CN115043938A - Antibody of SARS-CoV-2 and its mutant strain and application - Google Patents
Antibody of SARS-CoV-2 and its mutant strain and application Download PDFInfo
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- CN115043938A CN115043938A CN202210675885.XA CN202210675885A CN115043938A CN 115043938 A CN115043938 A CN 115043938A CN 202210675885 A CN202210675885 A CN 202210675885A CN 115043938 A CN115043938 A CN 115043938A
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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 mutant strains. In particular, the present application relates to antibodies or antigen-binding fragments against SARS-CoV-2 and mutants thereof, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the application also relates to the use of said antibody or antigen binding fragment. The antibodies or antigen-binding fragments of the present application can be used for the diagnosis, prevention and/or treatment of infection by SARS-CoV-2 and mutants thereof and/or diseases caused by said infection.
Description
Technical Field
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 mutant strains. In particular, the present application relates to antibodies or antigen-binding fragments against SARS-CoV-2 and mutants thereof, and compositions (e.g., diagnostic and therapeutic agents) comprising the antibodies. Furthermore, the application also relates to the use of said antibody or antigen binding fragment. The antibodies or antigen-binding fragments of the present application can be used for the diagnosis, prevention and/or treatment of infection by SARS-CoV-2 and mutants thereof and/or diseases caused by said infection.
Background
New crown pneumonia (COVID-19) caused by infection with a 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.
SARS-CoV-2 Spike glycoprotein (Spike, S) plays a key role in the pathogenesis and infection of novel coronaviruses. The mature S protein of SARS-CoV-2 virus is a highly glycosylated trimer consisting of 1260 amino acids (residues 14-1273) per protomer, with the S1 subunit consisting of 672 amino acids (residues 14-685) divided into four domains: one N-terminal domain (NTD), one C-terminal domain (CTD, also known as receptor binding domain, RBD), and two subdomains (SD1 and SD 2).
The SARS-CoV-2S glycoprotein is a conformational machine that mediates viral entry from a metastable, non-triggered state, through a pre-hairpin intermediate state to a stable, post-fusion state. Angiotensin converting enzyme 2(ACE2) has been shown to be the major receptor for SARS-CoV-2 (47-49). The detailed interaction between SARS-CoV-2 RBD and its receptor ACE has been revealed by the complex structure of the two. Structurally, an RBD consists of two subdomains: a core and an outer subdomain. The extension loop (residues 438-506) located on one side of the core subdomain presented a slightly recessed surface to support the ACE 2N-terminal helix (. alpha.1). Analysis of the interface between SARS-CoV-2 RBD and ACE2 shows that 17 residues in total in RBD are in contact with 20 amino acids in ACE2 to form a hydrophilic interaction network, which dominates the binding of virus to receptor.
The RBD of SARS-CoV-2S glycoprotein binds to the ACE2 receptor on the surface of target cells, initiating the process of membrane fusion and subsequent viral invasion of SARS-CoV-2. After binding of the S glycoprotein to ACE2 on the plasma membrane (plasma membrane invasion pathway) or subsequent endocytosis of the viral particle by the host cell (endosomal invasion pathway), a second cleavage occurs (S2' cleavage site) mediated by the cell surface serine protease TMPRSS2 or the endosomal cysteine proteases cathepsin B and L, respectively (cathepsins B and L). Protease cleavage at the S2' site releases the fusion peptide from the N-terminal region of the newly formed S2 subunit, further destabilizes the SARS-CoV-2S glycoprotein trimer, and can initiate the S2-mediated membrane fusion cascade. After the second cleavage, the fusion peptide at the N-terminus of S2 trimer was inserted into the host membrane to form a hairpin pre-intermediate state. Since the pre-hairpin intermediate state is extremely unstable, the S2 fusion protein refolds rapidly and irreversibly to a stable post-fusion state. These large conformational rearrangements draw the viral and host cell membranes closer together, eventually leading to membrane fusion. Therefore, the S protein determines the host range and tissue tropism of the virus. In view of the important role of the S protein in the virus invasion process and the characteristic that most of the S protein is exposed outside the virus envelope and can be directly recognized by the host immune system, the S protein is the main target of the host humoral immunity and cellular immunity and is the main action site of the antibody in the host. Thus, most of the current new crown vaccine strategies are based on the full-length S glycoprotein or its RBD domain as an immunogen.
However, SARS-CoV-2, as an RNA virus, has the characteristics of high mutation rate and fast evolution speed. Currently, Variants (VOCs) that have been designated as interesting by the world health organization (WTO); alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529). The initial variant BA.1 of Omicron was first discovered in Botzwara and south Africa at 11 months 2021 and rapidly replaced the Delta variant, becoming the most prevalent SARS-CoV-2 strain in the world. Another variation of Omicron, BA.2, spreads faster than the BA.1 variant, resulting in confirmed cases that exceed BA.1 cases by 2 months in 2022, becoming the major global epidemic variant at present. Even more terrible, Omicron is still rapidly mutating, producing a large number of new intrasubtype variants. The currently contemplated sub-variations of Omicron include at least XE, BA.2.12.1, BA.4 and BA.5.
Omicron has strong immune escape capability to vaccines and neutralizing antibodies. Studies have shown that the neutralizing activity of most monoclonal antibodies (mAbs) on BA.1 and BA.2 is completely or substantially lost. Among eleven approved or authorized mabs, REGN10987(imdevimab), REGN10933(casirivimab), LY-CoV555(bamlanivimab), CB6/LY-CoV016(etesevimab), S309(sotrovimab), CoV2-2130(cilgavimab), CoV2-2196 (tixagevimab), CT-P59 (regdanvivab), BRII-196 (amubervimab), BRII-198 (romlufevimab), and LY-CoV1404 (betelovimab), S309 retained most of the neutralizing activity (only about 2-fold reduction) for ba.1, but their activity was further escaped by ba.2. S309(sotrovimab) is a representative member of a class of neutralizing antibodies that target a highly conserved region of sarbecovirus, i.e., the core domain of the RBD (having more than 85% identical amino acid residues), and thus are generally shown to exhibit broad sarbecovirus neutralizing activity, although neutralizing activity is generally relatively modest. Ba.2 has S371F, D405N and R408S mutations, which may contribute to the escape capacity of such sarbecovirus neutralizing antibodies. In contrast, some neutralizing antibodies such as COV2-2130(cilgavimab) completely lost activity on BA.1, but restored activity on BA.2.
Disclosure of Invention
In the present application, the inventors developed human antibodies with superior properties, which are capable of neutralizing SARS-CoV-2 and its mutants (Omicron BA.1 mutant, Omicron BA.2 mutant, Beta mutant and Delta mutant), blocking or inhibiting the binding of SARS-CoV-2 and its mutants to the receptor ACE2, and are less likely to elicit an immunogenic response in human subjects. Therefore, the antibody or antigen-binding fragment provided by the application has the potential for detecting, diagnosing, preventing and/or treating SARS-CoV-2 infection or a disease caused by SARS-CoV-2 infection, and has great clinical value.
