Antibodies specific to the spike protein of SARS-CoV-2 and uses thereof

Abstract

The present disclosure relates to an antibody or antigen-binding fragment thereof that is specific for the spike protein of SARS-CoV-2. The present disclosure also relates to a pharmaceutical composition for treating and / or preventing a disease and / or disorder caused by coronavirus in a subject in need thereof, and a method for detecting coronavirus in a sample.

Description

[Technical field]

[0001] Priority information This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 266,008, filed December 27, 2021, the disclosure of which is incorporated herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .xml format and is incorporated herein by reference in its entirety. The .xml copy, created on December 26, 2022, is named "A1000-01000PCT_SeqListing_20221226.xml" and is 12 kilobytes in size.

[0003] The present disclosure relates to antibodies or antigen-binding fragments thereof that are specific for the spike protein of SARS-CoV-2 and uses thereof. [Background technology]

[0004] The COVID-19 epidemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading worldwide. This infection causes symptoms of direct cytopathic effects and excessive inflammatory responses in infected subjects. The lack of effective treatments causes high morbidity and mortality. The emergence of these newly identified viruses highlights the need for the development of novel antiviral strategies. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need to develop effective treatments for COVID-19. [Means for solving the problem]

[0006] The present disclosure provides novel neutralizing therapeutic or detectable anti-coronavirus spike protein (such as anti-SARS-CoV-2-spike protein) antibodies and their use for treating or preventing or detecting viral infection.

[0007] Thus, the present disclosure provides antibodies or antigen-binding fragments thereof that are specific for an epitope in the spike protein of a coronavirus (CoV), particularly SARS-CoV-2, particularly where the spike protein is fully glycosylated. Thus, the antibodies according to the present disclosure are useful for treating and / or preventing or detecting diseases and / or disorders caused by or associated with a CoV, particularly SARS-CoV-2. The antibodies of the present disclosure are also useful for detecting CoVs (particularly SARS-CoV-2).

[0008] In one embodiment, the present disclosure provides an antibody or antigen-binding fragment thereof specific for an epitope located in the spike protein of a CoV, wherein the spike protein is fully glycosylated.

[0009] In some embodiments of the present disclosure, the CoV described herein is alpha-CoV, beta-CoV, gamma-CoV, delta-CoV2 or omicron-CoV. In some embodiments, the CoV described herein includes, but is not limited to, SARS-CoV, MERS-CoV or SARS-CoV-2.

[0010] In one embodiment, the disclosure provides an antibody or antigen-binding fragment thereof specific for an epitope in a spike protein of a CoV, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region; The CDRs of the heavy chain variable region are CDRH1 having the amino acid sequence of GYTFTDYN (SEQ ID NO: 1) or GYTFTEYT (SEQ ID NO: 6) or a substantially similar sequence thereof, CDRH2 having the amino acid sequence of VNPNNGGT (SEQ ID NO: 2) or INPNNGGT (SEQ ID NO: 7) or a substantially similar sequence thereof, and CDRH3 having the amino acid sequence of ARGKGDY (SEQ ID NO: 3) or SRNYGYSYVYYFDN (SEQ ID NO: 8) or a substantially similar sequence thereof; The CDRs of the light chain variable region are The present invention provides an antibody or antigen-binding fragment thereof, comprising CDRL1 of ESVEYSGTSL (SEQ ID NO: 4) or ENIYSN (SEQ ID NO: 9) or a sequence substantially similar thereto, CDRL2 of the amino acid sequence of AAS or AAT or a sequence substantially similar thereto, and CDRL3 of the amino acid sequence of LQSRKVPYT (SEQ ID NO: 5) or QHFWGTPT (SEQ ID NO: 10) or a sequence substantially similar thereto.

[0011] In some embodiments, the amino acid sequence of CDRH1 is SEQ ID NO:1, or a substantially similar sequence thereof; the amino acid sequence of CDRH2 is SEQ ID NO:2, or a substantially similar sequence thereof; the amino acid sequence of CDRH3 is SEQ ID NO:3, or a substantially similar sequence thereof; and The amino acid sequence of CDRL1 is SEQ ID NO:4 or a substantially similar sequence thereof; the amino acid sequence of CDRL2 is AAS or a substantially similar sequence thereof; and the amino acid sequence of CDRL3 is SEQ ID NO:5 or a substantially similar sequence thereof.

[0012] In some embodiments, the amino acid sequence of CDRH1 is SEQ ID NO:6, or a substantially similar sequence thereof; the amino acid sequence of CDRH2 is SEQ ID NO:7, or a substantially similar sequence thereof; the amino acid sequence of CDRH3 is SEQ ID NO:8, or a substantially similar sequence thereof; and The amino acid sequence of CDRL1 is SEQ ID NO:9 or a substantially similar sequence thereof; the amino acid sequence of CDRL2 is AAT or a substantially similar sequence thereof; and the amino acid sequence of CDRL3 is SEQ ID NO:10 or a substantially similar sequence thereof.

[0013] In some embodiments of the present disclosure, the spike protein is fully glycosylated, hi one embodiment, the spike protein is monoglycosylated.

[0014] In some embodiments of the present disclosure, the epitope is located in the receptor binding domain (RBD), S1 region and / or S2 region of the spike protein. In some embodiments of the present disclosure, the antibody comprising SEQ ID NO: 1-5 is specific for an epitope located in the receptor binding domain of the spike protein. In some embodiments of the present disclosure, the antibody comprising SEQ ID NO: 6-10 is specific for an epitope located in the S2 region of the spike protein.

[0015] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof is a Fab fragment, a F(ab')2 fragment, an ScFv fragment, a monoclonal antibody, a chimeric antibody, a nanobody, a humanized antibody, or a human antibody.

