L of elution buffer (0.1M Glycine-HCl, pH 2.7) was added, mixed upside down, placed in a 2mL EP tube containing 30. Mu.L of neutralization buffer (1M Tris-HCl, pH 9.0), centrifuged at 100g for 30s, and repeated twice.

As shown in FIG. 1, the purification gave an electropherogram of the recombinantly expressed antibodies Amb1 and Amb2, each having a band around 50kD and 30kD, representing the heavy and light chains, respectively.

Binding Activity of Amb1 and Amb2 on RBD proteins of different novel coronavirus strains

1. ELISA detection

Coating novel coronavirus wild-type RBD protein (cat#z 03479, gensript) on an elisa plate with ph=9.60.1m NaHCO ₃ solution overnight at 4 ℃; blocking with 3% skimmed milk, incubating at 37deg.C for 1 hr, adding recombinant expressed Amb1 and Amb2 antibodies and ACE2-hFc recombinant protein (control), and incubating at 37deg.C for 1 hr; adding a secondary antibody of an enzyme-labeled anti-human Fc fragment, and incubating for 1h at 37 ℃; the color development liquid develops color, 2M H ₂SO₄ ends the reaction, and the enzyme label instrument detects OD450. The results show that the antibodies Amb1 and Amb2 have high binding activity, and the results show that the binding activity of the antibodies Amb1 and Amb2 on RBD proteins of novel coronavirus strains is higher than that of ACE 2-hFc.

2. BIAcore method for determining antibody affinity

The novel coronavirus wild type (cat#z 03479, gensript) and omacron ba.1 mutant RBD proteins (cat#z 03516, gensript) were diluted in 10mM NaAc and binding capacity of antibodies to CM5 chips was tried under different pH conditions. The pH value is set to four gradients of pH4.0, pH4.5, pH5.0 and pH5.5, and the pH with the best fixing effect is selected. The RBD was diluted to 10mM NaAc at optimal pH, immobilized on the chip surface, and the target coupling amount RU (Response Unit) of each antibody was determined according to the formula. Amb1 and Amb2 antibodies are used as mobile phases, the concentration gradient is set to be 2.6 nM-333 nM or 666nM, and response values generated by flowing Amb1, amb2 and ACE2-hFc with different concentrations through the chip surface are detected. The analysis was carried out at a constant temperature of 25℃and a flow rate of 30. Mu.L/min. The solution used for regenerating the chip surface was 100mM H ₃PO₄. Binding kinetic constants were calculated using BIAevaluation software version (Biacore, inc.) software to calculate affinity constants (K _D (M)) according to a 1:1 binding pattern.

As a result, as shown in FIG. 3, amb1 and Amb2 have affinity constants of 1.51X10 ^-7 and 1.64X10 ^-7, respectively, for wild-type RBD; the affinity constant of ACE2-hFc for wild-type RBD was 3.09×10 ^-7; amb1 and Amb2 have affinity constants of 2.05X10 ^-7 and 3.33X10 ^-7, respectively, for omacron ba.1 mutant RBD; the affinity constant of ACE2-hFc for Omicron ba.1 mutant RBD is 5.10 x 10 ^-7, which not only demonstrates that Amb1 and Amb2 have affinity for new coronaviruses and mutants thereof, and have higher affinity than the receptor ACE2 itself, have a blocking agent as a competitive blocking agent for the binding of viruses to the receptor ACE2, and thus find application in related drugs or formulations for preventing viral infections.

Pseudovirus neutralization assay

Preparation of pseudoviruses: 5-6X 10 ⁶ 293T cells were spread 1 day in advance in 6-well plates and cultured for 18-24 h, 2. Mu.g HIV-1 backbone plasmid pNL4-3. Luc.R-E-and 2. Mu. g S protein expression plasmids (SARS-CoV-2 wild-type, SARS-CoV-2 Omicron BA.1, SARS-CoV-2 BA.2, SARS-CoV-2 Beta or SARS-CoV-2 Delta) were co-transferred into 293T cells using 12. Mu.L FuGENE HD (cat#E2311, promega) transfection reagent according to the instructions. After 48h from the beginning of transfection, the culture medium in the 6-well plate is sucked out, added into a sterile 15mL centrifuge tube, centrifuged at 3000rpm for 15min, and the supernatant is taken to obtain the pseudoviruses of SARS-CoV-2 wild type, SARS-CoV-2 Omicron BA.1, SARS-CoV-2 BA.2, SARS-CoV-2 Beta or SARS-CoV-2 Delta, and frozen in a refrigerator at-80 ℃ for standby.

Viruses were transferred from-80℃to ice in advance for thawing, and serum-free DMEM was prepared. 293T-ACE2 cells (Yeasen next hol, cat. No. 41107ES 03) were seeded into 96-well cell culture plates at approximately 1.2X10- ⁴ cells/well one day in advance, and the infection was performed according to cell density the next day. The cell density is preferably about 50%. The antibodies were diluted with DMEM, the concentration of the first EP tube was 75.0. Mu.g/mL and the total volume was 100. Mu.L, followed by 80. Mu. LDMEM for each EP tube, 20. Mu.L of the previous tube was pipetted into the next tube, 5-fold dilution, and 10 gradients were set. Another new 96-well plate was prepared, and 96.0. Mu.L of pseudovirus plus 24.0. Mu.L of antibody (at which time the concentration of antibody was 15.0. Mu.g/mL) was gently mixed, incubated at 37℃for 1 hour, and 3 wells were additionally provided and 100. Mu.L of pseudovirus without antibody (96.0. Mu.L plus 24.0. Mu.L of DMEM) was added as a control. The culture medium in the 96-well cell culture plate is discarded, 100 mu L of the antibody pseudovirus mixed solution is added, the solution is changed after 6 hours, and the luciferase activity is detected after 48 hours. In detection, the plate is taken out in advance, placed at room temperature for 30min, and meanwhile, the detection reagent (cat#DD 1201-01, norflu) is taken out from the temperature of minus 20 ℃ for thawing, and 100 mu L of the detection reagent is added into each hole for uniform mixing and then cracking for more than 3 min. Then transferring the sample to a detection plate by a row gun, placing the sample on a GloMax micro-plate luminescence detector for detection, and performing data processing by GRAPHPADPRISM software.