Antibodies of the present application
In a first aspect, the present embodiments disclose an antibody or antigen-binding fragment thereof that specifically binds to the receptor binding Region (RBD) of the S protein of SARS-CoV-2 and mutants thereof, comprising:
(I) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:
(a) a VH CDR1, consisting of the sequence: SEQ ID NO.5, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.conservative substitutions, deletions or additions of 1, 2 or 3 amino acids),
(b) a VH CDR2, consisting of the sequence: SEQ ID NO.6, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(c) a VH CDR3, consisting of the sequence: SEQ ID No.7, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids); and/or the presence of a gas in the gas,
(II) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) as defined by the Kabat numbering system:
(d) a VL CDR1, consisting of the sequence: SEQ ID NO.11, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.conservative substitutions, deletions or additions of 1, 2 or 3 amino acids),
(e) a VL CDR2, consisting of the sequence: SEQ ID NO.12, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(f) a VL CDR3, consisting of the sequence: SEQ ID No.13, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids).
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the first aspect comprises:
(I)3 heavy chain CDRs: VH CDR1 of sequence SEQ ID NO.5, VH CDR2 of sequence SEQ ID NO.6 and VH CDR3 of sequence SEQ ID NO. 7; and/or, 3 light chain CDRs: VL CDR1 of sequence SEQ ID NO.11, VL CDR2 of sequence SEQ ID NO.12 and VL CDR3 of sequence SEQ ID NO. 13; or the like, or, alternatively,
(II) 3 CDRs defined according to the Kabat numbering system contained in the variable heavy chain region (VH) shown in SEQ ID No. 1; and/or, 3 CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as set forth in SEQ ID No. 3.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of 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: heavy chain framework region sequences derived from human rearranged antibody sequences, and light chain framework region sequences derived from human rearranged antibody sequences. In certain embodiments of the 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.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the first aspect comprises:
(I) a heavy chain variable region (VH) comprising an amino acid sequence selected from any one of the following (a) to (c):
(a) a sequence shown as SEQ ID NO. 1;
(b) a sequence having conservative substitutions, deletions or additions of one or several amino acids (e.g. conservative substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the sequence shown in SEQ ID No. 1; or
(c) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 1;
and
(II) a light chain variable region (VL) comprising an amino acid sequence selected from any one of the following (d) to (f):
(d) a sequence shown as SEQ ID NO. 3;
(e) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 3; or
(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 represented by SEQ ID No. 3.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO.1 and a VL comprising the sequence shown as SEQ ID NO. 3.
In a second aspect, the present embodiments disclose an antibody or antigen-binding fragment thereof that specifically binds to the receptor binding Region (RBD) of the S protein of SARS-CoV-2 and mutants thereof, comprising:
(I) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:
(a) a VH CDR1, consisting of the sequence: SEQ ID NO.8, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.conservative substitutions, deletions or additions of 1, 2 or 3 amino acids),
(b) a VH CDR2, consisting of the sequence: SEQ ID NO.9, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(c) a VH CDR3, consisting of the sequence: SEQ ID No.10, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids); and/or the presence of a gas in the gas,
(II) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) as defined by the Kabat numbering system:
(d) a VL CDR1, consisting of the sequence: SEQ ID NO.14, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.conservative substitutions, deletions or additions of 1, 2 or 3 amino acids),
(e) a VL CDR2, consisting of the sequence: SEQ ID NO.15, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(f) a VL CDR3, consisting of the sequence: SEQ ID No.16, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids).
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the second aspect comprises:
(I)3 heavy chain CDRs: VH CDR1 with sequence SEQ ID NO.8, VH CDR2 with sequence SEQ ID NO.9 and VH CDR3 with sequence SEQ ID NO. 10; and/or, 3 light chain CDRs: VL CDR1 of sequence SEQ ID NO.14, VL CDR2 of sequence SEQ ID NO.15 and VL CDR3 of sequence SEQ ID NO. 16; or the like, or, alternatively,
(II) 3 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, 3 CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as set forth in SEQ ID No. 4.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of 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: heavy chain framework region sequences derived from human rearranged antibody sequences, and light chain framework region sequences derived from human rearranged antibody sequences. In certain embodiments of the 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.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof of the second aspect comprises:
(I) a heavy chain variable region (VH) comprising an amino acid sequence selected from any one of the following (a) to (c):
(a) a sequence shown as SEQ ID NO. 2;
(b) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence shown in SEQ ID NO. 2; or
(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 represented by SEQ ID No. 2;
and
(II) a light chain variable region (VL) comprising an amino acid sequence selected from any one of the following (d) to (f):
(d) a sequence shown as SEQ ID NO. 4;
(e) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 4; or
(f) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 4.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence shown as SEQ ID NO.2 and a VL comprising the sequence shown as SEQ ID NO. 4.
In certain embodiments of the present application, the antigen binding fragment of the first or second aspect is selected from the group consisting of Fab, Fab ', (Fab') 2 Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb); and/or, the antibody is a rabbit derived antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
In certain embodiments of the application, the antibody or antigen-binding fragment thereof of the first or second aspect has one or more of the following characteristics:
(1) an RBD that specifically binds to SARS-CoV-2, or a mutant thereof, or an S protein or an RBD of an S protein thereof, or an S1 subunit or an S1 subunit thereof, including a SARS-CoV-2Omicron BA.1 mutant, an Omicron BA.2 mutant, a Beta mutant and a Delta mutant;
(2) blocking or inhibiting binding of SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof to the Ace2 receptor, and/or blocking or inhibiting infection of a cell by SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof;
(3) neutralizing the RBD of SARS-CoV-2, or a mutant thereof, or its S protein or S protein, or its S1 subunit or S1 subunit in vitro or in a subject (e.g., a human);
(4) preventing and/or treating infection of SARS-CoV-2, or a mutant strain thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit, or a disease associated with infection of SARS-CoV-2, or a mutant strain thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit (e.g. COVID-19).
Herein, an antibody or antigen-binding fragment thereof according to any aspect of the present application may include variants that differ from the antibody or antigen-binding fragment thereof from which it is derived only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids) amino acid residues, or that have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen-binding fragment thereof from which it is derived, and that substantially retain the above-described biological functions of the antibody or antigen-binding fragment thereof from which it is derived.
Derivatized antibodies
The antibody or antigen-binding fragment thereof of any aspect of the present application may be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of a monoclonal antibody or antigen-binding fragment thereof has no effect on its binding to SARS-CoV-2. Thus, the antibodies or antigen-binding fragments thereof disclosed immediately herein may also include such derivatized forms. For example, an antibody or antigen-binding fragment thereof of the present application can be functionally linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (e.g., avidin or polyhistidine tag) capable of mediating binding of the antibody or antigen-binding fragment to another molecule. In addition, the antibodies or antigen-binding fragments thereof of the present application may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of the antibody, for example to increase serum half-life.
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 (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., an isothiocyanate or a fluorescent protein), a radionuclide or biotin.
The detectable label described herein can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, 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 substances (e.g., chemiluminescent substances such as acridinium compounds), magnetic beads (e.g., Dynabeads), and the like @ ) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.
In certain embodiments of the present application, the detectable label can be suitable for use in immunological detection (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.).
In certain embodiments of the present application, a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the present application via a linker of varying length to reduce potential steric hindrance.
Preparation of antibodies
The antibodies according to any aspect of the present application may be prepared by various methods known in the art, for example by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy chain variable region and light chain variable region genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. The transfected host cells are then cultured under specific conditions and express the antibodies of the present application.