[0016] In some embodiments of the present disclosure, the antibodies or antigen-binding fragments thereof are multispecific.

[0017] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof comprises an Fc region or the antigen-binding fragment is fused to an Fc region, and the antibody or antigen-binding fragment thereof has N-glycans on the Fc region.

[0018] The present disclosure also provides a conjugate comprising an antibody or antigen-binding fragment thereof disclosed herein bound to the spike protein or fragment thereof of a CoV.

[0019] The present disclosure provides vectors encoding the antibodies or antigen-binding fragments thereof disclosed herein.

[0020] The present disclosure provides engineered cells that express the antibodies or antigen-binding fragments thereof disclosed herein or contain the vectors disclosed herein.

[0021] The present disclosure also provides a method for producing an antibody or antigen-binding fragment thereof disclosed herein, the method comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favoring expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown.

[0022] The present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof or conjugate disclosed herein, a pharma- ceutical acceptable carrier, and, optionally, an additional therapeutic agent. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 1-5.

[0023] The present disclosure provides a container or injection device comprising an antibody or antigen-binding fragment thereof or a conjugate disclosed herein. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 1-5.

[0024] The present disclosure provides a method for treating or preventing coronavirus infection in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof or conjugate disclosed herein. Alternatively, the present disclosure provides a pharmaceutical composition for use in treating or preventing coronavirus infection in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof or conjugate used herein, a pharma- ceutical acceptable carrier, and optionally an additional therapeutic agent. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 1-5.

[0025] The present disclosure provides a method for neutralizing coronavirus in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment thereof or conjugate disclosed herein. Alternatively, the present disclosure provides a pharmaceutical composition for use in neutralizing coronavirus in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof or conjugate used herein, a pharma- ceutically acceptable carrier, and optionally an additional therapeutic agent. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 1-5.

[0026] Examples of additional therapeutic agents include, but are not limited to, antiviral agents. In some embodiments of the present disclosure, the additional therapeutic agent is an anti-inflammatory agent or an antibody or antigen-binding fragment thereof specific for the spike protein of a CoV.

[0027] In some embodiments of the present disclosure, the subject is vaccinated.

[0028] In some embodiments of the present disclosure, the subject is administered one or more additional therapeutic agents.

[0029] In some embodiments of the present disclosure, the pharmaceutical composition is in a form suitable for injection. Alternatively, the present disclosure provides a method for administering an antibody or antigen-binding fragment thereof disclosed herein to a subject, comprising injecting the antibody or antigen-binding fragment into the subject. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 1-5.

[0030] In some embodiments of the present disclosure, the injection is subcutaneous, intravenous, or intramuscular. Alternatively, the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the subject's body.

[0031] The present disclosure provides a method for detecting coronavirus in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 6-10.

[0032] The present disclosure provides a kit for detecting coronavirus in a sample, the kit comprising an antibody or antigen-binding fragment thereof disclosed herein. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprises SEQ ID NOs: 6-10.

[0033] The present disclosure is described in detail in the following sections. Other features, objects, and advantages of the present disclosure can be found in the detailed description and claims. [Brief description of the drawings]

[0034] [Figure 1A] FIG. 1A shows that m37A12 can bind to the spike protein or RBD in an ELISA assay. [Figure 1B] FIG. 1B shows the binding avidity of full-length m37A12 IgG1 to the full-length spike protein as measured by biolayer interferometry. [Figure 2A] FIG. 2A shows that P36H3 can bind to the spike protein or S2. [Figure 2B] FIG. 2B shows the binding avidity of full-length P36H3 IgG1 to the full-length spike protein as measured by biolayer interferometry. [Figure 3A] FIG. 3A shows FACS analysis of m37A12 binding to 293T cells expressing WT and delta mutant spike proteins. [Figure 3B] Figure 3B shows the ability of m37A12 to neutralize WT or the indicated mutant SARS-CoV-2 pseudoviruses. [Figure 4] FIG. 4 shows FACS analysis of P36H3 binding to 293T cells expressing WT and delta mutant spike proteins. [Diagram 5] FIG. 5 shows FACS analysis of the binding capacity of m37A12 and p36H3 to 293T cells expressing Omicron BA.1 S protein. [Figure 6] FIG. 6 shows the neutralizing ability of m37A12 against Omicron BA.1 S protein pseudoviruses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] It is understood that the invention is not limited to the particular materials and methods described herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to limit the scope of the invention, which will be limited only by the scope of the appended claims.

[0036] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates to the contrary.

[0037] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity determining region (CDR) that is specific for or interacts with a particular antigen (SARS-CoV-2-spike protein). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM) that contain four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or VL). H The heavy chain constant region is made up of three domains: H1 , C H2 and C H3 Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L1 ) is included. V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-SARS-CoV-2 spike protein antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side comparative analysis of two or more CDRs.

[0038] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0039] As used herein, the terms "specific for" or "specifically binds to" mean that the antibody does not cross-react to any significant extent with other epitopes.

[0040] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0041] As used herein, the term "complementarity determining region" (CDR) refers to the discontinuous antigen-binding regions found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and these definitions include overlapping or subsets of amino acid residues when compared against each other.

[0042] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT, using default gap weighting. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0043] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, however available or known in the art.

[0044] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0045] As used herein, the term "nanobody" refers to antibodies that contain small single variable domains (VHHs of antibodies obtained from camelids and dromedaries. Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Calelus dromaderius) families, including New World members such as the Llama species (Lama paccos, Lama glama and Lama vicugna), have been characterized with respect to size, structural complexity and antigenicity to human subjects. Certain IgG antibodies from this family of mammals found in nature lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure with two heavy chains and two light chains in antibodies from other animals.

