Antibodies to SARS-CoV-2 and its mutants and their applications

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. Specifically, the present application relates to antibodies or antigen-binding fragments against SARS-CoV-2 and its mutants, and compositions (e.g., diagnostic agents and therapeutic agents) comprising the antibodies. In addition, the present application also relates to the use of the antibodies or antigen-binding fragments. The antibodies or antigen-binding fragments of the present application can be used to diagnose, prevent and / or treat SARS-CoV-2 and its mutants infection and / or diseases caused by the infection.

Description

Technical Field

[0001] 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. Specifically, the present application relates to antibodies or antigen-binding fragments against SARS-CoV-2 and its mutants, and compositions (e.g., diagnostic agents and therapeutic agents) comprising the antibodies. In addition, the present application also relates to the use of the antibodies or antigen-binding fragments. The antibodies or antigen-binding fragments of the present application can be used to diagnose, prevent and / or treat SARS-CoV-2 and its mutants infection and / or diseases caused by the infection. Background Art

[0002] COVID-19, caused by the novel coronavirus (SARS-CoV-2 or HCoV-2019), has become the most serious infectious disease in human history. The clinical features of COVID-19 include fever, dry cough and fatigue, respiratory failure, and even death.

[0003] The SARS-CoV-2 spike glycoprotein (Spike, S) plays a key role in the infection and pathogenesis of the new coronavirus. The mature S protein of the SARS-CoV-2 virus is a highly glycosylated trimer, each protomer of which consists of 1260 amino acids (residues 14-1273), of which the S1 subunit consists of 672 amino acids (residues 14–685) and is divided into four domains: an N-terminal domain (NTD), a C-terminal domain (CTD, also known as the receptor binding domain, RBD) and two subdomains (SD1 and SD2).

[0004] The SARS-CoV-2 S glycoprotein is a conformational machine that mediates viral entry by transforming from a metastable untriggered state, through a pre-hairpin intermediate, to a stable postfusion state. Angiotensin-converting enzyme 2 (ACE2) has been demonstrated to be the primary receptor for SARS-CoV-2 (47-49). Detailed interactions between the SARS-CoV-2 RBD and its receptor ACE have been revealed by the structure of the complex of the two. Structurally, the RBD consists of two subdomains: the core and the outer subdomains. An extended loop (residues 438-506) located on one side of the core subdomain presents a slightly concave surface to support the ACE2 N-terminal helix (α1). Analysis of the interface between the SARS-CoV-2 RBD and ACE2 showed that a total of 17 residues in the RBD contact 20 amino acids in ACE2, forming a hydrophilic interaction network that dominates the binding of the virus to the receptor.

[0005] The RBD of the SARS-CoV-2 S glycoprotein binds to the ACE2 receptor on the surface of the target cell, initiating the membrane fusion and subsequent viral invasion of SARS-CoV-2. After the S glycoprotein binds to ACE2 on the plasma membrane (plasma membrane invasion pathway) or the virus particle is subsequently endocytosed by the host cell (endosomal invasion pathway), a second cleavage (S2′ cleavage site) occurs, mediated by the cell surface serine protease TMPRSS2 or the endosomal cysteine ​​proteases cathepsins B and L, respectively. Protease cleavage at the S2′ site releases the fusion peptide from the N-terminal region of the newly formed S2 subunit, further destabilizing the SARS-CoV-2 S glycoprotein trimer and initiating 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 rapidly and irreversibly refolds to a stable post-fusion state. These huge conformational rearrangements bring the virus and host cell membranes closer, ultimately leading to membrane fusion. Therefore, the S protein determines the host range and tissue tropism of the virus. Given the crucial role of the S protein in the virus invasion process and the fact that most of it is exposed outside the viral envelope and can be directly recognized by the host immune system, the S protein is the main target of the host's humoral and cellular immunity and the main site of action of antibodies in the host. Therefore, most of the current COVID-19 vaccine strategies are based on the full-length S glycoprotein or its RBD domain as an immunogen.

[0006] However, as an RNA virus, SARS-CoV-2 has the characteristics of high mutation rate and rapid evolution. Currently, it has been designated as variants of concern (VOCs) by the World Health Organization (WHO); Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). Omicron's initial variant, BA.1, was first discovered in Botswana and South Africa in November 2021, and quickly replaced the Delta variant to become the most prevalent SARS-CoV-2 strain in the world. Another variant of Omicron, BA.2, spreads faster than the BA.1 variant, and the number of confirmed cases caused by it has exceeded BA.1 cases by the end of February 2022, becoming the current major global prevalent variant. What's more terrifying is that Omicron is still mutating rapidly, producing a large number of new intra-subtype variants. The Omicron subvariants that people are currently concerned about include at least XE, BA.2.12.1, BA.4 and BA.5.

[0007] Omicron has a strong immune escape ability against vaccines and neutralizing antibodies. Studies have shown that the vast majority of monoclonal antibodies (mAbs) completely or significantly lose their neutralizing activity against BA.1 and BA.2. Among the eleven approved or authorized monoclonal antibodies 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 against BA.1 (only reduced by about 2 times), 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, namely the core domain of RBD (with more than 85% of the same amino acid residues), and thus usually exhibit a wide range of sarbecovirus neutralizing activity, although the neutralizing activity is generally relatively low. The S371F, D405N and R408S mutations in 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 their activity against BA.1, but regained their activity against BA.2. Summary of the invention

[0008] In the present application, the inventors have developed human antibodies with excellent properties, which can neutralize SARS-CoV-2 and its mutants (Omicron BA.1 mutant, Omicron BA.2 mutant, Beta mutant and Delta mutant), block or inhibit the binding of SARS-CoV-2 and its mutants to receptor ACE2, and are not easy to induce immunogenic reactions in human subjects. Therefore, the antibodies or antigen-binding fragments provided in the present application have the potential to be used for the detection, diagnosis, prevention and / or treatment of SARS-CoV-2 infection or diseases caused by SARS-CoV-2 infection, and have great clinical value.

[0009] Antibodies of the present application

[0010] In a first aspect, the embodiments of the present application disclose an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to the receptor binding region (RBD) of the S protein of SARS-CoV-2 and its mutants, and the antibody or the antigen-binding fragment thereof comprises:

[0011] (I) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs) as defined according to the Kabat numbering system:

[0012] (a) VH CDR1, which consists of the following sequence: SEQ ID NO.5, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto,

[0013] (b) VH CDR2, which consists of the following sequence: SEQ ID NO. 6, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto, and

[0014] (c) VH CDR3, which consists of the following sequence: SEQ ID NO.7, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto; and / or,

[0015] (II) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs) as defined by the Kabat numbering system:

[0016] (d) VL CDR1, which consists of the following sequence: SEQ ID NO. 11, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto,

[0017] (e) VL CDR2, which consists of the following sequence: SEQ ID NO. 12, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto, and

[0018] (f) VL CDR3, which consists of the following sequence: SEQ ID NO. 13, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (eg, 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto.