Antigen-binding fragments according to any aspect of the present application may be obtained by hydrolysis of an intact antibody molecule (see Morimoto et al, J. biochem. Biophys. methods 24:107-117(1992) and Brennan et al, Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly from recombinant host cells (preparation of different fragments of SARS-CoV-2N protein and use in fluorescence chromatography, journal, Biotechnology journal, published time 2021.06.28). For example, Fab' fragments can be obtained directly from the host cell; fab 'fragments can be chemically coupled to form F (ab') 2 fragments (Carter et al, Bio/Technology,10:163-167 (1992)). Furthermore, Fv, Fab or F (ab') 2 fragments can also be isolated directly from the culture medium of the recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
To this end, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the present application, or a heavy chain variable region and/or a light chain variable region thereof. In certain embodiments of the present application, the isolated nucleic acid molecule encodes an antibody or antigen-binding fragment thereof of the present application, or a heavy chain variable region and/or a light chain variable region thereof.
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, cosmid, phage, or the like.
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 E.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 DG44), e.g., mouse cells, human cells, etc.).
In another aspect, there is provided a method of making an antibody or antigen-binding fragment thereof of the present application, comprising culturing a host cell of the present application under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.
Use of antibodies
In another aspect, the present embodiments disclose a kit comprising an antibody or antigen-binding fragment thereof provided herein.
In certain embodiments of the present application, the antibodies or antigen-binding fragments thereof provided herein further comprise a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., an isothiocyanate or a fluorescent protein), a radionuclide, or biotin.
To this end, the kits disclosed herein further comprise a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof. Optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., an isothiocyanate or a fluorescent protein), a radionuclide or biotin.
In another aspect, the present embodiments disclose a method for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or the S protein or RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof, in a sample, said mutant selected from the group consisting of a SARS-CoV-2Omicron ba.1 mutant, an Omicron ba.2 mutant, a Beta mutant, and a Delta mutant, said method comprising the use of an antibody or antigen-binding fragment thereof as disclosed herein.
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 antigen-binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide or biotin; for example, the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody that carries a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin).
In another aspect, the present embodiments disclose the use of the antibody or antigen-binding fragment thereof in the preparation of a kit for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof, in a sample, or for diagnosing whether a subject is infected with SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit, said mutant being selected from the group consisting of an Omicron ba.1 mutant, an Omicron ba.2 mutant, a Beta mutant, and a Delta mutant;
in certain embodiments of the present application, the kit detects the presence or level of SARS-CoV-2 in a sample by the methods described. In some embodiments, the sample is a blood sample (e.g., whole blood, plasma, or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).
In another aspect, the embodiments herein disclose a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as favipiravir, ridciclovir, interferon and the like.
In certain embodiments of the present application, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent in the pharmaceutical composition may be provided as separate components or as a mixed component 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 solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.
In another aspect, the present examples disclose a method for neutralizing the toxicity of SARS-CoV-2, or a mutant thereof, or the RBD of its S protein or S protein, or the RBD of its S1 subunit or S1 subunit in a sample. The method comprises contacting a sample comprising SARS-CoV-2, or a mutant thereof, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit with an antibody, or antigen-binding fragment thereof, disclosed in the examples herein.
In another aspect, the embodiments disclose the use of the antibody or antigen-binding fragment thereof for the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample, or for preventing or treating a subject' S SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit infection or a disease associated with SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or S1 subunit RBD infection (e.g., COVID-19).
In some embodiments, the subject is a mammal, e.g., a human;
in some embodiments, the antibodies or antigen-binding fragments thereof are used alone, or in combination with additional pharmaceutically active agents (e.g., additional antiviral agents, such as interferon, lopinavir, ritonavir, chloroquine phosphate, fabiravir, ridciclovir, and the like).
The antibody or antigen-binding fragment thereof disclosed herein, or the pharmaceutical composition of the present application, may be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present application should be sterile and stable under the conditions of manufacture and storage. One preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by the following methods: the recombinant proteins of the present application are incorporated in the necessary dosage in an appropriate solvent, and optionally, together with other desired ingredients (including, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filter sterilization. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for storage and use. Such sterile lyophilized powders may be dispersed in a suitable carrier, e.g., water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof, prior to use.
The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions disclosed herein, can be administered by any suitable method known in the art, including oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In a preferred embodiment, the antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the present application is administered by intravenous injection or bolus injection.
The pharmaceutical compositions disclosed herein can include a "therapeutically effective amount" or a "prophylactically effective amount" of an 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 onset of disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The "therapeutically effective amount" of an antibody or antigen-binding fragment thereof disclosed herein may vary depending on the following factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g. age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, etc.
In the examples herein, the dosage regimen may be adjusted to obtain the optimal desired response (e.g., a therapeutic or prophylactic response). For example, the dosage may be given in a single dose, may be given multiple times over a period of time, or may be reduced or increased proportionally with the exigencies of the therapeutic situation.
Definition of terms
In the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, for better understanding of the present application, definitions and explanations of related terms are provided below.
As used herein, "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)," formerly known as "novel coronavirus" or "2019-nCoV", belongs to the genus of its β -coronavirus and is an enveloped, single-stranded, positive-sense RNA virus. The genomic sequence of SARS-CoV-2 is known to those skilled in the art and can be found, for example, in GenBank: MN 908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). Like SARS-CoV, the SARS-CoV-2 Receptor is specifically bound to angiotensin transferase 2(ACE2) on host cells via Receptor Binding Domain (RBD) on S protein, and then is connected to viral membrane fusion and cell entry, and plays a crucial role in the process of viral infection of cells.
As used herein, the terms "novel coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by SARS-CoV-2 infection, which have the same meaning and are used interchangeably.
As used herein, the terms "S protein" and "spike protein" both refer to the surface spike protein of SARS-CoV-2, which has a Receptor Binding Domain (RBD) thereon, and both have the same meaning and are used interchangeably.
As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair comprising one light (L) and one heavy (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotype of an antibody can be defined as IgM, IgD, IgG, IgA, and IgE according to the difference of heavy chains. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH1, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 are composed of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus. The variable regions (VH and VL) of each heavy/light chain pair form the antibody binding sites, respectively. The assignment of amino acids to the various regions or domains follows either Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-; chothia et al (1989) Nature342: 878-883. The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.
As described herein, the terms: "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 may be defined according to various numbering systems known in the art, for example as defined in the Kabat numbering system (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md.,1991), the Chothia numbering system (Chothia & Lesk (1987) J.Mol.biol.196: 901-917; Chothia et al (1989) Nature342:878-883) or the IMGT numbering system (Lefranc et al, Dev.Comprant. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al, Dev. Complex. Immunol.27:55-77,2003).
As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch.7(Paul, W., ed., 2nd edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes.
As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, the "full-length heavy chain" means that it is composed of, in the direction from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full-length antibodies of the present application can be from a single species, e.g., human; chimeric antibodies or humanized antibodies are also possible. The full-length antibodies of the present application comprise two antigen-binding sites formed by VH and VL pairs, respectively, that specifically recognize/bind the same antigen.
As used herein, the term "Fd" means an antigen-binding fragment consisting of the VH and CH1 domains; the term "dAb fragment" means an antigen-binding fragment consisting of a VH domain (Ward et al, Nature 341: 544546 (1989)); the term "Fab fragment" means an antigen-binding fragment consisting of the VL, VH, CL and CH1 domains; the term "F (ab') 2 fragment" means an antigen-binding fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a fragment obtained by reducing the disulfide bond linking two heavy chain fragments of a F (ab') 2 fragment, consisting of one intact Fd fragment of the light and heavy chains (consisting of the VH and CH1 domains).