[0046] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0047] As used in this disclosure, the term "fully glycosylated" refers to the state of glycosylation on a CoV spike protein (particularly, the SARS-CoV-2 spike protein) in which all N-glycan sites in the CoV spike protein (particularly, the SARS-CoV-2 spike protein) are glycosylated with at least one sugar moiety.

[0048] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is a glycoantibody. As used herein, the term "glycoantibody" refers to a homogeneous population of monoclonal antibodies that have a single, uniform glycoform on the Fc region. Each glycoantibody in the homogeneous population is identical, binds to the same epitope, and contains the same Fc glycan with a well-defined glycan structure and sequence.

[0049] The term "homogeneous" in the context of the glycosylation profile of an Fc region is used interchangeably and is intended to mean a single glycosylation pattern represented by one desired N-glycan species and containing little or no minor precursor N-glycans. In one embodiment, the minor precursor N-glycans are less than about 2%.

[0050] As used herein, the term "glycan" refers to a polysaccharide, oligosaccharide, or monosaccharide. Glycans can be monomers or polymers of sugar residues and can be linear or branched. Glycans can contain natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6'sulfo N-acetylglucosamine, etc.). Glycans are also used herein to refer to the carbohydrate moiety of glycoconjugates such as glycoproteins, glycolipids, glycopeptides, glycoproteomes, peptidoglycans, lipopolysaccharides, or proteoglycans. Glycans usually consist of only O-glycosidic bonds between monosaccharides. For example, cellulose is a glycan (or more specifically, a glucan) composed of β-1,4 linked D-glucose, and chitin is a glycan composed of β-1,4 linked N-acetyl-D-glucosamine. Glycans can be homo- or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can be found attached to proteins, such as in glycoproteins and proteoglycans. Glycans are generally found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotes, but can also be found, although less commonly, in prokaryotes. N-linked glycans are found attached to the R-group nitrogen (N) of asparagine in sequons. Sequons are Asn-X-Ser or Asn-X-Thr sequences, where X is any amino acid except praline.

[0051] As used herein, the term "N-glycan" refers to an N-linked oligosaccharide attached by an N-acetylglucosamine (GlcNAc) bound to the amide nitrogen of an asparagine residue in an Fc-containing polypeptide.

[0052] In one preferred embodiment of the disclosure, the antibody or antigen-binding fragment thereof has glycol engineered N-glycans. The antibody comprises an Fc region or the antigen-binding fragment is fused to an Fc region and the antibody or antigen-binding fragment thereof has N-glycans on the Fc region.

[0053] As used in this disclosure, the term "therapeutic agent" refers to any compound, substance, drug, drug moiety or active ingredient having a therapeutic or pharmacological effect that is suitable for administration to a mammal, e.g., a human.

[0054] As used herein, the term "immunoconjugate" refers to an antigen binding protein, e.g., an antibody or antigen binding fragment, chemically or biologically linked to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antigen binding protein may be linked to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or therapeutic agent at any position along the molecule, so long as it is capable of binding its target (CoV-S). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the invention, the agent may be a second, different antibody that specifically binds to CoV-S. The type of therapeutic moiety that may be conjugated to an anti-CoV-S antigen binding protein (e.g., antibody or fragment) will take into account the condition to be treated and the desired therapeutic effect to be achieved.

[0055] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0056] The term "genetically engineered" or "genetic engineering" of a cell refers to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels in the cell. The genetic material can be in the form of DNA or RNA. Genetic material can be transferred into cells by a variety of means, including viral transduction and non-viral transfection. Once genetically engineered, the expression levels of certain genes in the cell can be altered, either permanently or temporarily.

[0057] The term "coronavirus" or "CoV" refers to any virus of the Coronaviridae family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a newly emerged coronavirus that is spreading rapidly to other parts of the globe. SARS-CoV-2 binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) via the viral spike protein. The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for viral membrane fusion.

[0058] The term "CoV-S," also referred to as "S" or "S protein," refers to the spike protein of a coronavirus and can refer to specific S proteins such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S.

[0059] The term "coronavirus infection" or "CoV infection" as used herein refers to an infection with a coronavirus, such as SARS-CoV-2, MERS-CoV or SARS-CoV. The term includes coronavirus respiratory infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and dysfunction), septic shock and death in severe cases.

[0060] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic agents that is administered to a mammal, such as a human, to prevent, treat, or eliminate a particular disease or pathological condition from which the mammal suffers.

[0061] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease.

[0062] As used herein, the terms "treatment", "treating" and the like encompass any treatment of disease in a mammal, particularly a human, and include (a) preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., halting its progression; and (c) relieving the disease, i.e., causing regression of the disease.

[0063] The terms "prevent" or "prophylaxis" are widely accepted in the art and, when used in reference to a condition, include administration of an agent prior to the onset of the condition to reduce the frequency or severity of symptoms of a medical condition in a subject compared to a subject not receiving the agent, or to delay the onset of symptoms of a medical condition in a subject.

[0064] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including, but not limited to, mice (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc. In particular, the subject is to be vaccinated.

[0065] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of a human, a physician, nurse, medical attendant, or individual; in the case of an animal, including a non-human mammal, a veterinarian) that a subject requires or would benefit from treatment. This judgment is within the area of ​​the caregiver's expertise, but is made based on a variety of factors, including knowledge that the subject is ill or will become ill as a result of a condition treatable by the compounds of the present disclosure.

[0066] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and may be used in a diagnostic or monitoring assay. This definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny.

[0067] "Neutralization" refers to the process by which a molecule (e.g., an antibody) inhibits to any detectable extent the activity of a coronavirus, e.g., inhibiting the ability of the coronavirus to bind to a receptor, to be cleaved by a protease, or to mediate viral entry into or replication in a host cell.