[0019] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the first aspect comprises:

[0020] (I) 3 heavy chain CDRs: VH CDR1 of SEQ ID NO.5, VHCDR2 of SEQ ID NO.6, VH CDR3 of SEQ ID NO.7; and / or, 3 light chain CDRs: VLCDR1 of SEQ ID NO.11, VL CDR2 of SEQ ID NO.12, VL CDR3 of SEQ ID NO.13; or,

[0021] (II) three CDRs defined according to the Kabat numbering system contained in the heavy chain variable region (VH) as shown in SEQ ID NO.1; and / or three CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as shown in SEQ ID NO.3.

[0022] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof described in the first aspect further comprises a framework region sequence derived from a human immunoglobulin. In certain embodiments of the present application, the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence. In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human rearranged antibody sequence, and a light chain framework region sequence derived from a human rearranged antibody sequence. In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

[0023] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the first aspect comprises:

[0024] (I) a heavy chain variable region (VH), comprising an amino acid sequence selected from any one of the following (a) to (c):

[0025] (a) the sequence shown in SEQ ID NO.1;

[0026] (b) a sequence having one or more conservative substitutions, deletions or additions of amino acids compared to the sequence shown in SEQ ID NO.1 (e.g., 1, 2, 3, 4 or 5 conservative substitutions, deletions or additions of amino acids); or

[0027] (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 shown in SEQ ID NO.1;

[0028] and

[0029] (II) a light chain variable region (VL), comprising an amino acid sequence selected from any one of the following (d) to (f):

[0030] (d) the sequence shown in SEQ ID NO.3;

[0031] (e) a sequence having one or more conservative substitutions, deletions or additions of amino acids compared to the sequence shown in SEQ ID NO. 3 (e.g., 1, 2, 3, 4 or 5 conservative substitutions, deletions or additions of amino acids); or

[0032] (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 shown in SEQ ID NO.3.

[0033] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO.1 and a VL comprising the sequence shown in SEQ ID NO.3.

[0034] In a second aspect, the embodiments of the present application disclose an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to the receptor binding region (RBD) of the S protein of SARS-CoV-2 and its mutants, and the antibody or the antigen-binding fragment thereof comprises:

[0035] (I) a heavy chain variable region (VH) comprising the following three complementarity determining regions (CDRs) as defined according to the Kabat numbering system:

[0036] (a) VH CDR1, which consists of the following sequence: SEQ ID NO. 8, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto,

[0037] (b) VH CDR2, which consists of the following sequence: SEQ ID NO. 9, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto, and

[0038] (c) VH CDR3, which consists of the following sequence: SEQ ID NO.10, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto; and / or,

[0039] (II) a light chain variable region (VL) comprising the following three complementarity determining regions (CDRs) as defined by the Kabat numbering system:

[0040] (d) VL CDR1, which consists of the following sequence: SEQ ID NO. 14, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto,

[0041] (e) VL CDR2, which consists of the following sequence: SEQ ID NO. 15, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto, and

[0042] (f) VL CDR3, which consists of the following sequence: SEQ ID NO. 16, or a sequence having one or more conservative substitutions, deletions or additions of amino acids (eg, 1, 2 or 3 conservative substitutions, deletions or additions of amino acids) compared thereto.

[0043] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof described in the second aspect comprises:

[0044] (I) 3 heavy chain CDRs: VH CDR1 of SEQ ID NO.8, VHCDR2 of SEQ ID NO.9, VH CDR3 of SEQ ID NO.10; and / or, 3 light chain CDRs: VLCDR1 of SEQ ID NO.14, VL CDR2 of SEQ ID NO.15, VL CDR3 of SEQ ID NO.16; or,

[0045] (II) three CDRs defined according to the Kabat numbering system contained in the heavy chain variable region (VH) as shown in SEQ ID NO. 2; and / or three CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as shown in SEQ ID NO. 4.

[0046] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof described in the second aspect further comprises a framework region sequence derived from a human immunoglobulin. In certain embodiments of the present application, the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence. In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human rearranged antibody sequence, and a light chain framework region sequence derived from a human rearranged antibody sequence. In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

[0047] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof according to the second aspect comprises:

[0048] (I) a heavy chain variable region (VH), comprising an amino acid sequence selected from any one of the following (a) to (c):

[0049] (a) the sequence shown in SEQ ID NO.2;

[0050] (b) a sequence having one or more conservative substitutions, deletions or additions of amino acids (e.g., 1, 2, 3, 4 or 5 conservative substitutions, deletions or additions of amino acids) compared to the sequence shown in SEQ ID NO. 2; or

[0051] (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 shown in SEQ ID NO. 2;

[0052] and

[0053] (II) a light chain variable region (VL), comprising an amino acid sequence selected from any one of the following (d) to (f):

[0054] (d) the sequence shown in SEQ ID NO.4;

[0055] (e) a sequence having one or more conservative substitutions, deletions or additions of amino acids compared to the sequence shown in SEQ ID NO. 4 (e.g., 1, 2, 3, 4 or 5 conservative substitutions, deletions or additions of amino acids); or

[0056] (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 shown in SEQ ID NO.4.

[0057] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown in SEQ ID NO.2 and a VL comprising the sequence shown in SEQ ID NO.4.

[0058] In certain embodiments of the present application, the antigen-binding fragment described in the first or second aspect is selected from Fab, Fab', (Fab')2, Fv, disulfide-bonded Fv, scFv, diabody and single-domain antibody (sdAb); and / or, the antibody is a rabbit antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody.

[0059] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof described in the first or second aspect has one or more of the following features:

[0060] (1) specifically binds to SARS-CoV-2, or its mutants, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit, wherein the mutants include SARS-CoV-2 Omicron BA.1 mutant, Omicron BA.2 mutant, Beta mutant, and Delta mutant;

[0061] (2) blocking or inhibiting the binding of SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit to Ace2 receptor, and / or blocking or inhibiting the infection of SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit to cells;

[0062] (3) neutralizing SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit in vitro or in a subject (e.g., human);

[0063] (4) Preventing and / or treating infection with SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit, or diseases associated with infection with SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit (e.g., COVID-19).

[0064] Herein, the antibodies or antigen-binding fragments thereof described in any aspect of the present application may include variants, wherein the variants differ from the antibodies or antigen-binding fragments thereof from which they are derived only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the antibodies or antigen-binding fragments thereof from which they are derived, and substantially retain the above-mentioned biological functions of the antibodies or antigen-binding fragments thereof from which they are derived.

[0065] Derivatized Antibodies

[0066] The antibodies or antigen-binding fragments thereof described in any aspect of the present application may be derivatized, for example, connected to another molecule (e.g., another polypeptide or protein). Generally speaking, the derivatization (e.g., labeling) of a monoclonal antibody or its antigen-binding fragment has no effect on its binding to SARS-CoV-2. Thus, the antibodies or antigen-binding fragments thereof just disclosed in the present application may also include such derivatized forms. For example, the antibodies or antigen-binding fragments thereof of the present application may be functionally connected (by chemical coupling, gene fusion, non-covalent connection or other means) to one or more other molecular groups, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and / or a protein or polypeptide (e.g., avidin or a polyhistidine tag) that can mediate the binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present application may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.