As used herein, the term "Fv" means an antigen-binding fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antigen-binding fragments that form an entire antigen-binding site. It is generally believed that the six CDRs confer antigen binding specificity on the antibody. However, even one variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the entire binding site.
As used herein, the term "Fc" means an antigen-binding fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody to the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but is not involved in antigen binding.
As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (linker) (see, e.g., Bird et al, Science 242: 423-. Such scFv molecules can have the general structure: NH 2-VL-linker-VH-COOH or NH 2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used (Holliger et al (1993), Proc. Natl. Acad. Sci. USA 90: 6444-. Other linkers useful in this application are described by Alfthan et al (1995), Protein Eng.8: 725-. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In certain embodiments of the present application, the scFv may form a di-scFv, which refers to two or more individual scfvs in tandem to form an antibody. In certain embodiments of the present application, a scFv can form (scFv)2, which refers to two or more individual scfvs connected in parallel to form an antibody.
As used herein, the term "diabodies" means that the VH and VL domains thereof are expressed on a single polypeptide chain, but that a linker that is too short is used to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of the other chain and generating two antigen binding sites (see, e.g., Holliger P. et al, Proc. Natl. Acad. Sci. USA 90: 6444-.
As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antigen-binding fragment consisting of a single, monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen to which the full-length antibody binds. Single domain antibodies are also known as nanobodies (nanobodies).
Each of the above antigen-binding fragments retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
Antigen-binding fragments of antibodies (e.g., the antigen-binding fragments described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of antibodies are specifically screened in the same manner as for intact antibodies.
As used herein, the term "Chimeric antibody" refers to an antibody in which a portion of its light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass) and another portion of its light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which nevertheless retains binding activity to an antigen of interest (u.s.p4,816, 567toyield 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" can include, for example, a human murine chimeric antibody, wherein the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody). In certain embodiments of the present application, the term "chimeric antibody" can include an antibody in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., an individual human antibody sequence) and the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human consensus germline antibody sequence).
To prepare chimeric antibodies, the variable region of one antibody can be linked to the constant region of another antibody (e.g., a human immunoglobulin) using methods known in the art. For example, DNA encoding VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene. The sequence of the Human heavy chain constant region gene is known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH Publication No.91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is typically preferably an IgG1 or IgG4 constant region. For example, the DNA encoding VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full-length light chain gene (as well as the Fab light chain gene). The sequence of the Human light chain constant region gene is known in the art (see, e.g., Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. department of Health and Human Services, NIH Publication No.91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is typically preferably a kappa constant region.
As used herein, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to have an amino acid sequence that has been modified to increase homology to the sequence of a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Typically, at least one or two but usually all three acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody are replaced by donor CDRs. The immunoglobulin providing the CDRs is referred to as the "donor" and the immunoglobulin providing the framework is referred to as the "acceptor". In one embodiment, the donor immunoglobulin is a non-human (e.g., murine) antibody and the acceptor framework may be a naturally occurring human framework or a sequence having about 85%, 90%, 95%, 99% or more identity thereto. Humanized antibodies generally retain the desired properties of the donor antibody, including antigen specificity, affinity, reactivity, and the like. The donor antibody can be a mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having a desired property (e.g., antigen specificity, affinity, reactivity, etc.).
To prepare humanized antibodies, the CDR regions of an immunized animal (e.g., a mouse) can be grafted into human framework sequences using methods known in the art (see, design of humanized antibody library of anti-liver cancer single chain antibodies based on homology modeling techniques [ J ]. J.Oncology, 2015, 16 th.ISSN: 1005-.
The chimeric or humanized antibodies of the present application can be prepared based on the sequence of a monoclonal antibody produced by immunizing an animal (e.g., a mouse). The DNA encoding the heavy and light chains can be obtained from a hybridoma of interest or a specific B cell from an immunized animal and engineered to contain human immunoglobulin sequences using standard molecular biology techniques.
As used herein, the term "germline antibody gene (germline antibody gene)" or "germline antibody gene segment (germline antibody gene segment)" refers to immunoglobulin-encoding sequences present in the genome of an organism that have not undergone a maturation process that can lead to genetic rearrangements and mutations that express specific immunoglobulins. Accordingly, the term "rearranged antibody sequence" refers to a sequence of a specific antibody that has been generated through a maturation process that results in genetic rearrangement and mutation of the expressed specific immunoglobulin. In the present application, the expression "heavy chain germline gene" means the germline antibody gene or gene segment encoding the immunoglobulin heavy chain, which includes the V gene (variable), the D gene (diversity), the J gene (conjugation), and the C gene (constant); similarly, the expression "light chain germline gene" refers to germline antibody genes or gene segments encoding immunoglobulin light chains, which include the V gene (va ria ble), the J gene (junction), and the C gene (constant). In the present application, the amino acid sequence encoded by the germline antibody gene or germline antibody gene segment is also referred to as the "germline sequence", the amino acid sequence encoded by the heavy chain germline gene is referred to as the heavy chain germline sequence, and the amino acid sequence encoded by the light chain germline gene is referred to as the light chain germline sequence. Germline antibody genes or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from specialized databases (e.g., IMGT, unsmig, NCBI, or VBASE 2).
As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include: a plasmid; phagemid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), or artificial chromosome (PAC) of P1 origin; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyomavirus (e.g., SV 40). A vector may 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 contain a replication initiation site.
As used herein, the term "host cell" refers to a cell which can be used for introducing a vector, and includes, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as between an antibody and the antigen against which it is directed(ii) a reaction between the two. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody is present in an amount less than about 10 -5 M, e.g. less than about 10 -6 M、10 -7 M、 10 -8 M、10 -9 M or 10 -10 M or less binds to the antigen with an affinity (KD).
As used herein, the term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, the antibody is present in an amount less than about 10 -5 The dissociation equilibrium constant (KD) of M binds to antigen.
As used herein, the term "neutralizing activity" refers to the functional activity of an antibody or antigen-binding fragment that binds to an antigenic protein on a virus, thereby preventing maturation of virus-infected cells and/or virus progeny and/or release of virus progeny, and the antibody or antigen-binding fragment having neutralizing activity can prevent amplification of the virus, thereby inhibiting or eliminating infection by the virus.
As used herein, the term "identity" is used to refer to the match of sequences between two proteins, two polypeptides, or two nucleic acids. When a position in both of the sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of the total 6 positions match). Typically, the comparison is made when the two sequences are aligned to yield maximum identity. Such alignments can be performed by using, for example, Needleman et al (1970) j.mol.biol.48: 443-453. The algorithm of E.Meyers and W.Miller (Compout.appl biosci., 4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) can also be used to determine percent identity between two amino acid sequences using a PAM120 weight residue table (weight residue table), a gap length penalty of 12, and a gap penalty of 4. In addition, percent identity between two amino acid sequences can be determined using Needleman and Wunsch (J.MoI biol.48: 444-.
As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., of similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., Brummell et al, biochem.32:1180-1187 (1993); Kobayashi et al Protein Eng.12(10):879-884 (1999); and Burks et al, Proc. Natl Acad. set USA 94:412-417(1997), which are incorporated herein by reference).