[0068] Coronaviruses (CoVs) infect humans and animals, causing a variety of diseases including respiratory, intestinal, renal and neurological diseases. CoVs use their spike glycoprotein (S), the primary target of neutralizing antibodies, to bind their receptors and mediate membrane fusion and viral entry. Coronavirus spike proteins are highly conserved among all human coronaviruses (CoVs) and are involved in receptor recognition, viral attachment and entry into host cells. Similarly, SARS-CoV-2 S protein is also highly conserved along with the S protein of CoVs. Upon binding of the S protein to the receptor, TM protease serine 2 (TMPRSS2), a type 2 TM serine protease located on the host cell membrane, promotes viral entry into the cell by activating the S protein. Once the virus enters the cell, viral RNA is released, polyproteins are translated from the RNA genome, and replication and transcription of the viral RNA genome occurs via proteolytic cleavage and assembly of the replicase-transcriptase complex. The viral RNA is replicated, structural proteins are synthesized, assembled, and packaged in the host cell, and then viral particles are released (Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 2015; 1282: 1-23).

[0069] The SARS-CoV-2-spike protein is a 1273 amino acid type I membrane glycoprotein that assembles into trimers that constitute spikes or peplomers on the surface of enveloped coronavirus particles. This protein has two essential functions, host receptor binding and membrane fusion, which are carried out by the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via the receptor binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence of SEQ ID NO: 11. Receptor binding by the S1 domain leads to a conformational change in the S2 domain that facilitates fusion between the viral envelope and the plasma membrane of its target cell. Examples of variants of SARS-CoV-2 spike protein include, but are not limited to, D614G:D614G; B.1.1.7:69-70 deletion, 144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; B.1.351:L18F, D80A, D215G, 242-244 deletion, R246I, K417N, E484K, N501Y, D614G and A701V. The term "CoV-S" includes protein variants of CoV spike protein isolated from different CoV isolates, as well as recombinant CoV spike protein or fragments thereof. The term also encompasses CoV spike protein or fragments thereof linked to a signal sequence, e.g., a histidine tag, mouse or human Fc, or ROR1.

[0070] The present disclosure develops antibodies or antigen-binding fragments thereof that are specific for epitopes in the CoV spike protein, particularly the SARS-CoV-2 spike protein.

[0071] In particular, an antibody or antigen-binding fragment thereof specific for an epitope in the spike protein of a CoV, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region CDR and a light chain variable region CDR, wherein the heavy chain variable region CDR comprises CDRH1, CDRH2 and CDRH3 regions, and the light chain variable region CDR comprises CDRL1, CDRL2 and CDRL3 regions; the CDRH1 region comprises the amino acid sequence of SEQ ID NO:1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of SEQ ID NO:2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of SEQ ID NO:3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the CDRL1 region comprises the amino acid sequence of SEQ ID NO:4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRL2 region comprises the amino acid sequence of AAS; and the CDRL3 region comprises the amino acid sequence of SEQ ID NO:5, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0072] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprising SEQ ID NOs: 1-5 is m31A12, which has high affinity and protection efficiency against various SARS-CoV-2 variants. m37A12 is a chimeric mAb that recognizes the RBD domain of the spike protein of SARS-CoV-2. m37A12 recognizes the spike protein of the SARS-CoV-2 Wuhan strain (WH01) and the SARS-CoV-2 Delta (B.1.617.2) variant with EC50 values ​​in the picomolar range in cell-based binding assays. In pseudovirus-based neutralization assays, m37A12 has neutralizing activity against SARS-CoV-2 Wuhan (WH01), D614G, alpha (B1.1.7) and delta (B.1.617.2) pseudoviruses with IC50 values ​​in the picomolar range.

[0073] In particular, an antibody or an antigen-binding fragment thereof specific for an epitope in the spike protein of CoV, the CDRH1 region comprises the amino acid sequence of SEQ ID NO:6, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of SEQ ID NO:7, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of SEQ ID NO:8, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the CDRL1 region comprises the amino acid sequence of SEQ ID NO:9, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRL2 region comprises the amino acid sequence of AAT; and the CDRL3 region comprises the amino acid sequence of SEQ ID NO:10, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0074] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof comprising SEQ ID NO:6-10 is P36H3.

[0075] Antibodies or antigen-binding fragments thereof according to the present disclosure can be full length (e.g., IgG1 or IgG4 antibodies) or can comprise only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments), and can be modified as needed to affect functionality.

[0076] In some embodiments of the disclosure, the antibody or antigen-binding fragment thereof is conjugated to an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment, conjugated to another moiety, e.g., a therapeutic moiety "immunoconjugate," such as a toxoid or an antiviral agent for treating coronavirus infection. In one embodiment of the invention, the anti-CoV-S antibody or fragment is conjugated to any of the additional therapeutic agents described herein. The disclosure also provides a conjugate comprising an antibody or antigen-binding fragment thereof disclosed herein bound to the spike protein or fragment thereof of SARS-CoV-2.

[0077] To determine whether an antibody "specifically binds to one or more amino acids" in a polypeptide or protein, various techniques known to those skilled in the art can be used. Exemplary techniques include routine cross-blocking assays, such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY), alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide to which an antibody specifically binds is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange to occur at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal.Chem.73:256A-265A.