[0067] In certain embodiments of the present application, the monoclonal antibody or antigen-binding fragment thereof of the present application carries a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds), a fluorescent dye (such as isothiocyanate or fluorescent protein), a radionuclide or biotin.

[0068] In this article, the detectable label described in the present application can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples thereof include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-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, Alexa750)), luminescent materials (e.g., chemiluminescent materials, such as acridinium ester compounds), magnetic beads (e.g., Dynabeads @ ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0069] In certain embodiments of the present application, the detectable label can be suitable for immunological detection (eg, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.).

[0070] In certain embodiments of the present application, the detectable label as described above can be linked to the antibody or antigen-binding fragment thereof of the present application via linkers of different lengths to reduce potential steric hindrance.

[0071] Antibody preparation

[0072] The antibodies described in any aspect of the present application can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy chain variable region and light chain variable region genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and the antibodies of the present application are expressed.

[0073] The antigen-binding fragments described in any aspect of the present application can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (Preparation of different fragments of SARS-CoV-2 N protein and application in fluorescence chromatography, Journal, Journal of Biotechnology, published on June 28, 2021). For example, Fab' fragments can 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)). In addition, Fv, Fab or F(ab')2 fragments can also be directly isolated from recombinant host cell culture fluid. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0074] To this end, the present application provides a kind of isolated nucleic acid molecule, which comprises the nucleotide sequence encoding the antibody or its antigen-binding fragment of the present application, or its heavy chain variable region and / or light chain variable region. In certain embodiments of the present application, the isolated nucleic acid molecule encodes the antibody or its antigen-binding fragment of the present application, or its heavy chain variable region and / or light chain variable region.

[0075] In another aspect, the present application provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the present application. In certain embodiments of the present application, the vector of the present application is, for example, a plasmid, a cosmid, a phage, etc.

[0076] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule of the present application or the vector of the present application. Such host cells include prokaryotic cells such as Escherichia coli cells (TG1), and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (such as mammalian cells (e.g., CHO-K1, CHO-S, CHO D44), such as mouse cells, human cells, etc.).

[0077] In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the present application is provided, which comprises culturing the host cell of the present application under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

[0078] Antibody Applications

[0079] On the other hand, an embodiment of the present application discloses a kit, which includes the antibody or antigen-binding fragment thereof provided by the present application.

[0080] In certain embodiments of the present application, the antibodies or antigen-binding fragments thereof provided herein also include a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (such as acridinium ester compounds), a fluorescent dye (such as isothiocyanate or fluorescent protein), a radionuclide or biotin.

[0081] To this end, the kit disclosed in the present application also includes a second antibody, which specifically recognizes the antibody or its antigen-binding fragment. Optionally, the second antibody also includes a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as an acridinium ester compound), a fluorescent dye (such as isothiocyanate or fluorescent protein), a radionuclide or biotin.

[0082] On the other hand, an embodiment of the present application discloses a method for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or an S protein or the RBD of the S protein, or an S1 subunit or the RBD of the S1 subunit in a sample, wherein the mutant is selected from SARS-CoV-2 Omicron BA.1 mutant, Omicron BA.2 mutant, Beta mutant and Delta mutant, and the method comprises using the antibody or antigen-binding fragment thereof disclosed in the present application.

[0083] In certain embodiments of the present application, the detection is an immunological detection, such as an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay; for example, the antibody or its antigen-binding fragment further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (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 using a second antibody carrying a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin to detect the antibody or its antigen-binding fragment.

[0084] On the other hand, the embodiments of the present application disclose the use of the antibody or its antigen-binding fragment in the preparation of a kit, wherein the kit is used to detect the presence or level of SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit in a sample, or to diagnose whether a subject is infected with SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit, wherein the mutant strain is selected from Omicron BA.1 mutant strain, Omicron BA.2 mutant strain, Beta mutant strain and Delta mutant strain;

[0085] In certain embodiments of the present application, the kit detects the presence or level of SARS-CoV-2 in a sample by the described method. In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), excrement, oral or nasal secretions, or bronchoalveolar lavage fluid from a subject (e.g., a mammal, preferably a human).

[0086] On the other hand, an embodiment of the present application discloses a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof disclosed in the present application, and a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition also comprises another pharmaceutically active agent, such as favipiravir, remdesivir, interferon, etc.

[0087] In certain embodiments of the present application, the antibody or antigen-binding fragment thereof in the pharmaceutical composition and the additional pharmaceutically active agent can 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 (such as an aqueous or non-aqueous suspension or solution). In some embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0088] On the other hand, the present application embodiment discloses a method for neutralizing the virulence of SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit in a sample. The method comprises contacting a sample containing SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit with an antibody or antigen-binding fragment thereof disclosed in the present application embodiment.

[0089] On the other hand, the embodiments of the present application disclose the use of the antibody or its antigen-binding fragment for preparing a drug, wherein the drug is used to neutralize the virulence of SARS-CoV-2 in a sample, or to prevent or treat SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit infection in a subject, or a disease associated with infection with SARS-CoV-2, or its mutant strain, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit (e.g., COVID-19).

[0090] In some embodiments, the subject is a mammal, such as a human;

[0091] 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, favipiravir, remdesivir, etc.).

[0092] The antibody or antigen-binding fragment thereof disclosed in the present application, or the pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present application should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by the following method: incorporating the recombinant protein of the present application in a suitable solvent at a required dose, and optionally, simultaneously incorporating other desired ingredients (including, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, a sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0093] Antibodies or their Fabs disclosed in the present application, or pharmaceutical compositions can be administered by any suitable method known in the art, including oral, oral, sublingual, eyeball, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, preferred routes of administration / modes are parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Technicians should understand that routes of administration and / or modes will change according to the intended purpose. In a preferred embodiment, antibodies or their Fabs or pharmaceutical compositions of the present application are administered by intravenous injection or push injection.

[0094] The pharmaceutical compositions disclosed in the present application may include a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody or antigen-binding fragment thereof of the present application. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent or delay the occurrence of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The "therapeutically effective amount" of the antibody or antigen-binding fragment thereof disclosed in the present application may vary depending on the following factors: the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0095] In the present application, the dosage regimen can be adjusted to obtain the best intended response (e.g., a therapeutic or preventive response). For example, a single dose can be administered, multiple doses can be administered over a period of time, or the dosage can be reduced or increased in proportion to the urgency of the treatment situation.

[0096] Definition of terms

[0097] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand this application, the definitions and explanations of the relevant terms are provided below.

[0098] As described herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "new coronavirus" or "2019-nCoV", belongs to the genus Beta coronavirus and is an enveloped single-stranded positive-sense RNA virus. The genome sequence of SARS-CoV-2 is known to those skilled in the art, and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 is the same as that of SARS-CoV. It specifically binds to the angiotensin-converting enzyme 2 (ACE2) on the host cell through the receptor binding domain (RBD) on the S protein, which leads to the membrane fusion and cell entry of the virus, and plays a vital role in the process of viral infection of cells.

