Specific antibodies spiking proteins of SARS-cov-2 and uses thereof

Abstract

To provide novel neutralizing therapeutic anti-coronavirus spike protein antibodies and their use for treating or preventing viral infections.SOLUTION: Provided is an antibody or an antigen-binding fragment thereof that specifically binds to a spike protein of SARS-CoV-2. Also provided are pharmaceutical compositions and methods for the treatment and / or prevention of a disease and / or disorder caused by a coronavirus in a subject in need thereof, as well as methods for detecting a coronavirus in a sample.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Priority information This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 251,472, filed October 1, 2021, the disclosure of which is incorporated herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted electronically in .xml format and is incorporated herein by reference in its entirety. The .xml copy, created on September 30, 2022, is named "G4590-14200PCT_SeqListing_20220930.xml" and is 10 kilobytes in size.

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

[0004] The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading worldwide. Infection causes symptoms of direct cytopathic effects and an exaggerated inflammatory response in infected subjects. The lack of effective treatments leads to 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 anti-coronavirus spike protein (such as anti-SARS-CoV-2 spike protein) antibodies and their use to treat or prevent viral infection.

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

[0008] The present disclosure also provides epitopes comprising portions located at amino acid residues 419 to 433 or amino acid residues 471 to 482 of SEQ ID NO: 8. In some embodiments, the epitope comprises portions located at amino acid residues 419 to 433 and amino acid residues 471 to 482 of SEQ ID NO: 8. Thus, the present disclosure provides antibodies that bind to the epitopes disclosed herein.

[0009] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to an epitope in a spike protein of a CoV, wherein the antibody or antigen-binding fragment thereof comprises a complementarity-determining region (CDR) of a heavy chain variable region and a complementarity-determining region of a light chain variable region, wherein the complementarity-determining region of the heavy chain variable region comprises CDRH1, CDRH2, and CDRH3 regions, and the complementarity-determining region of the light chain variable region comprises CDRL1, CDRL2, and CDRL3 regions; the CDRH1 region comprises the amino acid sequence of GYTFTEYT (SEQ ID NO: 1) or a sequence substantially similar thereto, the CDRH2 region comprises the amino acid sequence of INPNIGDT (SEQ ID NO: 2) or a sequence substantially similar thereto, and the CDRH3 region comprises the amino acid sequence of AREVYNYSFAY (SEQ ID NO: 3) or a sequence substantially similar thereto; The CDRL1 region comprises the amino acid sequence of QSLLYSSNQKNY (SEQ ID NO: 4) or a sequence substantially similar thereto, the CDRL2 region comprises the amino acid sequence of WAS or a sequence substantially similar thereto, and the CDRL3 region comprises the amino acid sequence of QQYYRYPLT (SEQ ID NO: 5) or a sequence substantially similar thereto.

[0010] In some embodiments of the present disclosure, the spike protein is fully glycosylated, hi one embodiment, the spike protein is monoglycosylated.

[0011] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is

[0012] [ka] or a sequence substantially similar thereto,

[0013] [ka] or a sequence substantially similar thereto.

[0014] In some embodiments of the present disclosure, the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0015] In some embodiments of the present disclosure, the antibodies are multispecific.

[0016] 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.

[0017] The present disclosure provides vectors encoding the antibodies or antigen-binding fragments thereof disclosed herein.

[0018] The present disclosure provides genetically engineered cells that express the antibodies or antigen-binding fragments thereof disclosed herein or contain the vectors disclosed herein.

[0019] The present disclosure also provides methods for producing the antibodies or antigen-binding fragments thereof disclosed herein, comprising: (a) introducing one or more polynucleotides encoding the antibodies or antigen-binding fragments into a host cell; (b) culturing the host cell under conditions favorable for expression of the one or more polynucleotides; and (c) optionally isolating the antibodies or antigen-binding fragments from the host cell and / or the medium in which the host cell is growing.

[0020] The present disclosure provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutically acceptable carrier, and optionally an additional therapeutic agent.

[0021] Therapeutic agents include, but are not limited to, antiviral agents. In some embodiments of the present disclosure, the therapeutic agent is an anti-inflammatory agent or an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of a CoV.

[0022] In some embodiments of the present disclosure, the subject is vaccinated.