[0078] By using routine methods known in the art, it can be easily determined whether an antibody specifically binds to the same epitope as a reference anti-SARS-CoV-2-spike protein antibody or competes for binding with a reference anti-SARS-CoV-2-spike protein antibody. For example, to determine whether a test antibody binds to the same epitope as a reference anti-SARS-CoV-2-spike protein antibody of the present disclosure, the reference antibody is bound to the SARS-CoV-2-spike protein. The ability of the test antibody to bind to the SARS-CoV-2-spike protein molecule is then evaluated. If the test antibody is able to bind to the SARS-CoV-2-spike protein after saturation binding by the reference anti-SARS-CoV-2-spike protein antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-SARS-CoV-2-spike protein antibody. On the other hand, if the test antibody is unable to bind to the SARS-CoV-2-spike protein molecule after saturation binding by the reference anti-SARS-CoV-2-spike protein antibody, then the test antibody may bind to the same epitope as that bound by the reference anti-SARS-CoV-2-spike protein antibody of the present disclosure. Further routine experiments (e.g., peptide mutations and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments of the present disclosure, two antibodies bind to the same (or overlapping) epitope if a 1-fold, 5-fold, 10-fold, 20-fold or 100-fold excess of one antibody inhibits binding of the other antibody by at least 50%, but preferably 75%, 90% or even 99%, as measured, for example, in a competitive binding assay. Alternatively, two antibodies are considered to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.Two antibodies are considered to have "overlapping epitopes" if only some of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0079] Antibodies also include antigen-binding fragments of intact antibody molecules. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. For example, the DNA can be sequenced and manipulated, either chemically or by using molecular biology techniques, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0080] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions (e.g., isolated complementarity determining regions (CDRs) such as CDR3 peptides) of antibodies or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0081] Antigen-binding fragments of antibodies typically contain at least one variable domain. A variable domain may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated with V H For antigen-binding fragments containing domains, V H Domain and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer, with the V H -V H , V H -V L Or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V H Or V L It may contain domains.

[0082] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) a V H -C H1 (ii) V H -C H2 (iii) V H -C H3 ;(iv)V H -C H1 -C H2 ;(v)V H -C H1 -C H2 -C H3 , (vi) V H -C H2 -C H3 (vii) V H -C L ;(viii)V L -C H1 ;(ix)V L -C H2 ;(x)V L -C H3 ;(xi)V L -CH1 -C H2 ;(xii)V L -C H1 -C H2 -C H3 ;(xiii)V L -C H2 -C H3 and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed herein, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. A hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable and / or constant domains within a single polypeptide molecule. Additionally, antigen-binding fragments of antibodies of the present disclosure may be linked to each other (e.g., by disulfide bonds) and / or to one or more monomeric V H Or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains.

[0083] The anti-SARS-CoV-2-spike protein antibodies disclosed herein may contain one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in the germline sequence of another mammal, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily produce a large number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, the V H and / or V LAll of the framework and / or CDR residues within the domain are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example only mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, the antibodies of the present disclosure may contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are either maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, e.g., improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present disclosure.

[0084] The present disclosure provides a method for the preparation of a medicament having one or more conservative substitutions, comprising administering to a subject a subject the medicament having one or more conservative substitutions, as disclosed herein. H , V L The present disclosure also includes anti-SARS-CoV-2 spike protein antibodies that include any variant of the V and / or CDR amino acid sequences disclosed herein. H , V L and / or V that has, e.g., 10 or less, 8 or less, 6 or less, 4 or less conservative amino acid substitutions compared to any of the CDR amino acid sequences. H , V L and / or an anti-SARS-CoV-2 spike protein antibody having a CDR amino acid sequence.

[0085] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof according to the present disclosure is a humanized antibody. To improve the binding affinity of the humanized antibody according to the present disclosure, some amino acid residues in the human framework regions are replaced by the corresponding amino acid residues in the species of the CDR, e.g., rodent.

[0086] The antibodies or antigen-binding fragments thereof of the present disclosure may be monospecific, bispecific or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. The anti-SARS-CoV-2-spike protein antibodies of the present disclosure may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or a fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity. For example, the present disclosure includes bispecific antibodies in which one arm of the immunoglobulin is specific for SARS-CoV-2-spike protein or a fragment thereof and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.

[0087] In another aspect, the disclosure provides an engineered cell that expresses the antibody or antigen-binding fragment thereof or contains the vector. The engineered cell can be an immune cell.

[0088] In one preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof may be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some aspects, the expression system is a mammalian cell expression system, such as a hybridoma, or a CHO cell expression system. Many such systems are widely available from commercial suppliers. H and V L In embodiments that include both regions, V H and VL The regions can be expressed using a single vector, for example in a bicistronic expression unit, or under the control of different promoters. H and V L The regions can be expressed using separate vectors. H Or V L The region may optionally include a methionine at the N-terminus.

[0089] Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells, for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3rd ed. 1997)).

[0090] An example of a method for producing an antibody or antigen-binding fragment includes: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favoring expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown.

[0091] A vector can be used to introduce a polynucleotide encoding an antibody or antigen-binding fragment of the present invention into a host cell. In one embodiment, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the invention is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0092] The present disclosure provides pharmaceutical compositions comprising the antibody or antigen-binding fragment or complex thereof. The pharmaceutical compositions of the present disclosure are formulated with suitable diluents, carriers, excipients and other factors that provide improved entry, delivery, tolerability, etc. The compositions may be formulated for a particular use, such as veterinary use or pharmaceutical use in humans. The form of the composition and the excipients, diluents and / or carriers used will depend on the intended use of the antibody and, in the case of therapeutic use, the mode of administration. A large number of suitable formulations can be found in a formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.