[0099] As used herein, the terms "new coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by infection with SARS-CoV-2. The two have the same meaning and can be used interchangeably.

[0100] As described in this article, the terms "S protein" and "spike protein" both refer to the surface spike protein of SARS-CoV-2 with a receptor binding domain (RBD) thereon. The two have the same meaning and can be used interchangeably.

[0101] As used herein, the term "antibody" refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair including a light chain (L) and a heavy chain (H). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotype of the antibody can be defined as IgM, IgD, IgG, IgA and IgE, depending on the heavy chain. Within the light and heavy chains, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions of high variability, called complementary determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site, respectively. The allocation of amino acids to various regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or the definitions of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0102] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0103] 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 that the full-length antibody binds to and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced 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, complementary determining region (CDR) fragments, scFv, diabodies, single domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.

[0104] As described 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 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 direction from N-terminal to C-terminal; and, when the full-length antibody is an IgE isotype, it optionally also includes 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 direction from N-terminal to C-terminal. 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 direction from N-terminal to C-terminal. Two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. The full-length antibody of the present application may be from a single species, such as humans; it may also be a chimeric antibody or a humanized antibody. The full-length antibody of the present application comprises two antigen-binding sites formed by a VH and a VL pair, respectively, and the two antigen-binding sites specifically recognize / bind to the same antigen.

[0105] As described herein, the term "Fd" means an antigen-binding fragment consisting of a VH and CH1 domains; the term "dAb fragment" means an antigen-binding fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989)); the term "Fab fragment" means an antigen-binding fragment consisting of a 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 a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in a F(ab')2 fragment, consisting of a complete light chain and the Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0106] As described herein, the term "Fv" means an antigen-binding fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antigen-binding fragment that can form a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a single variable region (e.g., a Fd fragment containing only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.

[0107] As described herein, the term "Fc" means an antigen-binding fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via disulfide bonds. The Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.

[0108] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a 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 Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in 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, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the present application, scFv may form a di-scFv, which refers to two or more single scFvs in series to form an antibody. In certain embodiments of the present application, scFv may form a (scFv)2, which refers to two or more single scFvs in parallel to form an antibody.

[0109] As used herein, the term "diabodies" means that the VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0110] As described herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, which refers to an antigen-binding fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen bound by a full-length antibody. Single-domain antibodies are also called nanobodies.

[0111] Each of the above antigen-binding fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0112] Antibody antigen-binding fragments (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0113] As described herein, the term "chimeric antibody" refers to a part of its light chain or / and heavy chain derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and the other part of the light chain or / and heavy chain derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the binding activity to the target antigen (USP4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)). In certain embodiments of the present application, the term "chimeric antibody" may include, for example, human-mouse chimeric antibodies, wherein the heavy chain and light chain variable regions of the antibody are derived from a first antibody (e.g., a mouse antibody), and the heavy chain and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody). In certain embodiments of the present application, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., an individual human antibody sequence), while the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human consensus germline antibody sequence).

[0114] To prepare chimeric antibodies, the variable region of one antibody can be connected to the constant region of another antibody (e.g., human immunoglobulin) using methods known in the art. For example, the DNA encoding VH is operably connected to another DNA molecule encoding the heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No.91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is generally preferably IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably connected to another DNA molecule encoding the light chain constant region CL to obtain a full-length light chain gene (and Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region, but is generally preferably a kappa constant region.

[0115] As described herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (acceptor antibody). Typically, at least one or two but usually all three (heavy and / or light immunoglobulin chains) of a humanized antibody are replaced by a donor CDR. The immunoglobulin that provides the CDR is called a "donor", and the immunoglobulin that provides the framework is called an "acceptor". In one embodiment, the donor immunoglobulin is a non-human (e.g., mouse) antibody, and the acceptor framework can be a naturally occurring human framework, or a sequence having about 85%, 90%, 95%, 99% or higher identity compared thereto. Humanized antibodies generally retain the expected properties of the donor antibody, including antigen specificity, affinity, reactivity, etc. The donor antibody can be a mouse, rat, rabbit, or non-human primate (eg, cynomolgus monkey) antibody having the desired properties (eg, antigen specificity, affinity, reactivity, etc.).

[0116] To prepare humanized antibodies, the CDR region of an immune animal (e.g., mouse) can be transplanted into a human framework sequence using methods known in the art (see, Design of humanized antibody library of anti-liver cancer single-chain antibody based on homology modeling technology [J]. Chinese Journal of Gerontology, 2015, Issue 16, ISSN: 1005-9202).

[0117] Chimeric antibodies or humanized antibodies of the present application can be prepared according to the sequence of the monoclonal antibody produced by immune animals (e.g., mice). DNA encoding heavy and light chains can be obtained from target hybridomas or specific B cells from immune animals, and engineered to include human immunoglobulin sequences using standard molecular biology techniques.

[0118] As used herein, the term "germline antibody gene" or "germline antibody gene segment" refers to a sequence encoding an immunoglobulin present in the genome of an organism that has not undergone a maturation process of genetic rearrangement and mutation that can lead to the expression of a specific immunoglobulin. Accordingly, the term "rearranged antibody sequence" refers to a sequence of a specific antibody that has undergone a maturation process of genetic rearrangement and mutation that can lead to the expression of a specific immunoglobulin. In the present application, the expression "heavy chain germline gene" refers to a germline antibody gene or gene segment encoding an immunoglobulin heavy chain, which includes a V gene (variable), a D gene (diversity), a J gene (joining) and a C gene (constant); similarly, the expression "light chain germline gene" refers to a germline antibody gene or gene segment encoding an immunoglobulin light chain, which includes a V gene (variable), a J gene (joining) and a 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 a "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, UNSW Ig, NCBI or VBASE2).

[0119] As described herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, including: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0120] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.

[0121] 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 to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (KD) binds to the antigen.

[0122] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Generally, antibodies bind to the antigen with a KD of less than about 10 -5 The dissociation equilibrium constant (KD) of M binding to antigen.

[0123] As described herein, the term "neutralizing activity" refers to the functional activity of an antibody or antigen-binding fragment that binds to antigenic proteins on a virus, thereby preventing virus-infected cells and / or the maturation of viral progeny and / or the release of viral progeny. An antibody or antigen-binding fragment with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0124] As described herein, the term "identity" is used to refer to the matching of sequences between two proteins, two polypeptides, or two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of the two DNA molecules is occupied by adenine, or a certain position in each of the two polypeptides is occupied by lysine), then each molecule is identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of the two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have a total of 50% identity (3 out of a total of 6 positions match). Typically, the two sequences are compared when they are aligned to produce maximum identity. Such an alignment can be achieved using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). 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 incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0125] As described herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid 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 USA 94: 412-417 (1997), which are incorporated herein by reference).