[0023] The present disclosure provides a container or injection device comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0024] The present disclosure provides use of an antibody or antigen-binding fragment thereof described herein in the manufacture of a medicament for treating or preventing infection with a coronavirus in a subject in need thereof. Additionally, the present disclosure provides a method for treating or preventing infection with a coronavirus in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein.

[0025] In some embodiments of the present disclosure, the CoV described herein is alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV 2. In some embodiments, the CoV described herein includes, but is not limited to, SARS-CoV, MERS-CoV, or SARS-CoV-2.

[0026] In some embodiments of the present disclosure, the pharmaceutical agent is administered in conjunction with one or more additional therapeutic agents, or the subject is administered one or more additional therapeutic agents.

[0027] The present disclosure provides a method for neutralizing coronavirus in a subject in need thereof, the method comprising administering to the subject an antibody or antigen-binding fragment thereof as disclosed herein.

[0028] The present disclosure provides a method of administering an antibody or antigen-binding fragment thereof disclosed herein to a subject, the method comprising injecting the antibody or antigen-binding fragment into the subject.

[0029] In some embodiments of the present disclosure, the pharmaceutical agent is injected subcutaneously, intravenously, or intramuscularly into the body of the subject, or the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the body of the subject.

[0030] 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.

[0031] The present disclosure provides a kit for detecting coronavirus in a sample, the kit comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0032] 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 explanation of the drawings]

[0033] [Figure 1A] FIG. 1 shows the identification of m31A7 mAb from Smg-immunized mice. [Figure 1B] FIG. 1 shows a comparison of heavy chains in spike-specific B cell repertoires induced by Smg and Sfg. [Figure 1C] Figure 1A shows a comparison of heavy chains in spike-specific B cell repertoires induced by Smg and Sfg. Figure 1A shows an overview of the single B cell screening platform. Single spike-specific B cells (CD3-CD19+spike+) from the spleens of immunized mice were sorted into 96-well plates by FACS. IgH and IgL gene transcripts from each single B cell were amplified by RT-PCR. After sequencing, cDNA derived from the variable regions of the IgH or IgL genes was subcloned into expression vectors containing human Ig heavy or light chain constant regions, respectively. Chimeric monoclonal antibodies were produced by Expi 293, and their binding to spike proteins was measured using spike-expressing 293T cells and FACS. Figure 1B shows the heavy chain distribution of the B cell repertoire. Less than 5% usage is indicated in white. Figure 1C shows that analysis of the heavy chain IgG repertoire of Sfg- or Smg-immunized mice showed IGHV1-9, IGHV1-18, and IGHV2-3 to be highly represented in the Smg group (p-values: 0.048, 1.6 × 10-11, and 0.005, respectively, using a chi-square test). [Figure 2A] Figure 1 shows the binding of m31A7 to SARS-CoV-2 spike protein. [Figure 2B] Figure 2 shows the binding of m31A7 to the SARS-CoV-2 spike protein. Figure 2A shows ELISA binding of m31A7 to the S1, S2, RBD, or S proteins. Figure 2B shows FACS analysis of m31A7 binding to HEK293T cells expressing SARS-CoV-2 WT S protein and mutants (including D614G, B.1.1.7 [alpha], B.1.351 [beta], and B.1.617.2 [delta]). [Figure 3A]FIG. 1 shows that m31A7 can neutralize various mutants of SARS-CoV-2 pseudoviruses. [Figure 3B] These figures demonstrate that m31A7 can neutralize various mutants of SARS-CoV-2 pseudoviruses. Figure 3A shows the neutralization of SARS-CoV-2 wild-type and mutant pseudoviruses by m31A7. Figure 3B shows the trace neutralization of SARS-CoV-2 wild-type and mutant pseudoviruses by m31A7 compared to the previously reported mAb EY6A (gray). Mutants (including D614G, B.1.1.7 [alpha], B.1.351 [beta], and B.1.617.2 [delta]) are indicated at the top of each panel. [Figure 4A] Figure 1 shows that m31A7 exhibits protective activity against SARS-CoV-2 antigen challenge in vivo. [Figure 4B] Figure 1 shows that m31A7 exhibits protective activity against SARS-CoV-2 antigen challenge in vivo. [Figure 4C] Figure 4 shows that m31A7 exhibits protective activity against SARS-CoV-2 challenge in vivo. Figure 4A shows the antibody injection and challenge schedule using K18hACE2 transgenic mice. Figure 4B shows the weight change after SARS-CoV-2 infection in PBS- or m31A7-treated mice. Figure 4C shows the body temperature change after SARS-CoV-2 infection in PBS- or m31A7-treated mice. *P<0.05. [Figure 5A] FIG. 1 shows the biophysical properties of m31A7. [Figure 5B] FIG. 1 shows the biophysical properties of m31A7. [Figure 5C-1] FIG. 1 shows the biophysical properties of m31A7. [Figure 5C-2] FIG. 1 shows the biophysical properties of m31A7. [Figure 5D]Biophysical properties of m31A7. Figure 5A shows the dissociation constants of m31A7 IgG and Fab to the S protein. Figure 5B shows the dissociation constants of m31A7 IgG and Fab to the BA.1 S protein. Figure 5C shows epitope mapping of the RBD by HDX-MS, showing potential m31A7-binding peptides measured at 15 seconds. Figure 5D shows a volcano plot of the change in deuterium uptake in the RBD upon addition of m31A7 IgG. Hits (ΔHDX > 5%, q value < 0.01) are shown in the upper right corner and highlighted in red. Unlabeled hits are redundant peptides with residues 471-482. *P < 0.05; **P < 0.01; ***P < 0.001. DETAILED DESCRIPTION OF THE INVENTION