The twenty conventional amino acids referred to herein are written following conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R.Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)) which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present application, amino acids are generally represented by single-letter and three-letter abbreviations as are well known in the art. For example, alanine can be represented by A or Ala.
As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by Gennaro AR,19th. ed. pennsylvania: mach Publishing Company,1995) and include: pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, the pH adjusting agent includes a phosphate buffer. Surfactants include cationic, anionic or nonionic surfactants, such as Tween-80. The ionic strength enhancer includes sodium chloride. Preservatives include various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents for maintaining osmotic pressure include sugars, NaCl, and the like. Agents that delay absorption include monostearate salts and gelatin. Diluents include water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include various antibacterial and antifungal agents, for example, thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning generally understood by those skilled in the art to be capable of stabilizing the desired activity of the active ingredient in a medicament, and include sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), glucose solutions (e.g., 5% glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), Ringer's solution, and any combination thereof.
As used herein, the term "preventing" refers to a method performed to prevent or delay the onset of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method performed in order to obtain a beneficial or desired clinical result. For purposes of this application, beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., no longer worsening) of the state of the disease, delay or slowing of disease progression, amelioration or palliation of the state of the disease, and remission (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to prolonging survival as compared to expected survival (if not treated).
As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
Advantageous effects of the present application
The present application provides antibodies or antigen-binding fragments having specific binding ability to SARS-CoV-2, or a mutant thereof, or an S protein thereof or an RBD of the S protein, or an RBD of the S1 subunit or the S1 subunit thereof, or to SARS-CoV-2, or a mutant thereof, or an RBD of the S protein or the S protein thereof, or an RBD of the S1 subunit or the S1 subunit thereof. Wherein the mutant strain of SARS-CoV-2 comprises SARS-CoV-2Omicron BA.1 mutant strain, SARS-CoV-2Omicron BA.2 mutant strain, SARS-CoV-2 Beta mutant strain and SARS-CoV-2Delta mutant strain. Specifically, these monoclonal antibodies bind to an epitope on the RBD region of the S protein of SARS-CoV-2 and neutralize SARS-CoV-2. The monoclonal antibodies of the present application are capable of inhibiting the binding of the RBD protein of SARS-CoV-2 to the receptor ACE 2.
Drawings
Fig. 1 shows protein electrophoresis results of antibodies Amb1 (left panel) and Amb2 (right panel) provided in the examples of the present application.
FIG. 2 shows the results of ELISA detection of the binding activity of the antibodies Amb1, Amb2 and the recombinant protein ACE2-hFc with wild-type neocoronavirus RBD protein, respectively, provided in the examples of the present application.
FIG. 3 is a graph showing the affinity determination curves of antibodies Amb1, Amb2 and recombinant protein ACE2 with wild-type new coronavirus RBD protein and new coronavirus Omicron BA.1 mutant strain, respectively, provided in the examples of the present application.
FIG. 4 shows the neutralization activity assay curves of antibodies Amb1 and Amb2 provided in the examples of the present application with Omicron BA.1 mutant strain, Omicron BA.2 mutant strain and wild-type novel coronavirus, respectively.
FIG. 5 shows the neutralization activity assay curves of antibodies Amb1 and Amb2 with Beta mutant and Delta mutant pseudoviruses, respectively, provided in the examples of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
Unless otherwise indicated, molecular biology experimental methods and immunoassays, as used herein, are essentially referred to in j.sambrook et al, molecular cloning: a laboratory manual, 2nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are described by way of example and are not intended to limit the scope of the claims to this application, as those skilled in the art will recognize.
Construction and screening of fully human anti-new coronavirus scFv antibody library
Human Peripheral Blood Mononuclear Cells (PBMC) isolated from 10mL of whole blood drawn from an inactivated vaccinee (three needles) were lysed using the one-step total RNA extraction reagent TRIzol to extract total RNA. Then, oligo-dT is used as a primer to perform reverse transcription PCR reaction, cDNA is prepared respectively, and the cDNA is mixed in equal volume. Antibody genes VH, Vkappa and Vlambda were amplified using Human Antibody signature primers (see Generation and Characterization of a Recombinant Human CCR5-specific Antibody: a phase display approach for a phage Antibody immunization, The Journal of biological chemistry, 275, 46, 36073-8), V kappa-linker-VH and V lambda-linker-VH were further obtained using overlap extension Polymerase Chain Reaction (PCR), and The fragments were separately digested with restriction enzyme SsI and ligated with T4DNA ligase into a previously cut phage display vector pComb3 XSS. After the connecting product is desalted by ethanol precipitation, the connecting product is transferred into TG1 bacterial competence by adopting an electrotransformation method, and the constructed library capacities are 1.5 multiplied by 10 respectively 9 And 2.2X 10 9 For subsequent screening. The procedure is based on reference (Barbas, C.F., III; Burton, D.R.; Scott, J.K., Silverman, G.J.Eds. (2001) phase Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York,736pages.)
Of phage antibody librariesEnrichment screening and induced expression of scFV antibody
The screening antigen used was a purified novel coronavirus wild-type RBD protein (RBD-WT, cat # Z03479, available from Gensript) and was screened as follows:
antigen was diluted with PBS solution at pH7.4, coating the wells of a 96-well plate with 100 μ L per well; coating was carried out overnight at 4 ℃. PBST was washed three times, 200. mu.L/well blocking buffer was added, and overnight at 4 ℃. PBST was washed 5 times, 200. mu.L per well, and library phage diluted to 1X 10 with blocking buffer 11 100. mu.L per well, 1.5h at 37 ℃. The plate was washed 5 times with PBST and 3 times with PBS, 100. mu.L of glycine-HCl pH 2.2 eluent was added to each well, and 15. mu.L of Tris-HCl pH9.1 was immediately added for neutralization. The remaining eluate was mixed with 5ml of log phase TG1 at 37 ℃ and 220rpm for 45 min. The above-mentioned bacterial solution was transferred to 20mL of 2YT/A +/G medium, cultured to logarithmic phase, infected with the helper Phage VCSM13 (Stratagene, USA), incubated at 37 ℃ for 1 hour, cultured with kanamycin (50. mu.g/mL final concentration) at 30 ℃ overnight with shaking to prepare Phage single-chain antibody, the plate was coated with SARS-CoV-2 RBD protein at 0.1. mu.g/well, and the secondary antibody was HRP-labeled anti-M13 antibody diluted at 1:2000 with PBS buffer (containing 5G/mL skim milk powder), subjected to Phage-ELISA and measured for OD450 value. This was repeated 3 times. Specific enrichment screening methods and procedures for induced expression of scFv fragments are referred to (Barbas, C.F., III; Burton, D.R.; Scott, J.K., Silverman, G.J. Eds. (2001) phase Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York,736 pages.).
Through the above tests, scFv segment 1, scFv segment 2, scFv segment 3 and scFv segment 4 were obtained. scFv segment 1 has the heavy chain variable region of the amino acid sequence set forth in SEQ ID NO.1 and the light chain variable region of the amino acid sequence set forth in SEQ ID NO. 3. scFv segment 2 has the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.2 and the heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 4. scFv segment 3 has the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO.1 and the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 4. scFv segment 4 heavy chain variable region having the amino acid sequence set forth in SEQ ID NO.2 and heavy chain variable region having the amino acid sequence set forth in SEQ ID NO. 3.