[0126] The compilation of the twenty conventional amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in this application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0127] As described herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an 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: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. For example, pH regulators 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, etc. Agents that maintain osmotic pressure include sugars, NaCl and the like. Agents that delay absorption include monostearate and gelatin. Diluents include water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0128] As described herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom in a subject. As described herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For the purposes of this application, a beneficial or desired clinical outcome includes alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to extending the survival period compared to the expected survival period (if not receiving treatment).

[0129] 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 prevention effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a disease treatment effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0130] Beneficial effects of this application

[0131] The present application provides antibodies or antigen-binding fragments that are infected with SARS-CoV-2, or its mutants, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit, or have specific binding ability with SARS-CoV-2, or its mutants, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit. Among them, the mutants of SARS-CoV-2 include SARS-CoV-2 Omicron BA.1 mutants, SARS-CoV-2 Omicron BA.2 mutants, SARS-CoV-2Beta mutants, and SARS-CoV-2 Delta mutants. Specifically, these monoclonal antibodies bind to epitopes on the RBD region of the S protein of SARS-CoV-2 and neutralize SARS-CoV-2. The monoclonal antibodies of the present application can inhibit the binding of the RBD protein of SARS-CoV-2 to the receptor ACE2. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1 The protein electrophoresis results of antibodies Amb1 (left figure) and Amb2 (right figure) provided in the examples of the present application.

[0133] Figure 2The ELISA test results of the binding activity of antibodies Amb1, Amb2 and recombinant protein ACE2-hFc provided in the examples of the present application with the wild-type SARS-CoV-2 RBD protein, respectively.

[0134] Figure 3 Affinity determination curves of the antibodies Amb1, Amb2 and recombinant protein ACE2 provided in the embodiments of the present application with the wild-type SARS-CoV-2 RBD protein and the RBD protein of the SARS-CoV-2 Omicron BA.1 mutant strain, respectively.

[0135] Figure 4 Neutralization activity test curves of antibodies Amb1 and Amb2 provided in the examples of the present application with Omicron BA.1 mutant, Omicron BA.2 mutant and wild-type novel coronavirus pseudovirus, respectively.

[0136] Figure 5 These are the neutralization activity test curves of the antibodies Amb1 and Amb2 provided in the examples of the present application with the Beta mutant and Delta mutant pseudoviruses, respectively. DETAILED DESCRIPTION

[0137] In order to make the purpose, technical solution and advantages of the present application more clear, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described here are only used to explain the present application and are not used to limit the present application.

[0138] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application are basically carried out with reference to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will appreciate that the embodiments describe the present application by way of example and are not intended to limit the scope of protection claimed in the present application.

[0139] Construction and screening of a fully human anti-COVID-19 scFv antibody library

[0140] The human peripheral blood mononuclear cells (PBMC) separated from 10 mL of whole blood extracted from the inactivated vaccine recipient (three injections) were lysed by one-step total RNA extraction reagent TRIzol to extract total RNA. Then oligo-dT was used as a primer to perform reverse transcription PCR reaction, and cDNA was prepared respectively, and the cDNA was mixed in equal volumes. Human antibody characteristic sequence primers (see Generation and Characterization of a Recombinant Human CCR5-specific Antibody: a phage display approach for rabbit antibody humanization, "The Journal of biological chemistry" 2000, 275, 46, 36073-8 pages) were used to amplify antibody genes VH, Vκ and Vλ, and Vκ-linker-VH and Vλ-linker-VH were further obtained by overlap extension polymerase chain reaction (PCR), and then single digestion with restriction endonuclease Sfi I was performed, and the digested fragments were connected to the pre-cut phage display vector pComb3XSS using T4 DNA ligase. The ligation products were desalted by ethanol precipitation and then transformed into TG1 competent bacteria by electroporation to construct libraries with a capacity of 1.5×10 9 and 2.2×10 9 The single chain antibody (scFv) library is used for subsequent screening. The basic reference of this process is (Barbas, CF, III; Burton, DR; Scott, JK, Silverman, GJ Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 736 pages.)

[0141] Enrichment and screening of phage antibody library and induced expression of scFV antibody

[0142] The screening antigen used was the purified wild-type RBD protein of the new coronavirus (RBD-WT, cat#Z03479, purchased from Gensript), and the screening was performed using the following method:

[0143] Dilute the antigen with PBS solution at pH 7.4 and coat the 96-well plate with 100 μL per well; coat overnight at 4°C. Wash three times with PBST, add 200 μL / well of blocking buffer, and coat overnight at 4°C. Wash five times with PBST, 200 μL per well, and dilute the library phage to 1×1011 , 100μL per well, 37℃ for 1.5h. Wash the plate 5 times with PBST, then 3 times with PBS, add 100μL pH 2.2 glycine-HCl eluent to each well, and immediately add 15μL pH 9.1 Tris-HCl to neutralize. Mix the remaining eluate with 5ml logarithmic phase TG1, 37℃, 220rpm, 45min. The above bacterial solution was transferred to 20ml 2YT / A+ / G medium, cultured to the logarithmic phase, and the helper phage VCSM13 (Stratagene, USA) was added for infection, incubated at 37°C for 1h, and kanamycin (final concentration of 50μg / mL) was added and shaken at 30°C overnight to prepare phage single-chain antibodies. The ELISA plate was coated with 0.1μg / well SARS-CoV-2 RBD protein, and the secondary antibody was HRP-labeled anti-M13 antibody diluted 1:2000 with PBS buffer (containing 5g / mL skim milk powder). Phage-ELISA identification was performed and the OD450 value was measured. The screening was repeated 3 times. For specific enrichment screening methods and the inducible expression process of scFv fragments, please refer to (Barbas, CF, III; Burton, DR; Scott, JK, Silverman, GJ Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 736 pages.).

[0144] After the above experiments, scFv segment 1, scFv segment 2, scFv segment 3 and scFv segment 4 were obtained. scFv segment 1 has a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as described in SEQ ID NO.3. scFv segment 2 has a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.2 and a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.4. scFv segment 3 has a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.1 and a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.4. scFv segment 4: has a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.2 and a heavy chain variable region with an amino acid sequence as described in SEQ ID NO.3.

[0145] Furthermore, the CDR sequences in scFv segment 1, scFv segment 2, scFv segment 3, and scFv segment 4 were 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, Bethesda, Maryland (1991), pages 647-669). Among them, the heavy chain CDR1 to 3 of SEQ ID NO. 1 are shown in SEQ ID NOs: 5 to 7, the heavy chain CDR1 to 3 of SEQ ID NO. 2 are shown in SEQ ID NOs: 8 to 10, the light chain CDR1 to 3 of SEQ ID NO. 3 are shown in SEQ ID NOs: 11 to 13, and the light chain CDR1 to 3 of SEQ ID NO. 4 are shown in SEQ ID NOs: 14 to 16.