[0034] It is understood that the present invention is not limited to the particular materials and methods described herein. It is also understood that the terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0035] Please note 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 otherwise.

[0036] The term "antibody" as used herein refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (SARS-CoV-2-spike protein). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (HCVR or VL) that encodes a heavy chain variable region (VHVR) and a VL. H The heavy chain constant region comprises three domains: C H1 , CH2 and C H3 Each heavy 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 ) included. V H and V L The V region 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 an anti-SARS-CoV-2 spike protein antibody (or antigen-binding portion thereof) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0037] 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 derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex.

[0038] As used herein, the term "specifically binds" means that the antibody does not cross-react to any significant extent with other epitopes.

[0039] As used herein, the term "epitope" refers to a site on an antigen to which an antibody binds.

[0040] As used herein, the term "complementarity-determining region" (CDR) refers to the discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., Mol. Biol. 262:732-745 (1996), and the definition includes overlapping or subsets of amino acid residues when compared with each other.

[0041] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward 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 with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, isoleucine; (2) aliphatic-hydroxyl side chains: serine, threonine; (3) amide-containing side chains: asparagine, glutamine; (4) aromatic side chains: phenylalanine, tyrosine, tryptophan; (5) basic side chains: lysine, arginine, histidine; (6) acidic side chains: aspartate and glutamate; 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, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution 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" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0042] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody may be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any reference available or known in the art.

[0043] 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.

[0044] "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.

[0045] As used in this disclosure, the term "fully glycosylated" refers to the state of glycosylation on a CoV spike protein (particularly a SARS-CoV-2 spike protein) in which all N-glycan sites within the CoV spike protein (particularly a SARS-CoV-2 spike protein) are glycosylated with at least one sugar moiety.

[0046] 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 suitable for administration to a mammal, e.g., a human.

[0047] 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, targeting or reporter moiety, enzyme, peptide, or protein, or therapeutic agent. The antigen-binding protein can be linked to the radioactive agent, cytokine, interferon, targeting or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule, so long as it is capable of binding to its target (CoV-S). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the present invention, the drug can be a second, different antibody that specifically binds to CoV-S. The type of therapeutic moiety that can be conjugated to an anti-CoV-S antigen-binding protein (e.g., antibody or fragment) will take into account the condition being treated and the desired therapeutic effect to be achieved.

[0048] 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.

[0049] The terms "genetically engineered" or "genetic engineering" of a cell refer 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 to cells by various means, including viral transduction and non-viral transfection. After being genetically engineered, the expression levels of specific genes in the cell can be altered permanently or temporarily.

[0050] The terms "coronavirus" or "CoV" refer to any virus in the Coronaviridae family, including, but not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to an emerging coronavirus that is rapidly spreading to other parts of the world. It 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.