Further, the CDR Sequences in scFv segment 1 and scFv segment 2 and scFv segment 3scFv segment 4 were also determined using the method described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, Public Health Service, national institutes of Health, Besserda, Maryland (1991), p.647-669). Wherein, the heavy chain CDR 1-3 of SEQ ID NO.1 is sequentially shown as SEQ ID NO. 5-7, the heavy chain CDR 1-3 of SEQ ID NO.2 is sequentially shown as SEQ ID NO. 8-10, the light chain CDR 1-3 of SEQ ID NO.3 is sequentially shown as SEQ ID NO. 11-13, and the light chain CDR 1-3 of SEQ ID NO.4 is sequentially shown as SEQ ID NO. 14-16.
Preparation of Whole antibody
Plasmids from the bacterial suspension of the positive clones were extracted using a plasmid extraction Kit (Qiagen Miniprep Kit, QIAGEN, Germany) and sequenced by the commercial company. The sequencing results were compared with the IgG gene sequences in the databases of V-Base gene library (http:// www.vbase2.org /), IMGT (http:// www.imgt.org /), V-QUEST (http:// www.imgt.org/IMGT _ vquest/share/textes /), Ig BLAST (http:// www.ncbi.nlm.nih.gov/igblast /), and the like. The whole antibody variable region gene sequence 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) is converted into an IgG1 whole antibody form, the whole gene is connected into a secretory mammal expression vector pHL-Sec (purchased from Addne, #99845 and modified from a classical mammal expression plasmid pCAGGS) by a seamless cloning method after being synthesized, a signal peptide sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) carried by the vector is replaced by a leader peptide sequence (Murine Ig kappa-chain leader sequence, METDTLLLWVLLLWVPWGSTGDJ) of a mouse kappa chain, and a stop codon is added behind the sequence. Thus, heavy chain expression plasmid pHL-HC and light chain expression plasmid pHL-LC were constructed.
The pHL-HC and pHL-LC with correct sequencing were subjected to endotoxin-free mass extraction. FreeStyle for HEK293F cells TM 293 expression Medium for suspension culture at 1.0X 10 day before transfection 6 cells/mL density were inoculated in disposable conical flasks, and the inoculated cells were placed at 37 ℃ in 5% CO 2 In the incubator, shaking culture was carried out at 125 rpm. Cells were counted on the day of transfection at a cell density of 2.0X 10 6 cells/mL (if diluted in medium beyond density), transient transfection was performed with viability greater than 95%. The DNA-PEI complex was prepared by mixing the heavy chain plasmid pHL-HC, the light chain plasmid pHL-LC and PEI (Polysciences Co.) at a ratio of 1:1:2.5 (wherein "1" means 1. mu.g/mL cells), added to the cell culture flask to be transfected, gently shaken, and placed at 37 ℃ with 5% CO 2 And continuing shaking culture in the incubator. After 4 days of transfection, supernatants were collected and cells were used daily for dynamic analysis of recombinant protein expression, and after 7 days of transfection, supernatants were collected.
Suction filtration was performed using a 0.22 μ M microfiltration membrane, and 1/10 volumes of 10 × pH adjusting buffer (0.5M Na) were added 2 HPO 4 pH7.4), the culture supernatant containing the recombinant antibody is passed directly through two 5mL HiTrap Protein G HP pre-cartridges in series at a flow rate of 5mL/min in a Protein purification system (AKTA pure25), after binding is completed at low flow rate, using a binding buffer (20mM Na. sup.) (HiTrap Protein G HP) 2 HPO 4 pH7.0) no longer changed in the UV absorbance at 280 nM. Then eluted with elution buffer (0.1M glycine-HCl, pH2.7), and 1/10 volumes of neutralization buffer (1M Tris-HCl, pH9.0) were added to the collection tubes. And (3) detecting the purification condition by SDS-PAGE coupled bromophenol blue staining, determining the peak of the target protein, collecting and concentrating the target protein, and further purifying by using an anion exchange column and a molecular sieve for the next step of experiment. Optionally, the supernatant is collected, centrifuged to remove cells and debris, and purified by a Protein G HP SpinTrap centrifugal small-amount antibody affinity chromatography column to prepare a small amount of monoclonal antibody. The purification method comprises the following steps: the centrifuged supernatant was mixed with binding buffer (20mM Na) 2 HPO 4 pH7.0) were mixed in equal volumes. Add 600. mu.L of binding buffer, centrifuge at 100g for 30s, and equilibrate the column. Add 600. mu.L of pre-equilibrated sample repeatedly, centrifuge at 100g for 30s, mix gently, stand for 4min, and bind the antibody to the column. Add 600. mu.L binding buffer, centrifuge at 100g for 30s, and wash off impurities. This was repeated twice. Adding 400. mu.L of elution buffer (0.1M Glycine-HCl, pH2.7), mixing by inversion, and placing in a medium containing 30. mu.L of neutralization buffer (1M Tris-HCl, pH9.0), 100g of the solution was centrifuged for 30s in a 2mL EP tube, and this was repeated twice.
As shown in FIG. 1, the electrophorograms of the purified recombinantly expressed antibodies Amb1 and Amb2 each showed a band around 50kD and 30kD, representing their heavy and light chains, respectively.
Binding Activity of Amb1 and Amb2 on RBD proteins of different novel coronavirus strains
1. ELISA detection
With pH 9.60.1M NaHCO 3 The solution coats a novel coronavirus wild type RBD protein (cat # Z03479, Gensript) on an enzyme label plate, and stays overnight at 4 ℃; sealing with 3% skimmed milk, incubating at 37 deg.C for 1h, adding recombinant expressed Amb1 and Amb2 antibodies and ACE2-hFc recombinant protein (control), and incubating at 37 deg.C for 1 h; adding enzyme-labeled anti-human Fc fragment secondary antibody, and incubating at 37 ℃ for 1 h; color developing solution for color development, 2M H 2 SO 4 The reaction was stopped and OD450 was detected by a microplate reader. The result shows that the antibody has high binding activity, and as shown in figure 1, Amb1 and Amb2 antibodies both have higher binding activity to the RBD protein of the novel coronavirus strain than ACE 2-hFc.