[0093] In some embodiments of the present disclosure, the pharmaceutical composition comprises an additional therapeutic agent, such as an antiviral agent. The antiviral agent may be an antibody against the S protein of SARS-CoV-2; an anti-inflammatory agent; an antibody against the NTD region of the S protein of SARS-CoV-2; an antibody against the HR1 region of the S protein of SARS-CoV-2; an antibody against the RBD region of the S protein of SARS-CoV-2; a SARS-CoV monoclonal antibody; a MERS-CoV monoclonal antibody; a SARS-CoV-2 monoclonal antibody; a peptide; a protease inhibitor; a PIKfyve inhibitor; a TMPRSS2 inhibitor; and a cathepsin inhibitor; a furin inhibitor; an antiviral peptide; an antiviral protein; or an antiviral chemical compound.For example, antivirals include 1A9;201;311mab-31B5;311mab-32D4;47D11;4A8;4C2;80R;apilimod;B38;camostat mesylate;casirivimab;CR3014;CR3022;D12;E-64D;EK1;EK1C4;H4;HR2P;IBP02;imdevimab;m336;MERS-27;MERS-4;MI-701;n3088;n3130;P2B-2F6;P2C-1F11;PI8;S230;S309;SARS-CoV-2 S HR2P fragment (aal 168~1203);tetrandrine;Viracept (nelfinavir mesylate);YM201636;a-1-PDX;Favipiravir;IFN-a;IFN-alb;IFN-a2a;Lopinavir-ritonavir;Q-Griffithin (Q-GRFT);and Griffithin;Oseltamivir;Zanamivir;Abacavir;Zidovudine;Zalcitabine;Didanosine;Stavudine;Efavirenz;Indinavir;Ritonavir;Nelfinavir;Amprenavir;Ribavirin;Remdesivir;Chloroquine;Hydroxychloroquine;rIFN-α-2a; The alpha-interferon species may be at least one selected from the group consisting of rIFN-β-lb; rIFN-γ; nIFN-α; nIFN-β; nIFN-γ; IL-2; PD-L1; anti-PD-L1; checkpoint inhibitors; interferon; interferon mixture; recombinant or natural interferon; alferon; alpha-interferon species; recombinant or natural interferon α; recombinant or natural interferon α2a; recombinant or natural interferon β; recombinant or natural interferon βlb; and recombinant or natural interferon γ. The alpha-interferon species may be a mixture of at least seven species of alpha-interferon produced by human leukocytes, the seven species being interferon α2; interferon α4; interferon α7; interferon α8; interferon α10; interferon α16; and interferon α17.

[0094] The dose of the antibody administered to the patient / animal may vary depending on the age and size of the patient / animal, the target disease, symptoms, route of administration, etc. The preferred dose is typically calculated according to body weight or body surface area. When the antibody of the present disclosure is used to treat symptoms or diseases associated with SARS-CoV-2 in adult patients / animals, it may be advantageous to administer the antibody of the present disclosure intravenously. Depending on the severity of the symptoms, the frequency and duration of treatment can be adjusted. Effective dosages and schedules for administering the antibody can be determined empirically, e.g., by monitoring the progress of the patient / animal by periodic evaluation and adjusting the dosage accordingly. Furthermore, interspecies scaling of dosage can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0095] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, such as, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, etc. (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active factors. Administration can be systemic or local.

[0096] The pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously using a container or injection device such as a standard needle and syringe. Furthermore, for subcutaneous delivery, a pen delivery device has easy application in delivering the pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is supplied pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is depleted of the pharmaceutical composition, the entire device is discarded.

[0097] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres. Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed close to the target of the composition, thereby requiring only a small portion of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0098] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying, for example, the antibody or salt thereof described herein in a sterile aqueous or oily medium that is usually used for injections. As the aqueous medium for injection, there are isotonic solutions containing glucose and other auxiliary agents, which may be used in combination with suitable solubilizing agents such as physiological saline, alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As the oily medium, for example, sesame oil, soybean oil, and the like, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol, are used. The injectable preparation thus prepared is preferably filled into a suitable ampoule.

[0099] Advantageously, the pharmaceutical compositions for oral or parenteral use are prepared in a dosage form of a unit dose adapted to the dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0100] The present disclosure provides a method for treating or preventing an infection by a coronavirus in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment or conjugate thereof disclosed herein. Alternatively, the present disclosure provides a pharmaceutical composition for use in treating or preventing an infection by a coronavirus in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding fragment or conjugate thereof used herein, a pharma- ceutical carrier, and optionally an additional therapeutic agent.

[0101] The present disclosure provides a method for neutralizing coronavirus in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment or conjugate thereof disclosed herein. Alternatively, the present disclosure provides a pharmaceutical composition for use in neutralizing coronavirus in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding fragment or conjugate thereof used herein, a pharma- ceutical acceptable carrier, and optionally an additional therapeutic agent.

[0102] The present disclosure provides a kit for detecting coronavirus in a sample, the kit comprising an antibody or antigen-binding fragment thereof.

[0103] The anti-SARS-CoV-2 spike protein antibodies of the present disclosure may also be used to detect and / or measure coronavirus or SARS-CoV-2 spike protein expressing cells in a sample, e.g., for diagnostic purposes. For example, the anti-SARS-CoV-2 spike protein antibodies or fragments thereof may be used to diagnose a condition or disease characterized by a coronavirus infection. An exemplary diagnostic assay for coronavirus may include, for example, contacting a sample obtained from a patient with an anti-SARS-CoV-2 spike protein antibody of the present disclosure, the anti-SARS-CoV-2 spike protein antibody being labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-SARS-CoV-2 spike protein antibody may be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S or 125The antibody may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0104] The following examples are provided to aid those skilled in the art in practicing the present disclosure. EXAMPLES

[0105] Materials and Methods FACS analysis and sorting of spike-specific B cells. mg Splenocytes isolated from immunized mice were incubated with 2 μg / ml S protein for 1 hour at 4° C., then washed and incubated with an antibody cocktail against CD19 (clone: ​​6D5, PE-Cy7 conjugate, BIOLEGEND™), CD3 (clone: ​​17A2, PE conjugate, BIOLEGEND™) and His (clone: ​​J095G46, APC conjugate, BIOLEGEND™) for 15 minutes at 4° C. Propidium iodide (BIOLEGEND™) was used to exclude dead cells. Live single spike-specific B cells (CD3 - CD19 + For repertoire analysis, S mg Or S fg Five spleens from immunized mice were pooled and stained before sorting.