[0051] 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.

[0052] As used herein, the term "coronavirus infection" or "CoV infection" refers to an infection caused by a coronavirus, such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory infections that predominate 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 in severe cases, death.

[0053] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent 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.

[0054] As used herein, the terms "therapeutically effective amount" or "effective amount" refer 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.

[0055] 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, but has not yet been diagnosed as having, the disease; (b) inhibiting the disease, i.e., halting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0056] The terms "preventing" or "prevention" are art-recognized and when used in reference to a condition, it includes administering an agent to reduce the frequency or severity of a medical condition in a subject prior to the onset of the condition, or to delay the onset of the condition, compared to a subject who is not administered the agent.

[0057] As used interchangeably herein, the terms "individual," "subject," "host," and "patient" refer to mammals, including but not limited to murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), etc. In particular, the subject is to be vaccinated.

[0058] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, nurse practitioner, or individual in the case of a human; a veterinarian in the case of an animal, including a non-human mammal) that a subject needs or would benefit from treatment. This judgment is within 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 a compound of the present disclosure.

[0059] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and can be used in a diagnostic or monitoring assay. The 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.

[0060] "Neutralization" refers to the process by which a molecule (e.g., an antibody) inhibits to any detectable extent the activity of a coronavirus, for example, 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.

[0061] Coronaviruses (CoVs) infect humans and animals, causing a variety of diseases, including respiratory, intestinal, renal, and neurological disorders. CoVs use their spike glycoprotein (S), the primary target of neutralizing antibodies, to bind to their receptors and mediate membrane fusion and viral entry. The coronavirus spike protein is highly conserved among all human coronaviruses (CoVs) and is involved in receptor recognition, viral attachment, and entry into host cells. Similarly, the SARS-CoV-2 S protein is also highly conserved with that of CoVs. Upon receptor binding, TM protease serine 2 (TMPRSS2), a type 2 TM serine protease located on the host cell membrane, activates the S protein, thereby promoting viral entry into the cell. 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 occur via proteolytic cleavage and assembly of the replicase-transcriptase complex. After viral RNA is replicated, structural proteins are synthesized, assembled, and packaged in the host cell, viral particles are released (Fehr AR, Perlman S, "Coronaviruses: an overview of their replication and pathogenesis," Methods Mol Biol. 2015;1282:1-23).

[0062] 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 attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via a 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 following amino acid sequence:

[0063] [ka]

[0064] Examples of SARS-CoV-2 spike protein variants 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 proteins isolated from different CoV isolates, as well as recombinant CoV spike proteins or fragments thereof. The term also encompasses CoV spike proteins or fragments thereof coupled to a signal sequence, such as a histidine tag, mouse or human Fc, or ROR1.

[0065] The present disclosure surprisingly discovered an epitope comprising a portion located at amino acid residues 419 to 433 or amino acid residues 471 to 482 of SEQ ID NO: 8. Thus, the present disclosure provides antibodies that bind to the epitopes disclosed herein.

[0066] The present disclosure develops antibodies or antigen-binding fragments thereof specific to epitopes in CoV spike proteins (particularly SARS-CoV-2 spike proteins).

[0067] In particular, the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) of a heavy chain variable region and a complementarity determining region of a light chain variable region, the complementarity determining region of the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining region of the light chain variable region comprising 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; and 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. 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 WAS or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; 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.

[0068] In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof is m31A7 and comprises the amino acid sequence of SEQ ID NO: 6, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. The CDRH1 region comprises the amino acid sequence of SEQ ID NO: 1, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the CDRH2 region comprises the amino acid sequence of SEQ ID NO: 4, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0069] Antibodies according to the present disclosure may be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2, or scFv fragments), and may be modified as needed to affect functionality.

[0070] In some embodiments of the present 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 (an "immunoconjugate"), e.g., a toxoid or antiviral agent, for treating coronavirus infection. In one embodiment of the present invention, the anti-CoV-S antibody or fragment is conjugated to any of the additional therapeutic agents described herein.