2. BIAcore method for determining antibody affinity
The novel coronavirus wild-type (cat # Z03479, Gensript) and the Omicron BA.1 mutant RBD protein (cat # Z03516, Gensript) were diluted in 10mM NaAc, attempting to bind the antibody to CM5 chips under different pH conditions. The pH value is set to four gradients of pH4.0, pH4.5, pH5.0, pH5.5, and the pH with the best fixing effect is selected. RBD was diluted to 10mM NaAc at optimal pH, immobilized on the chip surface, and the target coupling amount RU (response Unit) of each antibody was determined according to the formula. The Amb1 and Amb2 antibodies are used as mobile phases, the concentration gradient is 2.6 nM-333 nM or 666nM, and response values generated when different concentrations of Amb1, Amb2 and ACE2-hFc flow through the chip surface are detected. The analysis was carried out at a constant temperature of 25 ℃ and a flow rate of 30. mu.L/min. The solution used for regenerating the chip surface is 100mM H 3 PO 4 . The binding kinetics constants were calculated using BIA evaluation software version (Biacore, Inc.) in a 1:1 binding mode, and the affinity constants (K) D (M))。
As a result, as shown in FIG. 2, the affinity constants of Amb1 and Amb2 for wild-type RBD were 1.51X 10, respectively -7 And 1.64X 10 -7 (ii) a The affinity constant of ACE2-hFc to wild type RBD is 3.09 × 10 -7 (ii) a The affinity constants of Amb1 and Amb2 for the Omicron BA.1 mutant RBD were 2.05X 10 -7 And 3.33X 10 -7 (ii) a The affinity constant of ACE2-hFc to the Omicron BA.1 mutant RBD was 5.10X 10 -7 Not only does Amb1 and Amb2 show that the affinity of the new coronavirus and mutant strains thereof is higher than that of the receptor ACE2, but also the affinity of the new coronavirus and the mutant strains thereof is higher than that of the receptor ACE2, and the new coronavirus and mutant strains thereof are used as a blocking agent for competitively blocking the binding of the virus and the receptor ACE2, so that the new coronavirus and the mutant strains thereof can be applied to related medicines or preparations for preventing virus infection.
Pseudovirus neutralization assay
Preparation of pseudovirus: paving 5-6 multiplied by 10 days in advance 6 293T cells in 6-well plates, continued for 18-24h, using 12 u L FuGENE HD (cat # E2311, Promega) transfection reagent according to the instructions 2 u g HIV-1 skeleton plasmid pNL4-3 Luc. R-E-and 2 u g S protein expression plasmid (SARS-CoV-2 wild type, SARS-CoV-2Omicron BA.1, SARS-CoV-2 BA.2, SARS-CoV-2 Beta or SARS-CoV-2delta) were co-transferred into 293T cells. After 48h from the start of transfection, the culture medium in the 6-well plate is sucked out, added into a sterile 15mL centrifuge tube, centrifuged at 3000rpm for 15min, and the supernatant is taken, namely the pseudovirus of SARS-CoV-2 wild type, SARS-CoV-2Omicron BA.1, SARS-CoV-2 BA.2, SARS-CoV-2 Beta or SARS-CoV-2Delta, and frozen in a refrigerator at-80 ℃ for standby.
The virus was previously thawed on ice from-80 ℃ and serum-free DMEM was prepared. 293T-ACE2 cells (Yeasen Saint, cat # 41107ES03) were seeded one day in advance in 96-well cell culture plates at approximately 1.2X 10 4 Individual cells/well, infection was performed according to cell density the next day. The cell density is preferably about 50%. The antibody was diluted and the first EP tube antibody concentration was 75.0. mu.g/mL. 0 μ g/mL, total volume 100 μ L, following 80 μ L DMEM per EP tube, the previous 20 μ L was pipetted into the next tube, diluted 5-fold, setting up 10 gradients. A new 96-well plate was prepared, and pseudovirus 96.0. mu.L + antibody 24.0. mu.L (in this case, the antibody concentration was 15.0. mu.g/mL) was incubated at 37 ℃ for 1 hour,another 3 wells were set and 100. mu.L of pseudovirus (as described above) was added as a control. The culture medium in a 96-well cell culture plate is discarded, 100.0 mu L of antibody pseudovirus mixed solution is added, the solution is changed after 6h, and the luciferase activity is detected after 48 h. During detection, the plate is taken out in advance, placed at room temperature for 30min, meanwhile, the detection reagent is taken out from the temperature of minus 20 ℃ for unfreezing, and 100 mu L of detection reagent is added into each hole in the same volume for cracking for 3 min. After being mixed evenly, the mixture is transferred to a detection plate by a line gun and is placed in a GloMax 96 micropore plate luminescence detector for detection.
The results are shown in FIGS. 3 and 4, and Amb1 and Amb2 neutralize the IC of the wild type of the new coronavirus 50 0.1303 and 0.064. mu.g/mL respectively, Amb1 and Amb2 neutralized the IC of the mutant strain Omicron BA.1 50 0.02464 μ g/mL and 0.01549 μ g/mL, respectively, Amb1 and Amb2 neutralize the IC of Omicron BA.2 50 0.01126 μ g/mL and 0.02734 μ g/mL, respectively, Amb1 and Amb2 neutralize the IC of Beta mutants 50 0.02660. mu.g/mL and 0.04516. mu.g/mL, respectively. IC of Amb1 and Amb2 neutralized Delta mutant strains 50 1.220. mu.g/mL, and 0.4077. mu.g/mL, respectively.
In conclusion, the application discloses that the scFv of the antigen-binding fragment has high affinity activity to the RBD protein of the novel coronavirus, Amb1 and Amb2 prepared by genetic engineering means have strong neutralizing effect on wild type, Beta mutant, Delta mutant and pseudo-virus of Omicron BA.1 and BA.2, have the potential to cope with global new crown pandemic caused by the Ormcken variant and outbreak prevalence caused by continuously-appeared evasive vaccine and new strain of Ormcken neutralizing antibody, and have great clinical application value.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Sequence listing
<110> segmentally beneficial
Jiang Zhihua
Wang Hui
<120> SARS-CoV-2 and antibody of mutant strain and application
<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
Ala Ala Ser
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 (19)
1. An antibody or antigen-binding fragment thereof that specifically binds to the receptor binding Region (RBD) of the S protein of SAR S-CoV-2 and mutants thereof, comprising:
(I) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:
(a) a VH CDR1, consisting of the sequence: SEQ ID NO.5, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.substitutions, deletions or additions of 1, 2 or 3 amino acids),
(b) a VH CDR2, consisting of the sequence: SEQ ID NO.6, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(c) a VH CDR3, consisting of the sequence: SEQ ID No.7, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and/or the presence of a gas in the gas,
(II) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) as defined by the Kabat numbering system:
(d) a VL CDR1, consisting of the sequence: SEQ ID NO.11, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.substitutions, deletions or additions of 1, 2 or 3 amino acids),
(e) a VL CDR2, consisting of the sequence: SEQ ID NO.12, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(f) a VL CDR3, consisting of the sequence: SEQ ID No.13, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g. conservative substitutions, deletions or additions of 1, 2 or 3 amino acids).
2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
(I)3 heavy chain CDRs: VH CDR1 of sequence SEQ ID NO.5, VH CDR2 of sequence SEQ ID NO.6 and VH CDR3 of sequence SEQ ID NO. 7; and/or, 3 light chain CDRs: VL CDR1 of sequence SEQ ID NO.11, VL CDR2 of sequence SEQ ID NO.12 and VL CDR3 of sequence SEQ ID NO. 13; or the like, or, alternatively,
(II) 3 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, 3 CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as shown in SEQ ID No. 3.