[0146] Preparation of whole antibodies

[0147] Plasmids were extracted from the bacterial suspension of positive clones using a plasmid extraction kit (Qiagen Miniprep Kit, QIAGEN, Germany) and sent to a commercial company for sequencing. The sequencing results were compared with the 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 / ), and Ig BLAST (http: / / www.ncbi.nlm.nih.gov / igblast / ). The sequence of the variable region gene of the whole antibody obtained by sequencing (the coding sequence of the amino acid sequence shown in SEQ ID NO.1-4 is shown in SEQ ID NO.17-20 in sequence) was converted into the form of the whole IgG1 antibody. After the whole gene was synthesized, it was connected to the secretory mammalian expression vector pHL-Sec (purchased from Addgene, #99845, transformed from the classic mammalian expression plasmid pCAGGS) by seamless cloning. The signal peptide sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) on the vector was replaced by the leader peptide sequence of the mouse kappa chain (Murine Igκ-chain leadersequence, METDTLLLWVLLLWVPGSTGDJ), and a stop codon was added after the sequence. In this way, the heavy chain expression plasmid pHL-HC and the light chain expression plasmid pHL-LC were constructed respectively.

[0148] The pHL-HC and pHL-LC sequenced correctly were extracted in large quantities without endotoxin. HEK293F cells were cloned with FreeStyle TM 293 expression medium was used for suspension culture. One day before transfection, 1.0×10 6 The cells were inoculated into disposable conical shake flasks at a density of 2.0×10 cells / mL and placed in a 37°C, 5% CO2 incubator with shaking at 125 rpm. Cell counts were performed on the day of transfection. 6 cells / mL (if the density exceeds the limit, dilute with culture medium), and perform transient transfection when the activity is greater than 95%. Mix heavy chain plasmid pHL-HC, light chain plasmid pHL-LC and PEI (Polysciences) at a ratio of 1:1:2.5 (where "1" means 1 μg / mL cells) to prepare DNA-PEI complex, add to the cell culture bottle to be transfected, shake gently, and continue shaking culture in a 37°C, 5% CO2 incubator. After 4 days of transfection, collect supernatant and cells every day for dynamic analysis of the expression of recombinant protein. After 7 days of transfection, collect supernatant.

[0149] After suction filtration with a 0.22 μm microporous filter membrane, 1 / 10 volume of 10× pH adjustment buffer (0.5 M Na2HPO4, pH7.4) was added, and the culture supernatant containing the recombinant antibody was directly passed through two 5 mL HiTrap Protein G HP pre-packed columns in series at a flow rate of 5 mL / min in a protein purification system (AKTA pure25). After the binding was completed at a low flow rate, it was rinsed with binding buffer (20 mM Na2HPO4, pH7.0) until the ultraviolet absorbance at 280 nM did not change. Then, it was eluted with elution buffer (0.1 M glycine-HCl, pH2.7), and 1 / 10 volume of neutralization buffer (1 M Tris-HCl, pH9.0) was pre-added to the collection tube. The purification was detected by SDS-PAGE coupled with bromophenol blue staining, the peak where the target protein was located was determined, the target protein was collected and concentrated, and further purified with anion exchange column and molecular sieve for the next experiment. Optionally, the supernatant is collected, centrifuged to remove cells and their debris, and purified by Protein G HP SpinTrap centrifugal small amount antibody affinity chromatography column to prepare a small amount of monoclonal antibody. The purification method is as follows: the supernatant after centrifugation is mixed with equal volumes of binding buffer (20mM Na2HPO4, pH7.0). Add 600μL binding buffer, centrifuge at 100g for 30s, and balance the column. Repeat adding 600μL pre-equilibrated sample, centrifuge at 100g for 30s, gently mix, and let stand for 4min to bind the antibody to the column. Add 600μL binding buffer, centrifuge at 100g for 30s, and wash away impurities. Repeat twice. Add 400μL elution buffer (0.1M Glycine-HCl, pH2.7), invert and mix, place in a 2mL EP tube containing 30μL neutralization buffer (1M Tris-HCl, pH9.0), centrifuge at 100g for 30s, and repeat twice.

[0150] like Figure 1 As shown, the electrophoresis diagrams of the purified recombinantly expressed antibodies Amb1 and Amb2 show a band near 50KD and 30KD, representing the heavy chain and light chain, respectively.

[0151] Binding activity of Amb1 and Amb2 to RBD proteins of different novel coronavirus strains

[0152] 1. ELISA test

[0153] The wild-type RBD protein of the novel coronavirus (cat#Z03479, Gensript) was coated on the ELISA plate with pH=9.6 0.1M NaHCO3 solution, overnight at 4°C; blocked with 3% skim milk, incubated at 37°C for 1h, added recombinantly expressed Amb1 and Amb2 antibodies and ACE2-hFc recombinant protein (control), incubated at 37°C for 1h; added enzyme-labeled anti-human Fc fragment secondary antibody, incubated at 37°C for 1h; developed with colorimetric solution, terminated with 2M H2SO4, and detected OD450 with an ELISA reader. The results showed high binding activity, as shown in the following figure. Figure 1 As shown, both Amb1 and Amb2 antibodies showed higher binding activity to the RBD protein of the new coronavirus strain than ACE2-hFc.

[0154] 2. Determination of antibody affinity by BIAcore

[0155] The wild type of the new coronavirus (cat#Z03479, Gensript) and the RBD protein of the mutant strain of Omicron BA.1 (cat#Z03516, Gensript) were diluted in 10mM NaAc to try the binding ability of the antibody to the CM5 chip under different pH conditions. The pH value was set to four gradients of pH4.0, pH4.5, pH5.0, and pH5.5, and the pH with the best fixation effect was selected. The RBD was diluted in 10mM NaAc at the optimal pH value and fixed on the surface of the chip. The target coupling amount RU (Response Unit) of each antibody was determined according to the formula. Amb1 and Amb2 antibodies were used as the mobile phase, and the concentration gradient was set to 2.6nM~333nM or 666nM. The response values ​​generated by different concentrations of Amb1, Amb2 and ACE2-hFc flowing through the chip surface were detected. The analysis was carried out at a constant temperature of 25°C and a flow rate of 30μL / min. The solution used for chip surface regeneration was 100mM H3PO4. The binding kinetic constants were calculated using the BIAevaluation software version (Biacore, Inc.) according to the 1:1 binding model and the affinity constant (K D (M)).

[0156] The results are as follows Figure 2 As shown, the affinity constants of Amb1 and Amb2 for wild-type RBD are 1.51×10 -7 and 1.64×10 -7 ; The affinity constant of ACE2-hFc for wild-type RBD is 3.09×10 -7 The affinity constants of Amb1 and Amb2 for Omicron BA.1 mutant RBD were 2.05×10 -7 and 3.33×10-7 ; The affinity constant of ACE2-hFc for Omicron BA.1 mutant RBD is 5.10×10 -7 This not only shows that Amb1 and Amb2 have affinity for the new coronavirus and its mutant strains, but also that their affinity is higher than that of the receptor ACE2 itself. They can act as blockers that competitively block the binding of the virus to the receptor ACE2, and can therefore be used in related drugs or preparations to prevent viral infection.