[0071] Various techniques known to those skilled in the art can be used to determine whether an antibody "specifically binds to one or more amino acids" within a polypeptide or protein. Exemplary techniques include routine cross-blocking assays, alanine scanning mutation analysis, peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), and peptide truncation analysis, as described, for example, in "Antibodies" by Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Additionally, 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 amino acids within a polypeptide to which an antibody specifically binds is hydrogen / deuterium exchange, as detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, allowing 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, revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0072] Whether an antibody specifically binds to or competes for binding to the same epitope as a reference anti-SARS-CoV-2 spike protein antibody can be readily determined using routine methods known in the art. 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 allowed to bind to 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 with 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 with the reference anti-SARS-CoV-2 spike protein antibody, the test antibody may bind to the same epitope as 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 determine 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. These types of experiments 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, for example, a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, preferably 75%, 90%, or even 99%, as measured in a competitive binding assay. Alternatively, two antibodies are considered to bind 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.Two antibodies are considered to have "overlapping epitopes" if only the subset of amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0073] 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 proteolytic digestion or recombinant genetic engineering techniques, including, for example, 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. The DNA can be sequenced and manipulated, chemically or using molecular biology techniques, for example, to arrange 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.

[0074] 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 a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constraining FR3-CDR3-FR4 peptide. Other engineered molecules, such as 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 shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0075] Antigen-binding fragments of antibodies typically contain at least one variable domain, which 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 V related to domain H For antigen-binding fragments containing domains, V H Domain and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a dimer of monomeric V H or V L It may contain domains.

[0076] In certain embodiments, an antigen-binding fragment of an antibody may comprise 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) 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 -C H1 -CH2 ;(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. Hinge regions may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provide a flexible or semi-flexible link between adjacent variable and / or constant domains within a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present disclosure may be linked to each other 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 domain and constant domain configurations listed above, non-covalently associated (e.g., by disulfide bonds) with the domain.

[0077] 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 another mammalian germline sequence, 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 of skill in the art can readily produce numerous 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 a domain are mutated back to the residue 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, e.g., 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 that differs 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 within the framework and / or CDR regions, e.g., 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 antagonist or agonist 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.

[0078] The present disclosure also provides compounds of the V disclosed herein with one or more conservative substitutions. H , V L and / or CDR amino acid sequences. For example, the present disclosure provides anti-SARS-CoV-2 spike protein antibodies comprising any variant of the V and / or CDR amino acid sequences disclosed herein. H , V L and / or V with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer conservative amino acid substitutions compared to any of the CDR amino acid sequences. H , V L and / or an anti-EPHA10 antibody having the CDR amino acid sequence.

[0079] In some embodiments of the present disclosure, the antibody 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 region are replaced with the corresponding amino acid residues in the CDR species, e.g., rodent.

[0080] Antibodies of the present disclosure may be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of a single target polypeptide or may contain antigen-binding domains specific for two or more target polypeptides. Anti-SARS-CoV-2 spike protein antibodies of the present disclosure may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or other methods) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody with 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.

[0081] In another aspect, the present disclosure provides a genetically engineered cell that expresses the antibody or antigen-binding fragment thereof or comprises the vector. The genetically engineered cell can be an immune cell.

[0082] 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 Area and V L In embodiments that include both regions, V H Area and V LThe regions can be expressed using a single vector, for example, in a dicistronic expression unit, or under the control of different promoters. H Area and V L The regions may be expressed using separate vectors. H Area or V L The region may optionally include an N-terminal methionine.

[0083] 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 combinations of heavy and light chain gene products generate large pools of antibodies with different antigen specificities (see, e.g., Kuby, "Immunology," 3rd ed. 1997).

[0084] One 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 growing.

[0085] A polynucleotide encoding an antibody or antigen-binding fragment of the present invention can be introduced into a host cell using a vector. 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. 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 be replicated along with the host genome. Moreover, certain vectors are capable of directing 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 used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0086] The present disclosure provides pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof. The pharmaceutical compositions of the present disclosure are formulated with appropriate diluents, carriers, excipients, and other agents that provide improved entry, delivery, tolerance, etc. The compositions may be formulated for a particular use, such as veterinary use or human pharmaceutical use. The form of the composition and the excipients, diluents, and / or carriers used will depend on the intended use of the antibody and, for therapeutic use, the mode of administration. Many suitable formulations can be found in the 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 (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions of 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.