3. An antibody or antigen-binding fragment thereof that specifically binds to the receptor binding Region (RBD) of the S protein of SAR S-CoV-2 and mutants thereof, comprising:
(I) a heavy chain variable region (VH) comprising the following 3 Complementarity Determining Regions (CDRs) defined according to the Kabat numbering system:
(a) a VH CDR1, consisting of the sequence: SEQ ID NO.8, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.conservative substitutions, deletions or additions of 1, 2 or 3 amino acids),
(b) a VH CDR2, consisting of the sequence: SEQ ID NO.9, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(c) a VH CDR3, consisting of the sequence: SEQ ID No.10, or a sequence having substitution, deletion or addition of one or several amino acids (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids) thereto; and/or the presence of a gas in the gas,
(II) a light chain variable region (VL) comprising the following 3 Complementarity Determining Regions (CDRs) as defined by the Kabat numbering system:
(d) a VL CDR1, consisting of the sequence: SEQ ID NO.14, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g.substitutions, deletions or additions of 1, 2 or 3 amino acids),
(e) a VL CDR2, consisting of the sequence: SEQ ID NO.15, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids), and
(f) a VL CDR3, consisting of the sequence: SEQ ID No.16, or a sequence having conservative substitutions, deletions or additions of one or several amino acids compared thereto (e.g., conservative substitutions, deletions or additions of 1, 2 or 3 amino acids).
4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:
(I) the following 3 heavy chain CDRs: VH CDR1 of sequence SEQ ID NO.8, VH CDR2 of sequence SEQ ID NO.9 and VH CDR3 of sequence SEQ ID NO. 10; and/or, the following 3 light chain CDRs: VL CDR1 of sequence SEQ ID NO.14, VL CDR2 of sequence SEQ ID NO.15 and VL CDR3 of sequence SEQ ID NO. 16; or the like, or, alternatively,
(II) 3 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, 3 CDRs defined according to the Kabat numbering system contained in the light chain variable region (VL) as set forth in SEQ ID No. 4.
5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, further comprising a framework region sequence derived from a human immunoglobulin;
preferably, the human immunoglobulin is selected from a human rearranged antibody sequence or a human germline antibody sequence;
preferably, 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;
preferably, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region sequence derived from a human heavy chain germline sequence, and a light chain framework region sequence derived from a human light chain germline sequence.
6. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising:
(I) a heavy chain variable region comprising an amino acid sequence selected from any one of the following (a) to (c):
(a) an amino acid sequence shown as SEQ ID NO. 1;
(b) a sequence having conservative substitutions, deletions or additions of one or several amino acids (e.g. conservative substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids) compared to the sequence shown in SEQ ID No. 1; or
(c) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 1; and
(II) a light chain variable region comprising an amino acid sequence selected from any one of the following (d) to (f):
(d) a sequence shown as SEQ ID NO. 3;
(e) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 3; or
(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 represented by SEQ ID No. 3;
preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence shown as SEQ ID No.1 and a light chain variable region comprising the sequence shown as SEQ ID No. 3.
7. The antibody or antigen-binding fragment thereof of claim 3 or 4, comprising:
(I) a heavy chain variable region comprising an amino acid sequence selected from any one of the following (a) to (c):
(a) an amino acid sequence shown as SEQ ID NO. 2;
(b) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence shown in SEQ ID NO. 2; or
(c) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence set forth in SEQ ID No. 1; and
(II) a light chain variable region comprising an amino acid sequence selected from any one of the following (d) to (f):
(d) a sequence shown as SEQ ID NO. 4;
(e) a sequence having conservative substitution, deletion or addition of one or several amino acids (e.g., conservative substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids) as compared with the sequence represented by SEQ ID NO. 4; or
(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 represented by SEQ ID No. 4;
preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the sequence shown as SEQ ID No.2 and a light chain variable region comprising the sequence shown as SEQ ID No. 4.
8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', (Fab') 2 Fv, disulfide-linked Fv, scFv, diabody (diabody), and single domain antibody (sdAb); and/or the antibody is rabbit source antibodyA body, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.
9. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics:
(1) an RBD that specifically binds to SARS-CoV-2, or a mutant thereof, or an S protein or an RBD of an S protein thereof, or an S1 subunit or an S1 subunit thereof, including an Omicron BA.1 mutant, an Omicron BA.2 mutant and a Beta mutant;
(2) blocking or inhibiting binding of SARS-CoV-2, or a mutant thereof, 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 infection of a cell by SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit;
(3) neutralizing the RBD of SARS-CoV-2, or a mutant thereof, or its S protein or S protein, or its S1 subunit or S1 subunit in vitro or in a subject (e.g., a human);
(4) preventing and/or treating infection of SARS-CoV-2, or a mutant strain thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit, or a disease associated with infection of SARS-CoV-2, or a mutant strain thereof, 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 of any one of claims 1-7, or a heavy chain variable region and/or a light chain variable region thereof.
11. A vector comprising the nucleic acid molecule of claim 10; preferably, the vector is a cloning vector or an expression vector.
12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.
13. A method of making the antibody or antigen-binding fragment thereof of any one of claims 1-7, the method comprising culturing the host cell of claim 12 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.
14. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7; for example, the antibody or antigen-binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., an isothiocyanate or a fluorescent protein), a radionuclide or biotin; for example, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound), a fluorescent dye (e.g., an isothiocyanate or a fluorescent protein), a radionuclide or biotin.
15. A method for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or its RBD of S1 subunit in a sample, said mutant selected from the group consisting of SARS-CoV-2Omicron ba.1 mutant, Omicron ba.2 mutant, Beta mutant, and Delta mutant, said method comprising using the antibody or antigen-binding fragment thereof of any one of claims 1 to 6;
preferably, 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 antigen-binding fragment thereof further comprises a detectable label, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide or biotin; for example, the method further comprises detecting the antibody or antigen-binding fragment thereof using a second antibody that carries a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium compound), a fluorescent dye (e.g., fluorescein isothiocyanate or a fluorescent protein), a radionuclide, or biotin).
16. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 in the preparation of a kit for detecting the presence or level of SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof, in a sample, or for diagnosing whether a subject is infected with SARS-CoV-2, or a mutant thereof, or the S protein or the RBD of the S protein, or the S1 subunit or the RBD of the S1 subunit thereof, said mutant selected from the group consisting of an Omicron ba.1 mutant, an Omicron ba.2 mutant, a Beta mutant, and a Delta mutant;
preferably, the kit detects the presence or level of SARS-CoV-2 in the sample by the method of claim 15;
preferably, the sample is a blood sample (e.g., whole blood, plasma or serum), fecal matter, oral or nasal secretions, or alveolar lavage fluid from a subject (e.g., a mammal, preferably a human).
17. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-7, and a pharmaceutically acceptable carrier and/or excipient; preferably, the pharmaceutical composition further comprises additional pharmaceutically active agents, such as fapivavir, redciclovir, interferon and the like.
18. A method for neutralizing the virulence of SARS-CoV-2, or a mutant thereof, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit in a sample comprising contacting a sample comprising SARS-CoV-2, or a mutant thereof, or its S protein or the RBD of the S protein, or its S1 subunit or the RBD of the S1 subunit with the antibody or antigen-binding fragment thereof of any one of claims 1-7.
19. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 for the preparation of a medicament for neutralizing the virulence of SARS-CoV-2 in a sample, or for preventing or treating a subject' S SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or RBD of S1 subunit infection or a disease associated with SARS-CoV-2, or a mutant thereof, or its S protein or RBD of S protein, or its S1 subunit or S1 subunit RBD infection (e.g., COVID-19);
preferably, the subject is a mammal, e.g., a human;
preferably, the antibodies or antigen-binding fragments thereof are used alone or in combination with additional pharmaceutically active agents (e.g., additional antiviral agents such as interferon, lopinavir, ritonavir, chloroquine phosphate, fabavir, ridciclovir, and the like).
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