[0106] Single B cell screening. Primers were designed based on previous publications. Reactions were then performed at 50°C for 30 min, 95°C for 15 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, and a final incubation at 72°C for 10 min. A semi-nested second round PCR was performed using KOD One PCR Master Mix (TOYOBO™) with 1 μl of unpurified first round PCR product at 98°C for 2 min, followed by 45 cycles of 98°C for 10 s, 55°C for 10 s, 68°C for 10 s, and a final incubation at 68°C for 1 min. The PCR products were then analyzed on a 1.5% agarose gel and sequenced. IgV and L genes were identified by searching the IMGT website (http: / / imgt.org / IMGT_vquest / input). Genes were then amplified from the second round PCR products using single gene-specific V and L gene primers containing restriction sites for cloning into vectors containing human IgH or IgL expression backbones. Chimeric IgH and IgL expression constructs were co-transfected into Expi293 for antibody production.

[0107] Binding of antibodies to 293T surface expressing S protein. 293T cells were transfected with pcDNA6 / spike-P2A-eGFP. Transfected cells were selected under 10 μg / ml blasticidin for 2-3 weeks. Selected cells were then sorted by FACSAria II to identify eGFP. + The expressing cells were obtained. These cells were maintained in DMEM containing 10% FBS and 10 μg / ml blasticidin. 2-3 × 10 5The cells were incubated in FACS buffer on ice for 1 hour. The cells were then washed three times with FACS buffer before being stained with BV421 mouse anti-human IgG (BD BIOSCIENCES™, 562581, 1:100) for 20 minutes on ice and washed twice with FACS buffer. The percentage of positive cells was quantified using a FACS Canto II and the data was analyzed with FlowJo. The S protein variants used here are: WH01 spike: original S protein; D614G: D614G; B.1.1.7: 69-70 deletion, 144 deletion, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H; B.1.351: L18F, D80A, D215G, 242-244 deletion, R246I, K417N, E484K, N501Y, D614G, A701V and Omicron BA.1.

[0108] Affinity and avidity determination using Octet (Biolayer Interferometry). Fab fragments were prepared by using Pierce Fab Micro Preparation Kit (THERMOFISHER SCIENTIFIC™) according to the manufacturer's instructions. Briefly, m31A7 IgG (250 μg) was digested by incubation with immobilized papain resin at 37° C. for 8 hours. Fab was then purified by Protein A column. Purified m31A7 IgG or Fab fragments were loaded onto Protein G or FAB2G biosensors (Molecular Devices, FORTEBIO™) at 10 or 6.7 μg / ml of kinetic buffer (0.01% endotoxin-free BSA, 0.002% Tween-20, 0.005% NaN3 in PBS), respectively. Association and dissociation of S protein (SARS-CoV-2 WH01) by both IgG or Fab was performed in kinetics buffer at the indicated concentrations for 5 and 15 min, respectively. A 1:1 global fit model (OCTET™) was used to calculate the K D values ​​were calculated.

[0109] Preparation of mono-GlcNAc-IgG. 1 mg of IgG (e.g., m37A12 or P36H3) produced in 293S cells was incubated with Endo H (5000 units) in sodium phosphate buffer (20 mM, pH 7.4, 0.5 mL) for 16 hours at room temperature. Complete cleavage of the N-glycans on the heavy chain was confirmed by SDS-PAGE. Mono-GlcNAc-IgG was purified on a Protein A affinity column. Fractions containing mono-GlcNAc-IgG were combined and concentrated by centrifugal filtration (Amicon Ultra centrifugal filter, MILLIPORE™).

[0110] Transglycosylation of mono-GlcNAc IgG with glycan oxazoline. A mixture of 100 pg EndoS D233Q and 1 mg mono-GlcNAc IgG in 20 mM Tris buffer (pH 7.4) was added to 1 mg FSCT-oxazoline as previously described (Lo, HJ et al. J Am Chem Soc 141, 6484-6488). The solution was incubated at 37° C. for 60 min. The reaction mixture was then purified on a Protein A affinity column.