[0087] 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 compound. For example, the antiviral agent may be at least one selected from the group consisting of:1A9;201;311mab-31B5;311mab-32D4;47D11;4A8;4C2;80R;apilimod;B38;mostat 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-Griffithsin (Q-GRFT); and Griffithsin; Oseltamivir; Zanamivir; Abacavir; Zidovudine; Zalcitabine; Didanosine; Stavudine; Efavirenz; Indinavir; Ritonavir; Nelfinavir; Amprenavir; Ribavirin; Remdesivir; Chloroquine; Hydroxychloroquine; rIFN-alpha-2 a; rIFN-beta-lb; rIFN-gamma; nIFN-alpha; nIFN-beta; nIFN-gamma; IL-2; PD-L1; anti-PD-L1; checkpoint inhibitors; interferon; interferon mixtures; recombinant or natural interferons; alferon; alpha-interferon species; recombinant or natural interferon alpha; recombinant or natural interferon alpha 2a; recombinant or natural interferon beta; recombinant or natural interferon beta lb; and recombinant or natural interferon gamma. The alpha-interferon species can be a mixture of at least seven species of alpha-interferons produced by human leukocytes, the seven species being interferon alpha 2, interferon alpha 4, interferon alpha 7, interferon alpha 8, interferon alpha 10, interferon alpha 16, and interferon alpha 17.

[0088] The dose of an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. When the antibody of the present disclosure is used to treat a condition or disease associated with EPHA10 in an adult patient, it may be advantageous to administer the antibody of the present disclosure intravenously. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering antibodies can be determined empirically. For example, the patient's progress can be monitored by periodic evaluation, and the dosage can be adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0089] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, or receptor-mediated endocytosis (see, e.g., 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, for example, 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 agents. Administration can be systemic or local.

[0090] The pharmaceutical compositions 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, pen delivery devices are readily useful for delivering the pharmaceutical compositions of the present disclosure. Such pen delivery devices 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 emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0091] In certain circumstances, pharmaceutical compositions can be delivered in controlled-release systems. 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 near the target of the composition, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, "Medical Applications of Controlled Release," supra, vol. 2, pp. 115-138). Other controlled-release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0092] Injectable preparations include intravenous, subcutaneous, intradermal, and intramuscular injections, as well as infusions. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or salt thereof described herein in a sterile aqueous or oily medium conventionally used for injections. Aqueous media for injection include, for example, saline, isotonic solutions containing glucose or other auxiliary agents, and the like. These may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The injection preparations prepared in this manner are preferably filled into appropriate ampoules.

[0093] Preferably, the above-mentioned pharmaceutical compositions for oral or parenteral use are prepared in a dosage form suitable for a dosage of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0094] 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.

[0095] The present disclosure provides a method for neutralizing coronavirus in a subject in need thereof, the method comprising administering an antibody or antigen-binding fragment thereof to the subject.

[0096] The present disclosure provides a kit for detecting coronavirus in a sample, the kit comprising an antibody or antigen-binding fragment thereof.

[0097] The anti-SARS-CoV-2 spike protein antibodies of the present disclosure can 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, an anti-SARS-CoV-2 spike protein antibody or a fragment thereof can be used to diagnose a condition or disease characterized by coronavirus infection. An exemplary diagnostic assay for coronavirus can include, for example, contacting a sample obtained from a patient with an anti-SARS-CoV-2 spike protein antibody of the present disclosure, where the anti-SARS-CoV-2 spike protein antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-SARS-CoV-2 spike protein antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C, 32 P, 35 S, or 125 The 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, luciferase, etc. 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).

[0098] The following examples are provided to aid those skilled in the art in practicing the present disclosure. [Example]

[0099] Materials and Methods FACS analysis and sorting of spike-specific B cells. mgSplenocytes isolated from immunized mice were incubated with 2 μg / ml S protein for 1 h at 4°C, then washed and incubated with a cocktail of antibodies against CD19 (clone: ​​6D5, PE-Cy7 conjugate, Biolegend), CD3 (clone: ​​17A2, PE conjugate, Biolegend), and His (clone: ​​J095G46, APC conjugate, Biolegend) for 15 min at 4°C. Dead cells were excluded using propidium iodide (Biolegend). Live single spike-specific B cells (CD3 - CD19 + ) were sorted by BD FACSAria II into 96-well PCR plates (Applied Biosystems) containing 10 μl / well of capture buffer (10 mM Tris-HCl, pH 8, and 5 U / μl RNasin (Promega)). For repertoire analysis, S mg or S fg Five spleens from immunized mice were pooled and stained before sorting.