As shown in FIGS. 4 and 5, the wild-type IC ₅₀ for Amb1 and Amb2 neutralized mutant Omicron BA.1 was 0.1303. Mu.g/mL and 0.064. Mu.g/mL, respectively, the IC ₅₀ for Amb1 and Amb2 neutralized mutant Omicron BA.1 was 0.02464. Mu.g/mL and 0.01549. Mu.g/mL, respectively, the IC ₅₀ for Amb1 and Amb2 neutralized Omicron BA.2 was 0.01126. Mu.g/mL and 0.02734. Mu.g/mL, respectively, and the IC ₅₀ for Amb1 and Amb2 neutralized Beta mutant was 0.02660. Mu.g/mL and 0.04516. Mu.g/mL, respectively. IC ₅₀ for Amb1 and Amb2 and Delta mutants was 1.220 μg/mL and 0.4077 μg/mL, respectively.

In conclusion, the antigen binding fragment scFv disclosed by the application has high affinity activity on RBD proteins of novel coronaviruses, amb1 and Amb2 prepared by genetic engineering means have strong neutralization effects on pseudoviruses of wild type, beta mutant, delta mutant, omicron BA.1 and BA.2, have potential to cope with outbreak epidemic caused by Omicker variant strains and constantly occurring novel Omicker variant strains capable of evading vaccines and neutralizing antibodies, and have great clinical application value.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2 and mutants thereof, comprising:

2. An antibody or antigen-binding fragment thereof that specifically binds to the Receptor Binding Domain (RBD) of the S protein of SARS-CoV-2 and mutants thereof, comprising:

(a) An amino acid sequence as shown in SEQ ID NO. 2;

(d) A sequence shown in SEQ ID NO. 4;

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, further comprising a framework region sequence derived from a human immunoglobulin;

Alternatively, the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence;

optionally, the antibody or antigen binding fragment thereof comprises: heavy chain framework region sequences derived from human rearranged antibody sequences, and light chain framework region sequences derived from human rearranged antibody sequences;

Optionally, the antibody or antigen binding fragment thereof comprises: heavy chain framework region sequences derived from human heavy chain germline sequences and light chain framework region sequences derived from human light chain germline sequences;

Alternatively, the antigen binding fragment is selected from the group consisting of Fab, fab ', (Fab') ₂, fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb);

Alternatively, the antibody is a rabbit antibody, chimeric antibody, humanized antibody, bispecific antibody, or multispecific antibody.

4. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3, or a heavy chain variable region and/or a light chain variable region thereof.

5. A vector comprising the nucleic acid molecule of claim 4; alternatively, the vector is a cloning vector or an expression vector.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3; for example, the antibody or antigen binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., isothiocyanate or fluorescent protein), a radionuclide, or biotin; for example, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; 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), a fluorescent dye (e.g., isothiocyanate or fluorescent protein), a radionuclide, or biotin.

8. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-3, and a pharmaceutically acceptable carrier and/or excipient.

9. The pharmaceutical composition of claim 8, further comprising an additional pharmaceutically active agent selected from at least one of interferon, lopinavir, ritonavir, chloroquine phosphate, fampica Weirui darunavir.

10. Use of the antibody or antigen binding fragment thereof of any one of claims 1-3 in the preparation of a kit for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or an S protein or RBD of an S protein, or an S1 subunit or RBD of an S1 subunit in a sample, or for diagnosing whether a subject is infected with SARS-CoV-2, or a mutant thereof, or an RBD of an S protein or an S protein, or an RBD of an S1 subunit or an S1 subunit thereof, said mutant selected from the group consisting of an Omicron ba.1 mutant, an Omicron ba.2 mutant, a Beta mutant and a Delta mutant; or for neutralizing toxicity of SARS-CoV-2, or a mutant strain thereof, or an RBD of S protein or S protein thereof, or an RBD of S1 subunit or S1 subunit thereof in a sample; or for the preparation of a medicament;

optionally, the kit is for detecting the presence or level of SARS-CoV-2 in a sample;

Alternatively, the assay is an immunological assay, such as an enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay; for example, the antibody or antigen binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin; for example, the method further comprises detecting the antibody or antigen binding fragment thereof using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin;

Optionally, 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, optionally a human);

Optionally, the step of neutralizing comprises contacting a sample comprising SARS-CoV-2, or a mutant thereof, or an RBD of S protein or S protein thereof, or an S1 subunit thereof, or an RBD of S1 subunit, with the antibody or antigen-binding fragment thereof of any one of claims 1-3;

Alternatively, the medicament is for neutralizing toxicity of SARS-CoV-2 in a sample, or for preventing or treating SARS-CoV-2, or a mutant thereof, or an S protein or RBD of an S protein, or an S1 subunit or RBD infection of an S1 subunit thereof, or a disease associated with SARS-CoV-2, or a mutant thereof, or an RBD of an S protein or S protein, or an RBD infection of an S1 subunit or S1 subunit thereof, in a subject (e.g., COVID-19);

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

Alternatively, the antibody or antigen binding fragment thereof is used alone or in combination with another pharmaceutically active agent (e.g., another antiviral agent such as interferon, lopinavir, ritonavir, chloroquine phosphate, fampride Weirui darcy, etc.).