[0157] Pseudovirus neutralization assay

[0158] Preparation of pseudovirus: lay 5-6×10 6 293T cells were placed in 6-well plates and cultured for 18-24 hours. 2 μg HIV-1 backbone plasmid pNL4-3.luc.RE- and 2 μg S protein expression plasmid (SARS-CoV-2 wild type, SARS-CoV-2 Omicron BA.1, SARS-CoV-2 BA.2, SARS-CoV-2 Beta or SARS-CoV-2delta) were co-transfected into 293T cells using 12 μL FuGENE HD (cat#E2311, Promega) transfection reagent according to the instructions. From the start of transfection to 48 hours later, the culture medium in the 6-well plate was aspirated and added to a sterile 15 mL centrifuge tube, centrifuged at 3000 rpm for 15 minutes, and the supernatant was taken, which was the pseudovirus 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 -80 ° C refrigerator for use.

[0159] Transfer the virus from -80℃ to ice to thaw in advance and prepare serum-free DMEM. One day in advance, inoculate 293T-ACE2 cells (Yeasen, Cat. No. 41107ES03) in a 96-well cell culture plate with an inoculation volume of approximately 1.2×10 4cells / well, and infect according to the cell density the next day. The cell density is better at about 50%. Dilute the antibody, the antibody concentration of the first EP tube is 75.0μg / mL. 0μg / mL, the total volume is 100μL, and each subsequent EP tube is 80μL DMEM, and 20μL of the previous tube is pipetted to the next tube, 5-fold dilution, and a total of 10 gradients. Prepare another new 96-well plate, take 96.0μL of pseudovirus + 24.0μL of antibody (the antibody concentration is 15.0μg / mL at this time), incubate at 37℃ for 1h, and set up 3 wells to add 100μL of pseudovirus (as described above) as a control. Discard the culture medium in the 96-well cell culture plate, add 100.0μL of antibody pseudovirus mixture, change the medium after 6h, and detect luciferase activity after 48h. During the test, take out the plate in advance and place it at room temperature for 30 minutes. At the same time, take out the test reagent from -20℃ and thaw it. Add 100μL of the same volume of test reagent to each well and lyse it for 3 minutes. After mixing, transfer it to the test plate with a spray gun and place it in the GloMax 96 microplate luminescence detector for detection.

[0160] The results are as follows Figure 3 , 4 As shown, Amb1 and Amb2 neutralize the IC of the wild-type SARS-CoV-2. 50 The IC values ​​of Amb1 and Amb2 for neutralizing mutant Omicron BA.1 were 0.1303 μg / mL and 0.064 μg / mL, respectively. 50 The IC values ​​of Amb1 and Amb2 for neutralizing Omicron BA.2 were 0.02464 μg / mL and 0.01549 μg / mL, respectively. 50 The IC values ​​of Amb1 and Amb2 for neutralizing the Beta mutant were 0.01126 μg / mL and 0.02734 μg / mL, respectively. 50 The IC values ​​of Amb1 and Amb2 for neutralizing the Delta mutant strain were 0.02660 μg / mL and 0.04516 μg / mL, respectively. 50 They are 1.220 μg / mL and 0.4077 μg / mL respectively.

[0161] In summary, the antigen-binding fragment scFv disclosed in the present application has high affinity activity for the RBD protein of the new coronavirus, and Amb1 and Amb2 prepared by genetic engineering have strong neutralizing effects on wild-type, Beta mutant, Delta mutant, Omicron BA.1 and BA.2 pseudoviruses. It has the potential to respond to the global new coronavirus pandemic caused by the Omicron variant strain and the outbreak caused by the emerging new Omicron variant strains that can evade vaccines and neutralizing antibodies, and has significant clinical application value.

[0162] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application. Sequence Listing <110> Duan Liangwei Jiang Zhihua Wang Hui <120> Antibodies to SARS-CoV-2 and its mutants and their applications <160> 20 <170> SIPOSequenceListing 1.0 <210> 1 <211> 124 <212> PRT <213> Artificial Sequence <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Thr Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Gly Tyr Ser Gly Tyr Gly Ala Ala Tyr Tyr Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 121 <212> PRT <213> Artificial Sequence <400> 2 Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Asn Ser 20 25 30 Ser Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Ser Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Gly Val Tyr Tyr 85 90 95 Cys Ala Ser Trp Leu Tyr Gly Asp Pro Ile Ser Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 3 <211> 111 <212> PRT <213> Artificial Sequence <400> 3 Gln Ser Val Leu Thr Gln Ala Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Lys Gly Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <400> 4 Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Arg Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Val Glu Val Lys 100 105 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <400> 5 Gly Gly Thr Phe Ser Ser Tyr Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <400> 6 Ile Ile Pro Ile Leu Gly Ile Ala 1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <400> 7 Ala Arg Glu Arg Gly Tyr Ser Gly Tyr Gly Ala Ala Tyr Tyr Phe Asp 1 5 10 15 Tyr <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Gly Gly Ser Ile Ser Asn Ser Ser Tyr Tyr 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <400> 9 Ile Tyr Tyr Ser Gly Ser Thr 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <400> 10 Ala Ser Trp Leu Tyr Gly Asp Pro Ile Ser Phe Asp Tyr 1 5 10 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Ser Ser Asn Ile Gly Ala Gly Tyr Asp 1 5 <210> 12 <211> 3 <212> PRT <213> Artificial Sequence <400> 12 Gly Asn Asn 1 <210> 13 <211> 11 <212> PRT <213> Artificial Sequence <400> 13 Gln Ser Tyr Asp Ser Ser Leu Ser Lys Gly Val 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <400> 14 Gln Ser Ile Ser Ser Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <400> 15 Wing Wing Being 1 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Gln Gln Ser Tyr Ser Thr Pro Leu Thr 1 5 <210> 17 <211> 372 <212> DNA <213> Artificial Sequence <400> 17 gaggtgcagc tgttagagtc tgggactgag gtgaagaagc ctgggtcctc agtgaaggtc 60 tcctgcaagg cttctggagg caccttcagc agctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tccttggtat agcaaactac 180 gcacagaagt tccagggcag agtcacgatt accgcggaca aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagaacgt 300 ggatatagtg gctacggggc ggcttactac tttgactact ggggccaggg aaccctggtc 360 accgtctcct ca 372 <210> 18 <211> 363 <212> DNA <213> Artificial Sequence <400> 18 gaggtgcagc tgttggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc aatagtagtt actactgggg ctggatccgc 120 cagcccccag ggaaggggct ggagtggatt gggagtatct attatagtgg gagcacctac 180 tacaacccgt ccctcaagag tcgagtcacc atatcagtag acacgtccaa gaaccagttc 240 tccctgaagc tgagctctgt gaccgccgcg gacacgggcg tgtattactg tgcgagttgg 300 ttatacggtg acccaatttc ctttgactac tggggccagg gaaccctggt caccgtctcc 360 tca 363 <210> 19 <211> 333 <212> DNA <213> Artificial Sequence <400> 19 cagtctgtgc tgactcaggc gccctcagtg tctggggccc cagggcagag tgtcaccatc 60 tcctgcactg ggagcagctc caacatcggg gcaggttatg atgtacactg gtaccagcag 120 ctcccaggaa cagcccccaa actcctcatc tatggtaaca acaatcggcc ctcaggggtc 180 cctgaccgat tctctggctc caagtctggc acctcagcct ccctggccat cactgggctc 240 caggctgagg atgaggctga ttattactgc cagtcctatg acagtagcct gagcaaaggg 300 gtgttcggcg gagggaccaa gctgaccgtc cta 333 <210> 20 <211> 321 <212> DNA <213> Artificial Sequence <400> 20 gacattgagc tcacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatcg gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300 gggaccaggg tggaagtcaa g 321