[0100] Single B cell screening. Primers were designed based on a previous publication. The reaction was then performed at 50°C for 30 minutes, 95°C for 15 minutes, followed by 40 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, with a final incubation at 72°C for 10 minutes. Semi-nested second-round PCR was performed using 1 μl of unpurified first-round PCR product with KOD One PCR Master Mix (TOYOBO) at 98°C for 2 minutes, followed by 45 cycles of 98°C for 10 seconds, 55°C for 10 seconds, and 68°C for 10 seconds, with a final incubation at 68°C for 1 minute. 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. The chimeric IgH and IgL expression constructs were co-transfected into Expi293 for antibody production.

[0101] Antibody binding to the S protein expressed on the surface of 293T cells. 293T cells were transfected with pcDNA6 / Spike-P2A-eGFP. The transfected cells were selected for 2-3 weeks under 10 μg / ml blasticidin. The selected cells were then sorted by FACSAria II to identify the eGFP-expressing S protein. + Expressing cells were obtained. These cells were maintained in DMEM containing 10% FBS and 10 μg / ml blasticidin. 2-3 × 10 5Cells were incubated with serially diluted antibodies in FACS buffer on ice for 1 hour. Cells were then washed three times with FACS buffer, then stained with BV421 mouse anti-human IgG (BD Biosciences, 562581, 1:100) on ice for 20 minutes and washed twice with FACS buffer. The percentage of positive cells was quantified using a FACS Canto II, and data were analyzed using FlowJo. The S protein variants used here are as follows: 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 and A701V; BA.1.

[0102] Affinity and avidity determination using Octet (Biolayer Interferometry). Fab fragments were prepared using the 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 using a Protein A column. Purified m31A7 IgG or Fab fragments were loaded onto a Protein G or FAB2G biosensor (Molecular Devices, ForteBio) at 10 or 6.7 μg / ml in kinetic buffer (0.01% endotoxin-free BSA, 0.002% Tween-20, 0.005% NaN3 in PBS). Association and dissociation of S protein (SARS-CoV-2 WH01) by both IgG and Fab were performed for 5 and 15 minutes, respectively, in the kinetic buffer at the indicated concentrations. D Values ​​were calculated using a 1:1 global fit model (Octet).

[0103] result: We now demonstrate the monoglycosylation state (S) of SARS-CoV-2. mg The present inventors also aimed to isolate spike-specific monoclonal antibodies from mice immunized with S mg Immunized mice and fully glycosylated spike (S fg ) immunized mice and compared the spike-specific B cell repertoire. mg Selection of S protein-specific B cells from immunized mice identified the monoclonal antibody (mAb) m31A7 from an IGHV1-18 amplified clone (Fig. 1A). mg and S fg Heavy chain repertoire analysis of spike-specific B cells isolated from immunized mice revealed that several heavy chain loci, including IGHV1-9, IGHV1-18, and IGHV2-3, are involved in the S mg Selective expansion in immunized mice was demonstrated (Figures 1B and 1C). This m31A7 mAb interacts with the full-length S protein, S1 and RBD, but not S2 (Figure 2A), and binds to HEK293T cells expressing S proteins from different SARS-CoV-2 variants (Figure 2B). Furthermore, m31A7 exhibits subpicomolar IC50 values ​​up to 1000-fold higher than the reported human mAb EY6A. 50m31A7 was shown to neutralize various pseudovirus mutants (WT, D614G, alpha, beta, and delta) (D. Zhou et al., "Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient," Nat Struct Mol Biol 27, 950-958 (2020)) (Figures 3A and 3B). Prevention studies also demonstrated good in vivo efficacy of m31A7 in K18hACE2 transgenic mice (Figure 4A) in maintaining both body weight and temperature after challenge with SARS-CoV-2 virus (Figures 4B and 4C). Biolayer interferometry (BLI) analysis measured the dissociation constant of m31A7 and its Fab binding to the S protein at 34.9 pM and 0.22 nM, respectively (Figure 5A). The binding activity of full-length m31A7 IgG1 to full-length Omicron BA.1 S protein was measured by biolayer interferometry and is shown in Figure 5B. Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed the binding region on the RBD (Figures 5C and 5D). fg The extremely low usage of IGHV1-18 in the B cell repertoire (Figures 1A and 1B) is due to the S fg These results suggest that m31A7 or related antibodies may not be elicited.