[0111] result: In this example, the inventors demonstrated that the monoglycosylated form (S mg A spike-specific monoclonal antibody was isolated from mice immunized with S mg Sorting of S protein-specific B cells from immunized mice led to the identification of two monoclonal antibodies (mAbs), m37A12 and P36H3, from IGHV1-18 amplified clones. The m37A12 mAb interacts with the wild-type (WT) full-length S protein, S1, and RBD (Figure 1A). The EC 50 (M) is 5.084 x 10 -11 and the EC of RBD 50 (M) is 8.000 x 10 -11 Our biolayer interferometry (BLI) analysis revealed that the KD of m31A7 for the full-length WT spike protein is 4.2 × 10 -10We showed that P36H3 appears to be a marker for inflammatory bowel disease (Fig. 1B). P36H3 binds to the S2 domain of the WT spike protein (Fig. 2A). The EC 50 (M) is 1.622 x 10 -10 and EC of S2 50 (M) is 2.316 x 10 -11 BLI analysis revealed that the KD of P36H3 relative to the full-length WT spike protein was 7.02 × 10 -12 M (Figure 2B). Furthermore, FACS analysis showed that m37A12 was -11 The results show that the antibody binds to both WH01 (WT) and HEK293T cells expressing the delta spike protein with an EC50 of 100 M (Figure 3A). 50 (M) is 2.469 x 10 -10 Delta's EC 50 (M) is 2.042 x 10 -11 Importantly, m37A12 inhibited various pseudovirus variants, (WT, D614G, and delta), with IC at subpicomolar concentrations. 50 It was shown that WH01 was neutralized by IC 50 (M) is 8.788 x 10 -11 Alpha's IC 50 (M) is 6.764 x 10 -11 ;Beta IC 50 (M) is 2.557 x 10 -9 and the gamma IC 50 (M) is 2.424 x 10 -9 Delta's IC 50 (M) is 1.504 x 10 -10 On the other hand, FACS analysis showed that P36H3 was -10 The EC50 of WH01 was 1.0 M or higher, indicating that it bound to both WH01 (WT) and HEK293T cells expressing the delta spike protein (Figure 4). 50 (M) is 2.227 x 10 -10 Delta's EC 50 (M) is 3.737 x 10 -10 FACS analysis showed that m37A12 and p36H3 were 7.734 × 10 -9M(m37A12) or 5.213×10 -9 EC of M(p36H3) 50 We show that m37A12 binds to HEK293T cells expressing the OmicronBA.1 spike protein at 5.993 × 10 -8 IC 50 and was shown to neutralize the pseudovirus Omicron BA.1 mutant (Fig. 6 ).

[0112] While the present disclosure has been described in conjunction with specific embodiments set forth, many alternatives, modifications and variations thereon will be apparent to those skilled in the art, and all such alternatives, modifications and variations are deemed to fall within the scope of the present disclosure.

Claims

1. An antibody or antigen-binding fragment thereof specific for an epitope in a spike protein of a CoV, the antibody or antigen-binding fragment thereof comprising a complementarity determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region; The CDRs of the heavy chain variable region comprise a CDRH1 having an amino acid sequence of SEQ ID NO: 1 or 6 or a substantially similar sequence thereof, a CDRH2 having an amino acid sequence of SEQ ID NO: 2 or 7 or a substantially similar sequence thereof, and a CDRH3 having an amino acid sequence of SEQ ID NO: 3 or 8 or a substantially similar sequence thereof; and The CDRs of the light chain variable region include a CDRL1 having an amino acid sequence of SEQ ID NO: 4 or 9 or a substantially similar sequence thereof, a CDRL2 having an amino acid sequence of AAS or AAT or a substantially similar sequence thereof, and a CDRL3 having an amino acid sequence of SEQ ID NO: 5 or 10 or a substantially similar sequence thereof; An antibody or an antigen-binding fragment thereof.

2. the amino acid sequence of CDRH1 is SEQ ID NO:1 or a sequence substantially similar thereto; the amino acid sequence of CDRH2 is SEQ ID NO:2 or a sequence substantially similar thereto; the amino acid sequence of CDRH3 is SEQ ID NO:3 or a sequence substantially similar thereto; and the amino acid sequence of CDRL1 is SEQ ID NO:4 or a sequence substantially similar thereto; the amino acid sequence of CDRL2 is AAS or a sequence substantially similar thereto; the amino acid sequence of CDRL3 is SEQ ID NO:5 or a sequence substantially similar thereto; or the amino acid sequence of CDRH1 is SEQ ID NO:6 or a sequence substantially similar thereto; the amino acid sequence of CDRH2 is SEQ ID NO:7 or a sequence substantially similar thereto; the amino acid sequence of CDRH3 is SEQ ID NO:8 or a sequence substantially similar thereto; and The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of CDRL1 is SEQ ID NO: 9 or a substantially similar sequence thereof; the amino acid sequence of CDRL2 is AAT or a substantially similar sequence thereof; and the amino acid sequence of CDRL3 is SEQ ID NO: 10 or a substantially similar sequence thereof.

3. The antibody or antigen-binding fragment thereof may be a Fab fragment, F(ab') ２ The antibody or antigen-binding fragment thereof of claim 1, which is a fragment, an ScFv fragment, a monoclonal antibody, a chimeric antibody, a nanobody, a humanized antibody or a human antibody.

4. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is multispecific.

5. A conjugate comprising the antibody or antigen-binding fragment thereof of claim 1 bound to the spike protein or fragment thereof of SARS-CoV-2.

6. A vector encoding the antibody or antigen-binding fragment thereof of claim 1.

7. 7. A genetically engineered cell expressing the antibody or antigen-binding fragment thereof of claim 1 or containing the vector of claim 6.

8. 5. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favoring expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown.

9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the conjugate according to claim 5, a pharma- ceutical acceptable carrier, and optionally a further therapeutic agent.

10. 10. The pharmaceutical composition of claim 9, wherein the therapeutic agent is an antiviral agent, an anti-inflammatory agent, or an antibody or antigen-binding fragment thereof specific for the spike protein of SARS-CoV-2.

11. A pharmaceutical composition for use in treating or preventing infection by coronavirus and / or neutralizing coronavirus in a subject in need thereof, comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the conjugate according to claim 5, a pharma- ceutical acceptable carrier, and optionally a further therapeutic agent.

12. 12. The pharmaceutical composition of claim 11, wherein the additional therapeutic agent is an antiviral agent, an anti-inflammatory agent, or an antibody or antigen-binding fragment thereof specific for the spike protein of SARS-CoV-2.

13. The pharmaceutical composition of claim 11 , wherein the subject is vaccinated.

14. The pharmaceutical composition according to claim 11, wherein the pharmaceutical composition is in a form suitable for injection.

15. 5. A method for detecting coronavirus in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

16. A kit for detecting coronavirus in a sample, comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4.

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