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to the receptor binding domain (RBD) of the S protein of SARS-CoV-2 and its mutants, wherein the antibody or the antigen-binding fragment thereof comprises: three complementarity determining regions (CDRs) of the heavy chain variable region (VH) defined according to the Kabat numbering system: VH CDR1 as shown in SEQ ID NO.5, VH CDR2 as shown in SEQ ID NO.6, and VH CDR3 as shown in SEQ ID NO.7; and Three complementarity determining regions (CDRs) of the light chain variable region (VL) are defined according to the Kabat numbering system: VL CDR1 as shown in SEQ ID NO.11, VL CDR2 as shown in SEQ ID NO.12 and VL CDR3 as shown in SEQ ID NO.

13.

2. An antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to the receptor binding domain (RBD) of the S protein of SARS-CoV-2 and its mutants, wherein the antibody or the antigen-binding fragment thereof comprises: The heavy chain variable region (VH) as shown in SEQ ID NO.1; and The light chain variable region (VL) is shown in SEQ ID NO.

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

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, comprising: a heavy chain framework region sequence derived from a human rearranged antibody sequence, and a light chain framework region sequence derived from a human rearranged antibody sequence.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, comprising: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.

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

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody is a rabbit antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a multispecific antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein The antibody or antigen-binding fragment thereof has one or more of the following characteristics: (1) specifically binds to SARS-CoV-2, or its mutants, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit, wherein the mutants include the Omicron BA.1 mutant, the Omicron BA.2 mutant, and the Beta mutant; (2) blocking or inhibiting the binding of SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit to Ace2 receptor, and / or blocking or inhibiting the infection of SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit to cells; (3) neutralizing SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit in vitro or in a subject (e.g., human); (4) Preventing and / or treating infection with SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit, or diseases associated with infection with SARS-CoV-2, or its mutants, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit (e.g., COVID-19).

10. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, or the heavy chain variable region and / or light chain variable region thereof. A vector comprising the nucleic acid molecule according to claim 10 . The vector according to claim 11 , which is a cloning vector or an expression vector.

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

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the method comprising culturing the host cell according to claim 13 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.

15. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

16. The kit according to claim 15, wherein the antibody or antigen-binding fragment thereof further comprises a detectable label, wherein the detectable label is selected from at least one of an enzyme, a chemiluminescent agent, a fluorescent dye, a radionuclide or biotin.

17. The kit according to claim 15, further comprising a second antibody, which specifically recognizes the antibody or its antigen-binding fragment; the second antibody further comprises a detectable label, which is selected from at least one of an enzyme, a chemiluminescent reagent, a fluorescent dye, a radionuclide or biotin.

18. The kit according to claim 16 or 17, wherein the enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

19. The kit according to claim 16 or 17, wherein the chemiluminescent reagent is selected from acridinium ester compounds.

20. The kit according to claim 16 or 17, wherein the fluorescent dye is selected from at least one of isothiocyanate and fluorescent protein.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a kit for detecting the presence or level of at least one of SARS-CoV-2, or its mutant strains, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit in a sample, wherein the mutant strain is selected from SARS-CoV-2 Omicron BA.1 mutant, Omicron BA.2 mutant, Beta mutant and Delta mutant, and the method for using the kit comprises using the antibody or antigen-binding fragment thereof.

22. The use according to claim 21, wherein the detection is an immunological detection.

23. The use according to claim 22, wherein the immunological detection is selected from at least one of enzyme immunoassay, chemiluminescent immunoassay, fluorescent immunoassay or radioimmunoassay.

24. The use according to claim 22, wherein the enzyme immunoassay is selected from ELISA.

25. The use according to claim 21, wherein the antibody or antigen-binding fragment thereof further comprises a detectable label, and the detectable label is selected from at least one of an enzyme, a chemiluminescent agent, a fluorescent dye, a radionuclide or biotin.

26. The use according to claim 21, wherein the method for using the kit further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody carrying a detectable label, wherein the detectable label is selected from at least one of an enzyme, a chemiluminescent agent, a fluorescent dye, a radionuclide or biotin.

27. The use according to claim 25 or 26, wherein the enzyme is selected from at least one of horseradish peroxidase and alkaline phosphatase.

28. The use according to claim 25 or 26, wherein the chemiluminescent reagent is selected from acridinium ester compounds.

29. The use according to claim 25 or 26, wherein the fluorescent dye is selected from at least one of isothiocyanate and fluorescent protein.

30. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a kit for diagnosing whether a subject is infected with SARS-CoV-2, or a mutant thereof, or at least one of its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit, wherein the mutant is selected from the group consisting of an Omicron BA.1 mutant, an Omicron BA.2 mutant, a Beta mutant, and a Delta mutant.

31. The use according to any one of claims 21 to 30, wherein the sample is at least one of a blood sample, excrement, oral or nasal secretions, or alveolar lavage fluid from a subject.

32. The use according to claim 31, wherein the subject is a mammal.

33. The use according to claim 32, wherein the subject is a human.

34. The use according to claim 31, wherein the blood sample is selected from at least one of whole blood, plasma or serum.

35. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier and / or excipient.

36. The pharmaceutical composition of claim 35, further comprising an additional pharmaceutically active agent.

37. The pharmaceutical composition of claim 36, wherein the additional pharmaceutically active agent is selected from at least one of favipiravir, remdesivir or interferon.

38. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 for the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample, or for preventing or treating infection with SARS-CoV-2, or a mutant thereof, or its S protein or RBD of the S protein, or its S1 subunit or RBD of the S1 subunit in a subject, or a disease associated with infection with at least one of SARS-CoV-2, or a mutant thereof, or its S protein or RBD of the S protein, or its S1 subunit or RBD of the S1 subunit.

39. The use according to claim 38, wherein the disease is COVID-19.

40. The use according to claim 38, wherein the subject is a mammal.

41. The use according to claim 40, wherein the subject is a human.

42. The use according to claim 38, wherein the antibody or antigen-binding fragment thereof is used alone.

43. The use according to claim 38, wherein the antibody or antigen-binding fragment thereof is used in combination with another pharmaceutically active agent.

44. The use according to claim 43, wherein the additional antiviral agent is selected from at least one of interferon, lopinavir, ritonavir, chloroquine phosphate, favipiravir or remdesivir.

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