[0104] While the present disclosure has been described in conjunction with specific embodiments set forth, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are deemed to be within the scope of the present disclosure.

Claims

1. An antibody or antigen-binding fragment thereof specific for an epitope in the spike protein of CoV, wherein the epitope comprises a portion located at amino acid residues 419 to 433 or amino acid residues 471 to 482 of SEQ ID NO:

8.

2. the antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) of a heavy chain variable region and a complementarity determining region of a light chain variable region, the complementarity determining region of the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the complementarity determining region of the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions; the CDRH1 region comprises the amino acid sequence of SEQ ID NO: 1 or a sequence substantially similar thereto, the CDRH2 region comprises the amino acid sequence of SEQ ID NO: 2 or a sequence substantially similar thereto, and the CDRH3 region comprises the amino acid sequence of SEQ ID NO: 3 or a sequence substantially similar thereto; The antibody or antigen-binding fragment thereof of claim 1, wherein the CDRL1 region comprises the amino acid sequence of SEQ ID NO: 4 or a sequence substantially similar thereto, the CDRL2 region comprises the amino acid sequence of WAS or a sequence substantially similar thereto, and the CDRL3 region comprises the amino acid sequence of SEQ ID NO: 5 or a sequence substantially similar thereto.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the spike protein is fully glycosylated.

4. The antibody or antigen-binding fragment thereof of claim 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 or a sequence substantially similar thereto, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or a sequence substantially similar thereto.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.

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

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CoV is alpha-CoV, beta-CoV, gamma-CoV, or delta-CoV2.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the CoV is SARS-CoV, MERS-CoV or SARS-CoV-2.

9. A complex comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8 bound to the spike protein of SARS-CoV-2 or a fragment thereof.

10. 10. The conjugate of claim 9, wherein the spike protein is fully glycosylated.

11. A vector encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

12. A genetically engineered cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

13. A genetically engineered cell comprising the vector of claim 11.

14. 9. A method for producing the antibody or antigen-binding fragment of any of claims 1 to 8, 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 favorable for 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 growing.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, a pharmaceutically acceptable carrier, and optionally a further therapeutic agent.

16. 16. The pharmaceutical composition of claim 15, wherein the therapeutic agent is an antiviral agent.

17. 16. The pharmaceutical composition of claim 15, wherein the therapeutic agent is an anti-inflammatory agent or an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2.

18. A container or injection device comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

19. 10. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 7 in the manufacture of a medicament for the treatment or prevention of infection by coronavirus in a subject in need thereof.

20. 20. The use according to claim 19, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV and MERS-CoV.

21. 20. The use of claim 19, wherein the medicament is administered in combination with one or more additional therapeutic agents.

22. 22. The use of claim 21, wherein the one or more additional therapeutic agents is an antiviral agent.

23. 23. The use of claim 22, wherein the therapeutic agent is an anti-inflammatory agent or an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2.

24. 20. The use according to claim 19, wherein the subject is vaccinated.

25. 20. The use of claim 19, wherein the medicament is for neutralizing coronavirus in a subject.

26. 20. The use according to claim 19, wherein the medicament is injected into the body of the subject subcutaneously, intravenously or intramuscularly.

27. 9. A method for detecting coronavirus in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

28. A kit for detecting coronavirus in a sample, said kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

29. An epitope comprising a portion located at amino acid residues 419 to 433 or amino acid residues 471 to 482 of SEQ ID NO:

8.

30. 1. A method for producing spike-specific monoclonal antibodies or binding fragments thereof immunized with the monoglycosylated form (Smg) of SARS-CoV-2, said method comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing said host cells under conditions favorable for expression of said 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 growing. Including, d) the antibody or binding fragment thereof is specific for an epitope in the spike protein of CoV, and the epitope comprises a portion located at amino acid residues 419 to 433 or amino acid residues 471 to 482 of SEQ ID NO: 8; method.

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